Antibodies to platelet FcγRIIa and related methods of use

Abstract

Described is an antibody that interacts with FcγRIIa.Described is a method for detecting the amount of FcγRIIa on platelets in sample using antibody against FcγRIIa.Described is a method for treating selected subjects with risk of thrombosis with antithrombotic therapy, said method comprises administering to selected subjects antithrombotic agents that are aspirin, cilostazol, or adenosine diphosphate (ADP) receptor antagonist and / or protease-activated receptor (PAR) antagonist, wherein antibody against FcγRIIa is used to detect the amount of FcγRIIa in sample, thereby determining the level of FcγRIIa expressed on platelets, and selecting subject.In certain methods, due to the elevated level of FcγRIIa expression, subject is identified as being at risk of thrombosis and as being in need of antithrombotic therapy. TIFF2025528831000016.tif114166

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 371,636, filed August 16, 2022, the contents of which are incorporated herein by reference in their entirety.

[0002] Reference to the Electronic Sequence Listing The contents of the electronic sequence listing (179429.00023.xml; size: 21,565 bytes; and creation date: August 15, 2023) are incorporated herein by reference in their entirety. [Background technology]

[0003] background Increased platelet reactivity contributes to the greater risk of thrombosis, which is the proximate cause of heart attack and stroke.Given the negative health effects of thrombosis, assays for platelet reactivity should be useful for identifying individuals at risk of thrombosis and for selecting appropriate treatment regimens.However, current assays for platelet reactivity have not demonstrated their performance and are sensitive to commonly used drugs and other therapies.Therefore, there is a need for new assays for platelet reactivity, and related innovations, techniques and drugs. Summary of the Invention

[0004] overview As described below, the present disclosure features antibodies that bind to FcγRIIa, which are useful for assaying platelet reactivity, as well as assays that feature the use of these antibodies. Disclosed herein are monoclonal antibodies that can recognize and bind to FcγRIIa. The monoclonal antibodies can bind to and interfere with, modulate, and / or inhibit the binding interaction between FcγRIIa and other molecules.

[0005] Further compositions and methods for quantifying FcγRIIa on platelets and thereby assaying platelet reactivity are also disclosed. Platelet reactivity is a powerful marker of medical risk, but available tests for platelet reactivity to date have been sensitive to external factors and reproducibility concerns and have not proven useful as indicators of medical risk. Generally, a blood sample is drawn from an individual, and a suitable anticoagulant is added to the in vitro sample. Then, the platelets in the sample are fixed by adding a suitable fixative. Subsequently, an antibody that binds to FcγRIIa (e.g., a primary antibody conjugated with a detectable label) and / or a secondary conjugated antibody is added to the sample. The sample can be analyzed using flow cytometry. Platelets can be identified by their characteristic size, and the mean fluorescence intensity, which reflects the surface expression of FcγRIIa, can be quantified. In addition to mean fluorescence, quantitative measurements can be determined, such as the copy number of FcγRIIa, for example, the number of FcγRIIa molecules per platelet, or an increase in the level above a previous level or above a reference level. Expression of FcγRIIa at or above a predefined threshold can be used to identify patients with elevated FcγRIIa expression and increased platelet reactivity.

[0006] In one aspect, this method is based on the quantification of the surface protein FcγRIIa on platelets without platelet activation.In another aspect, this method can be performed on fixed platelets.In another aspect, this method avoids the results being affected by assay conditions (for example, anticoagulants used during blood collection) and / or patient treatment.In another aspect, this method is performed so that neither anticoagulants nor P2Y12 antagonists change the expression of FcγRIIa on platelets, and therefore do not change the test results.

[0007] In one aspect, a method for identifying a subject (for example, a human) with increased platelet reactivity is disclosed, which comprises determining the level of FcγRIIa on platelets from the subject; and comparing the level of FcγRIIa on platelets with a reference value, wherein an increased level compared to the reference value indicates that the subject has increased platelet reactivity.The level of FcγRIIa on platelets can be determined as mean fluorescence intensity (MFI) measurement.In addition, MFI measurement can be normalized and converted into a measurement of the approximate or actual copy number of FcγRIIa on platelets in sample.

[0008] In another aspect, a method is disclosed for identifying a subject (e.g., a human) at increased risk of thrombosis, the method comprising determining the level of FcγRIIa expressed on platelets from the subject; and comparing the level of FcγRIIa on the platelets with a reference value, wherein an increased level compared to the reference indicates that the subject has an increased risk of thrombosis.

[0009] In yet another aspect, a method for determining platelet reactivity is disclosed, the method comprising determining the level of FcγRIIa expressed on platelets from a subject and comparing the level of FcγRIIa on the platelets with a reference value, wherein an increased level compared to the reference is indicative of increased platelet reactivity.

[0010] In yet another aspect, disclosed is a method for selecting antithrombotic therapy for a subject, comprising determining the level of FcγRIIa expressed on platelets from the subject, and comparing the level of FcγRIIa with a reference value, wherein an increased level compared to the reference value indicates the need for preventive therapy, antithrombotic therapy, or additional antithrombotic therapy.In one embodiment, the antithrombotic therapy is one or more drugs selected from the group consisting of aspirin, cilostazol, prasugrel, ticagrelor, clopidogrel, and vorapaxar.

[0011] In yet another aspect, a kit for determining platelet reactivity is disclosed, comprising an FcγRIIa-specific reagent and instructions for use of the kit in the method of any of the above aspects.

[0012] In yet another aspect, a method for inhibiting platelet activation is disclosed, comprising administering to a subject in need thereof an effective amount of an agent that inhibits FcγRIIa activation, thereby inhibiting platelet activation. In one embodiment, the agent is any one or more of a small molecule, an inhibitory nucleic acid, and an antibody or its antigen-binding fragment. In another embodiment, the inhibitory nucleic acid is any one or more of an antisense molecule, an shRNA, and an siRNA. In one embodiment, the inhibitory nucleic acid reduces the FcγRIIa level in megakaryocytes. In another embodiment, if platelets obtained from the subject have an increased FcγRIIa level compared to the reference value, the subject is determined to be in need thereof.

[0013] In yet another aspect, a test device for detecting FcγRIIa in a liquid sample is disclosed, the device having a liquid-permeable material defining the following portions in capillary communication: a) a first portion which is a site for applying a liquid sample, comprising a liquid-permeable medium and an FcγRIIa-binding conjugate; b) a second portion which comprises the liquid-permeable medium; and c) a third portion which is a site for detecting binding of the FcγRIIa-binding conjugate at the test site, the third portion comprising a liquid-permeable medium having FcγRIIa immobilized to the medium at the test site.

[0014] In a related aspect, a method for determining platelet reactivity is disclosed, which involves using a test device of the present disclosure to determine the level of FcγRIIa expressed on platelets and comparing the level of FcγRIIa on the platelets with a reference value, wherein an increased level compared to the reference indicates increased platelet reactivity. In another related aspect, the present disclosure provides a method for detecting FcγRIIa in a liquid sample, which involves a) applying the liquid sample to a device of the present disclosure; and b) detecting the presence or absence of an FcγRIIa-binding conjugate at a test site, wherein the absence of an FcγRIIa-binding conjugate at the test site identifies the presence of FcγRIIa in the sample, and the presence of an FcγRIIa-binding conjugate at the test site identifies the absence of FcγRIIa in the sample.

[0015] In a related aspect, a kit is disclosed that includes a test device according to the present disclosure. In various embodiments, the kit includes instructions for using the device for detecting an analyte. In other embodiments, the kit includes a means for measuring a liquid sample and a test vial.

[0016] In yet another aspect, a composition or kit for identifying and treating a subject with increased platelet reactivity is provided, the composition comprising an FcγRIIa-specific reagent and instructions for using the reagent to measure the level of FcγRIIa in a biological sample of the subject (wherein the subject is identified as having increased platelet reactivity by a level of FcγRIIa greater than about 7,500 copies per platelet); and (b) a therapeutic reagent that is one or more of prasugrel, ticagrelor, clopidogrel, and vorapaxar.

[0017] In various embodiments of the above aspects, or any other aspect of the disclosure provided herein, the reference value is a measured or predicted level of FcγRIIa on the surface of platelets from disease-free, low-risk, or otherwise healthy individuals. In one embodiment, the reference value is at or about 1,500 copies of FcγRIIa per platelet, and the increased level is at or about 1,600, 1,700, 1,800, 1,900, 2,000, 2,100, 2,200, 2,300, 2,400, or 2,500 copies of FcγRIIa per platelet, or more. The increased level can be any level greater than 1,600, 1,700, 1,800, 1,900, 2,000, 2,100, 2,200, 2,300, 2,400, or 2,500 copies of FcγRIIa per platelet, or higher. In another embodiment, the level of FcγRIIa is determined using an FcγRIIa-specific reagent. In another embodiment, the FcγRIIa-specific reagent is an antibody or an antigen-binding fragment thereof. In another embodiment, the level of platelet FcγRIIa is determined using an assay selected from the group consisting of flow cytometry, immunoassay, ELISA, Western blotting, and radioimmunoassay. In another embodiment, the level of FcγRIIa is determined using a fluorometric or colorimetric assay. In yet other embodiments, the level of FcγRIIa is determined using flow cytometry.

[0018] In still other embodiments, the reference value represents the average level of FcγRIIa on platelets from a set of disease-free, low-risk, or otherwise healthy subjects. An increased level can be indicated by a level measured in a patient that is increased by at least about 1.5-fold, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, and 2.5-fold, or more, including 5-fold to 10-fold or 10-fold to 25-fold, compared to a reference value determined based on average values ​​from disease-free, low-risk, or otherwise healthy patients. In various embodiments, the level of FcγRIIa is determined using an FcγRIIa-specific reagent. In certain embodiments, the FcγRIIa-specific reagent or FcγRIIa-binding conjugate is an antibody or antigen-binding fragment thereof. In various embodiments, the antithrombotic therapy is one or more of aspirin, cilostazol, prasugrel, ticagrelor, clopidogrel, and vorapaxar, or a combination thereof.

[0019] The reference value of a set of disease-free, low-risk, or otherwise healthy subjects can be determined, for example, by measuring the level of FcγRIIa as MFI, the copy number of FcγRIIa, or other, and for example, by determining the average for a set of 2-50, 5-30, 10-20, or at least 5, or at least 10, 15, 20, or 25 subjects (including up to 30, 40, 50, and including the range of 30, 40, 50), or more, and the subjects have a medical history indicating that they are disease-free, low-risk, or otherwise healthy subjects, including those who have no history of heart disease, cardiac events, or cardiac therapy, or who otherwise have no symptoms that are associated with or may be associated with an increased risk of future cardiac events, including stroke and thrombosis.In one embodiment, a low-risk subject is a subject who does not exhibit a detectable disease state.In another embodiment, a low-risk subject is a subject who has had no more than one known cardiac event, including heart attack.

[0020] In various embodiments of any of the aspects delineated herein, the first portion of the test device further contains a control conjugate; and the third portion of the test device contains a control conjugate-binding substance present in a control site for detecting binding of the control conjugate. In additional embodiments, the analyte-binding conjugate and the control conjugate coat the surface of the liquid-permeable membrane in the first portion. In other embodiments, no coating is present in the sample application site. In additional embodiments, the test device further includes a fourth portion acting as a wick, the fourth portion including a sorbent material. In other embodiments, the second portion of the test device includes a liquid-permeable material acting as a filter to remove particulate matter. In still other embodiments, the first portion of the test device contains a conjugate that specifically binds to platelets. In various embodiments, the conjugate that specifically binds to platelets is one or more of an antibody against glycoprotein (GP) IIb, GP IIIa, GP V, GP Ib, GP IX, a lysosomal membrane protein, and platelet endothelial cell adhesion molecule (PECAM). In certain embodiments, the conjugate that specifically binds to platelets is one or more of anti-CD41, anti-CD41a, anti-CD61, anti-CD42d, anti-CD42b, anti-CD42a, anti-CD63, and anti-CD31. In still other embodiments, the second portion of the test device comprises an agent that alters the composition of the liquid when the liquid contacts the second portion.

[0021] Compositions and methods for assaying platelet reactivity, as well as therapeutic agents that act through FcγRIIa binding, are provided. The compositions and products described herein were isolated or otherwise produced in accordance with the examples provided below. Other features and advantages of the present disclosure will be apparent from the detailed description and claims.

[0022] In one aspect of the present invention, an isolated antibody or antigen-binding fragment thereof that specifically binds to FcγRIIa is provided. The isolated antibody or antigen-binding fragment thereof comprises at least one heavy chain complementarity-determining region (CDRH) and at least one light chain complementarity-determining region (CDRL). In some embodiments, the CDRH is selected from SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, and the CDRL is selected from SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, wherein SEQ ID NO: 11 encodes the amino acid sequence DTS. In some embodiments, the CDRH is selected from SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, and the CDRL is selected from SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, wherein SEQ ID NO: 21 encodes the amino acid sequence WAS.

[0023] In some embodiments, at least two heavy chain complementarity determining regions (CDRHs) and at least two light chain complementarity determining regions (CDRLs) are provided, wherein the CDRHs are selected from SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, and the CDRLs are selected from SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, or wherein the CDRHs are selected from SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17, and the CDRLs are selected from SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:22.

[0024] Some embodiments include an antibody or antibody-binding fragment thereof comprising a CDRH1 region comprising SEQ ID NO: 5, a CDRH2 region comprising SEQ ID NO: 6, and a CDHR3 region comprising SEQ ID NO: 7; and a CDRL1 region comprising SEQ ID NO: 10, a CDRL2 region comprising SEQ ID NO: 11, and a CDRL3 region comprising SEQ ID NO: 12, and a sequence at least 90% identical to SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12. Some embodiments include an antibody or antibody-binding fragment thereof comprising a CDRH1 region comprising SEQ ID NO: 15, a CDRH2 region comprising SEQ ID NO: 16, and a CDHR3 region comprising SEQ ID NO: 17; and a CDRL1 region comprising SEQ ID NO: 20, a CDRL2 region comprising SEQ ID NO: 21, and a CDRL3 region comprising SEQ ID NO: 22, as well as a sequence at least 90% identical to SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22.

[0025] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region encoded by SEQ ID NO: 4 and a light chain complementarity determining region encoded by SEQ ID NO: 9, and a sequence 90% identical to SEQ ID NO: 4 and SEQ ID NO: 9. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region encoded by SEQ ID NO: 14 and a light chain complementarity determining region encoded by SEQ ID NO: 19, and a sequence 90% identical to SEQ ID NO: 14 and SEQ ID NO: 19.

[0026] In some embodiments, the antibody or antibody-binding fragment thereof specifically binds to FcγRIIa on platelets. In some embodiments, the antibody or antibody-binding fragment thereof is linked to a detectable label. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody or antibody-binding fragment thereof is produced using recombinant antibody technology, nucleic acid aptamer technology, or non-immunoglobulin protein scaffold technology. In some embodiments, the antibody or antibody-binding fragment thereof specifically binds to FcγRIIa on platelets that have been fixed with a fixative solution before binding to the antibody or antibody-binding fragment thereof.

[0027] In some embodiments, a method for detecting FcγRIIa is provided, comprising binding FcγRIIa to an antibody or antibody-binding fragment thereof described herein. In some embodiments, a blood sample is treated with a fixative before or simultaneously with the introduction of an antibody or antibody-binding fragment thereof described herein into the blood sample. In some embodiments, the fixative is combined with the blood sample up to two days after collection. In some embodiments, the fixative is diluted with a solution comprising a buffer and glycerol before being combined with the blood sample. In some embodiments, the fixative is diluted with a solution comprising a buffer and glycerol before being combined with the blood sample. In some embodiments, the buffer is phosphate-buffered saline (PBS), and the glycerol constitutes approximately 2% to 7% of the total volume of the buffer-glycerol solution. In some embodiments, the glycerol is 5% of the total volume of the buffer-glycerol solution.

[0028] In some embodiments, the bound FcγRIIa and antibody complexes are detected via flow cytometry to measure the level of FcγRIIa in the sample. In some embodiments, the measured level of FcγRIIa in the sample is normalized based on a comparison of the known level of a fluorescent marker with the measured level of fluorescence in the sample.

[0029] Provided is a method for detecting the presence of FcγRIIa in a blood sample, comprising the steps of treating the blood sample with an anticoagulant, adding a fixative to the blood sample, separating and washing platelets from the blood sample, incubating the platelets with an antibody, including any one of the antibodies or antigen-binding fragments described herein, and performing an analysis to quantify FcγRIIa in the sample. In some embodiments, the analysis is selected from the group consisting of flow cytometry, immunoassay, ELISA, Western blotting, and radioimmunoassay. In some embodiments, the method described herein further comprises reducing the concentration of fixative in the blood sample and storing the blood sample for up to 1-14 days, including 1-14 days, before incubating the platelets with the antibody.

[0030] In a second aspect, the invention described herein provides a method of preparing a blood sample, the method comprising treating the blood sample with an anticoagulant, adding a fixative to the blood sample, subsequently diluting the concentration of the fixative in the blood sample, and storing the blood sample for a period of up to and including 1 to 14 days. In some embodiments of the method, the fixative is added to a concentration of 5% and subsequently diluted to 1.25%.

[0031] Another aspect of the present invention provides a method for detecting FcγRIIa, comprising the step of binding FcγRIIa to an antibody, wherein the antibody comprises at least one heavy chain complementarity determining region (CDRH) and at least one light chain complementarity determining region (CDRL). In some embodiments, the CDRH is selected from SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, and the CDRL is selected from SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, wherein SEQ ID NO: 11 encodes the amino acid sequence DTS. In some embodiments, the CDRH is selected from SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, and the CDRL is selected from SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, wherein SEQ ID NO: 21 encodes the amino acid sequence WAS. In some embodiments, FcγRIIa is detected on platelets from a blood sample.

[0032] In some aspects, the method further comprises comparing the level of detection of FcγRIIa between a blood sample from a human having or suspected of having heart disease, thrombosis, coronary artery disease, kidney disease, or myocardial infarction and a control human blood sample without heart disease, thrombosis, coronary artery disease, kidney disease, or myocardial infarction.

[0033] In some aspects, a method of detecting platelet reactivity in a subject is provided, the method comprising detecting platelet reactivity using an antibody or antibody-binding fragment thereof described herein. [Brief explanation of the drawings]

[0034] [Figure 1A]Figures 1A-1E show Western blots and graphs demonstrating FcγRIIa phosphorylation as a result of platelet activation. Platelets (2 × 108 in 0.5 ml) from low-risk subjects isolated by gel filtration were exposed to selected intervals of time with or without agonist (Figure 1E) or with thrombin (50 nM) (Figure 1A), convulxin (10 ng / ml) (Figure 1B), ADP (25 μM) (Figure 1C), and PAF (100 nM) (Figure 1D) prior to preparation of platelet lysates. FcγRIIa was isolated by immunoprecipitation, and Western blots were probed with anti-phosphotyrosine antibody (4G10), stripped, and reprobed with anti-FcγRIIa to confirm equal loading (data not shown). The fluorescence intensity of the bands was quantified using the Li-Cor system. Results (n = 3–5 per condition) are peak intensities that do not change with adjustment of image contrast or region of interest size. Results were compared to those obtained without agonist (Student's t-test, *p<0.05, **p<0.01). The inset shows a representative gel probed with an anti-phosphotyrosine antibody. In addition to the expected band at 45 kDa, bands at 55 kDa, 75 kDa, and 90 kDa showed evidence of phosphorylation. Activation by each agonist resulted in phosphorylation of FcγRIIa after 3 minutes. Results with thrombin and PAF were evident earlier than those with ADP. [Figure 1B] See legend to Figure 1A. [Figure 1C] See legend to Figure 1A. [Figure 1D] See legend to Figure 1A. [Figure 1E] See legend to Figure 1A. [Figure 2A]Figures 2A and 2B are Western blots and gels. Figure 2A is a Western blot of anti-phosphotyrosine immunoprecipitates showing that FcγRIIa is phosphorylated after platelet activation by thrombin. Tyrosine-phosphorylated proteins (Figure 2A) were isolated by immunoprecipitation using an anti-phosphotyrosine antibody (4G10) from platelet lysates (2 × 10 platelets) exposed to thrombin (50 nM) for 3 minutes without agonist. Western blots of immunoprecipitated proteins in lanes 1 and 2 and unselected platelet lysate in lane 3 were probed with anti-FcγRIIa. Anti-FcγRIIa identified tyrosine-phosphorylated proteins at 45 kDa, 55 kDa, 75 kDa, and 90 kDa. Figure 2B is a representative gel of lipid raft preparations showing results from non-activated (control) and activated (10 ng / ml convulxin) platelets (4 x 108 in 0.5 ml). After 1.5 minutes, platelets were lysed and lipid rafts were prepared (sucrose gradient). Lipid rafts were identified using cholera toxin B (CTB). FcγRIIa was immunoprecipitated, and Western blots were probed with anti-phosphotyrosine antibody (4G10), stripped, and reprobed with anti-FcγRIIa. Bands were identified using chemiluminescence. [Figure 2B] See legend to Figure 2A. [Figure 3]This is a set of micrographs showing the intracellular localization of CD36 and FcγRIIa before and during platelet activation. Expression of FcγRIIa and CD36 by human platelets (top panel). PRP was prepared, and platelets were activated with thrombin (50 nM), convulxin (10 ng / ml), ADP (25 μM), and PAF (100 nM). After fixation with Optilyse (1.5% formaldehyde), platelets were pretreated with 1% BSA and then incubated with mouse anti-CD36 and goat anti-FcγRIIa for 1 hour. After three washes in HT buffer, secondary antibodies were used to identify CD36 (Alexa 488 anti-mouse IgG) and FcγRIIa (Alexa 555 anti-goat IgG). Platelets were imaged using a Zeiss LSM 510 META confocal / scanning laser microscope. Representative platelets for each condition were imaged using differential interference contrast (phase contrast) on the right. Fluorescence imaging was used to identify CD36 (green signal) and FcγRIIa (red signal). Colocalization is confirmed by the yellow signal. Platelet activation was associated with the colocalization of FcγRIIa with lipid rafts. The effects of an FcγRIIa antagonist (Fab fragment of IV.3) and a GP IIb-IIIc antagonist (tirofiban) on platelet FcγRIIa expression (lower panel). Non-activated and thrombin-activated platelets were pretreated with IV.3 (100 μg / ml) or tirofiban (0.5 μg / ml). Representative platelets exhibited reduced FcγRIIa clustering following pretreatment with IV.3. Magnification: 2,000x. [Figure 4]Figures 4A and 4B are graphs and Western blots showing FcγRIIa expression (Figure 4A) and FcγRIIa phosphorylation (Figure 4B) in response to thrombin. Patients taking aspirin but no other antiplatelet or anticoagulant agents who had a previous myocardial infarction or coronary revascularization were screened to identify three patients with elevated FcγRIIa expression (mean fluorescence intensity ≥ 2). FcγRIIa expression on platelets in low-risk subjects was found to be less than 1.5 units (n = 5). To confirm the results obtained using flow cytometry, Western blot analysis was performed using platelet lysates (Figure 4A insert). Platelets (2 × 108 in 0.5 ml) isolated by gel filtration were activated with thrombin (50 nM). Phosphorylation of FcγRIIa was quantified as described for Figures 1A–1E. Patients with increased expression of FcγRIIa exhibited greater phosphorylation after activation (p<0.05). Results are mean±SD. [Figure 5A]Figures 5A–5D are graphs showing the effect of a selective FcγRIIa antagonist (Fab IV.3) on platelet activation and aggregation. Platelet activation was assessed by flow cytometry via PAC-1 binding (reflecting GP IIb-IIIa activation, Figure 5A) or P-selectin surface expression (Figure 5B). Whole blood from low-risk subjects (n = 6) was added to reaction tubes containing fluorochrome-conjugated antibodies, selected agonists, and either control or antagonists. Neither equimolar concentrations of non-immune IgG nor the Fab of non-immune IgG attenuated platelet activation (data not shown). IV.3 attenuated activation induced by each agonist, an effect that was most pronounced when activation was confirmed by PAC-1 (Figure 5A). Platelet aggregation was assessed by transmitted light aggregometry (Figure 5C). Aggregation induced in platelet-rich plasma (stirred and warmed to 37°C) with collagen, thrombin receptor agonist peptide (TRAP), adenosine diphosphate (ADP), and platelet-activating factor (PAF) (n = 3 for each agonist) was inhibited by IV.3. Figure 5D shows the effect of tirofiban (0.5 g / ml) on platelet activation (from n = 3 subjects) as determined by surface expression of P-selectin. Consistent with its mechanism of action, tirofiban abolished PAC-1 binding to platelets. Results are means ± SD. Differences were determined using a paired Student's t-test. [Figure 5B] See legend to Figure 5A. [Figure 5C] See legend to Figure 5A. [Figure 5D] See legend to Figure 5A. [Figure 6]Figures 6A and 6B are graphs showing the effect of a nonselective antagonist of SRC kinase (PP2) on platelet activation. Platelet activation was assessed by flow cytometry via PAC-1 binding (Figure 6A) or P-selectin surface expression (Figure 6B). Whole blood from low-risk subjects (n = 7) was added to reaction tubes containing fluorochrome-conjugated antibodies, selected agonists, and either control conditions (vehicle alone, dimethyl sulfoxide) or antagonists (PP2, 20 μM). Inhibition of tyrosine phosphorylation by PP2 inhibited activation induced by each agonist. Results are mean ± SD of the percentage inhibition (1 - [PP2 / control]). Differences between results from PP2 and control conditions were confirmed using a paired Student's t-test. [Figure 7] 1 is a graph showing that FcγRIIa levels on platelets from patients with end-stage renal disease (ESRD) correlate with platelet reactivity. [Figure 8A] Figures 8A and 8B are graphs showing the levels of FcγRIIa on platelets from patients with coronary artery disease (CAD) or end-stage renal disease (ESRD) compared to low-risk controls. Figure 8A is a graph showing that platelets from patients with coronary artery disease (CAD) or end-stage renal disease (ESRD) have higher levels of FcγRIIa compared to low-risk controls. Figure 8B is a graph showing that platelets from patients with coronary artery disease (CAD) and who have had one or more myocardial infarctions (MI) have higher levels of FcγRIIa compared to low-risk controls. [Figure 8B] See legend to Figure 8A. [Figure 9] 1 is a table showing a list of inflammation-related cytokines and growth factors that lead to FcγRIIa expression. [Figure 10] Figure 1 shows that IFNγ increases FcγRIIa expression by megakaryocytes. The top graph demonstrates that IFNγ increases FcγRIIa expression by cells exhibiting megakaryocyte characteristics. The bottom graph of the figure shows evidence of stem cell differentiation into megakaryocytes. [Figure 11] Figure 1 shows that IFNγ increases FcγRIIa expression by human stem cell-derived megakaryocytes. The top graph demonstrates that IFNγ increases FcγRIIa expression by cell lines exhibiting megakaryocytic characteristics. The bottom graph of the figure shows evidence of differentiation of the cells into megakaryocytes. [Figure 12] 1 shows the effect of IFNγ on the expression of FcγRIIa on platelets. [Figure 13] 1 shows the effect of IFNγ on FcγRIIa expression in monocytic and myeloid cell lines. [Figure 14] FIG. 1 is a schematic diagram illustrating the interrelationship between FcγRIIa, platelet activation, and atherogenesis. [Figure 15] 1 is a graph showing that FcγRIIa-specific antibodies activate platelets. [Figure 16] 1 provides a series of Western blots showing FcγRIIa phosphorylation in thrombin-activated platelets. [Figure 17] The phosphorylation status of FcγRIIa immunoprecipitated from ADP-activated platelets is shown. [Figure 18] 1 is a diagram of lipid rafts. [Figure 19] 1 is a diagram showing the cytoskeletal rearrangements that occur during platelet activation. [Figure 20] 1 shows the effect of coagulation factor XIII on platelet activation. [Figure 21] 1 illustrates the role of FcγRIIa in platelet activation. [Figure 22] 1 is a table summarizing clinical events in patients with elevated platelet FcγRIIa expression. [Figure 23] 1 shows the results of a study demonstrating the effect of time between blood draw and antibody incubation. [Figure 24] Results are shown for tests where the fixative was diluted prior to storage and subsequent antibody incubation. [Figure 25] 1 shows the results of a study on the storage of blood samples from patients with elevated FcγRIIa expression. [Figure 26] 1 shows a study demonstrating the effect of varying the time for fixation of blood samples. [Figure 27] 1 shows the results of a study demonstrating the effect of varying fixative dilution levels after 15 minutes of fixation with 0.5% formaldehyde. [Figure 28] 1 shows the results of a study demonstrating the effect of varying the initial concentration of fixative on blood samples fixed with formaldehyde for 15 minutes within 2 hours of sample collection. [Figure 29] 1 shows the results of a study on two different subjects comparing a commercially available FcγRIIa antibody with the 5G1 antibody described herein. [Figure 30] It has been shown that platelets can be fixed for up to two days after blood is drawn from a subject. [Figure 31] The stability of the fixed sample over time is shown. [Figure 32] Figure 1 shows the stability of samples over time after fixation in fixation buffer diluted with PBS-glycerol. DETAILED DESCRIPTION OF THE INVENTION

[0035] Detailed Description of the Disclosure The present disclosure features compositions and methods that are useful for determining platelet reactivity in a subject's biological sample, for identifying subjects at increased risk of thrombosis, and for selecting appropriate therapy for such high-risk subjects, including subjects at high risk for a subsequent cardiovascular event.

[0036] The present disclosure is based, at least in part, on the discovery that FcγRIIa contributes to (e.g., amplifies) platelet activation, and thus greater expression of FcγRIIa increases the degree of platelet activation; that the FcγRIIa protein level per platelet correlates with disease status (e.g., the FcγRIIa protein level per platelet increases 2-5 fold in subjects with atherosclerosis and diabetes, and increases 2-10 fold in subjects with end-stage renal disease); and that FcγRIIa levels are useful for identifying subjects with an increased tendency to develop thrombosis and related thrombotic diseases, and for selecting appropriate therapies for such subjects. In particular, increased platelet reactivity is useful for identifying subjects who may benefit from more aggressive drug treatment (e.g., treatment with more potent antiplatelet agents such as clopidogrel, prasugrel, ticagrelor, or vorapaxar).

[0037] Thus, the present disclosure provides methods for measuring FcγRIIa, including flow cytometry- and immunoassay-based methods, diagnostic methods employing FcγRIIa as a marker of platelet reactivity, and methods for selecting appropriate therapeutic agents for subjects identified as having increased platelet reactivity compared to a reference, because FcγRIIa increases platelet reactivity and is therefore a marker of elevated platelet reactivity. Advantageously, the diagnostic methods of the present disclosure can be used on biological samples (e.g., blood, serum, and plasma) obtained from subjects being treated with antiplatelet or anticoagulant agents.

[0038] In certain aspects, the present disclosure provides test devices, such as lateral flow devices, which include a liquid-permeable medium that allows a liquid sample (e.g., blood, serum, plasma) to flow through the device. The test devices of the present disclosure can be used to detect an analyte of interest (FcγRIIa) via a detectably labeled reactive substance capable of specifically interacting with the analyte (FcγRIIa). The test devices described herein are particularly suitable for detecting an antigen of interest using an antibody that specifically binds to the antigen and conventional immunoassay techniques.

[0039] FcγRIIa As reported in more detail below, when platelet activation induces cytoskeletal rearrangements, FcγRIIa was found to cluster in cytoskeletal lipid rafts. When FcγRIIa is crosslinked with fibrinogen and coagulation factor XIII, clustering leads to phosphorylation. Phosphorylation of FcγRIIa leads to downstream phosphorylation and ultimately to calcium release, which enhances platelet activation. Consistent with the association of FcγRIIa with membrane cytoskeletal proteins during activation, results from confocal microscopy and lipid raft preparations demonstrated clustering of FcγRIIa restricted to membrane cytoskeletal lipid rafts. The results presented herein indicate that reorganization of membrane cytoskeletal proteins during activation is involved in the clustering of FcγRIIa, which appears to promote crosslinking of FcγRIIa with fibrinogen and factor XIII. Crosslinking with fibrinogen and factor XIII leads to phosphorylation by SRC kinases (e.g., Lyn).

[0040] Furthermore, fibrinogen and factor XIII coimmunoprecipitated with FcγRIIa from activated platelets and increased platelet activation. Inhibition of fibrinogen binding to GP IIb-IIIa did not abolish the amplification of fibrinogen-induced activation. Furthermore, platelet activation induced by activating anti-FcγRIIa antibodies was not attenuated by tirofiban. These results indicate that the interaction between FcγRIIa and GP IIb-IIIa is sufficient, but probably not essential, for FcγRIIa to contribute to platelet activation.

[0041] The fibrinogen-induced amplification of platelet activation was abolished by antibody IV.3 Fab, a specific inhibitor of FcγRIIa, but not by tirofiban. In contrast, platelet activation induced by coagulation factor XIII was abolished by both IV.3 and tirofiban. Without being bound by any particular theory, this finding is consistent with the hypothesis that cross-linking of FcγRIIa homodimers with fibrinogen or anti-FcγRIIa antibodies, as well as cross-linking of heterodimers (FcγRIIa and GP IIb-IIIa) by coagulation factor XIII, leads to phosphorylation of FcγRIIa, which amplifies platelet activation.

[0042] Inhibition of FcγRIIa phosphorylation appeared to be less effective at higher thrombin concentrations. This observation is consistent with previous results (Canobbio I, et al., Cell Signal 2006; 18:861-70) and indicates that FcγRIIa phosphorylation is not essential for platelet activation. Phosphorylation of FcγRIIa appears to amplify platelet activation in much the same way that the release of thromboxane A2 and ADP during the activation process amplifies the degree of platelet activation (Murray R, et al., Proc Natl Acad Sci USA 1989; 86:124-8; and Storey RF, et al., Platelets 2001; 12:443-7). These results are consistent with the greater platelet reactivity observed when platelet expression of FcγRIIa is increased (Calverley DC, et al., Atherosclerosis 2002; 164:261-7; Canobbio I, et al., Cell Signal 2006; 18:861-70; and Serrano FA, et al., Thromb J 2007; 5:7).

[0043] Coagulation factor XIII has a potent effect on platelet activation, increasing the degree of activation nearly fourfold. These results indicate that the effect of factor XIII is mediated by FcγRIIa. Furthermore, inhibition of SRC kinase (a downstream kinase) with PP2 attenuated platelet activation. Given the essential role of Fcγ in GP VI-mediated activation, the effect was most pronounced in convulxin-induced activation.

[0044] "FcγRIIa" refers to low affinity immunoglobulin gamma Fc region receptor II-a. An exemplary amino acid sequence of FcγRIIa is GenBank Accession No. NP_001129691.1: (SEQ ID NO 1) It is available as TIFF2025528831000002.tif50128.

[0045] An exemplary nucleic acid sequence encoding FcγRIIa is GenBank Accession No. NM_001136219.1: (SEQ ID NO 2) It is available as TIFF2025528831000003.tif132144.

[0046] By "FcγRIIa-specific agent" is meant any small molecule compound, antibody, nucleic acid molecule, or polypeptide, or fragment thereof, that specifically binds to FcγRIIa.

[0047] "Platelet reactivity" refers to the susceptibility of platelets to activation and clotting. Platelet reactivity tests are based on the distinction between low and high platelet reactivity. Platelet activity or reactivity includes, but is not limited to, platelet adhesion and aggregation. Platelet reactivity is a determining factor in thrombosis.

[0048] Increased platelet FcγRIIa expression has been associated with a clinical phenotype of increased platelet reactivity and evidence of an increased risk of thrombosis. Greater platelet FcγRIIa expression has been observed in blood from patients with prior stroke, myocardial infarction, and unstable angina. Somewhat more compelling evidence of a prothrombotic phenotype is provided by the association of greater platelet FcγRIIa expression with a greater risk of subsequent thrombotic events in patients with end-stage renal disease (El-Shahawy M, et. al., Am J Kidney Dis. 2007; 49:127-34). Among patients with a myocardial infarction (MI), high platelet FcγRIIa expression (≥11,000 copies / platelet) was associated with a greater risk of MI, stroke, and death (odds ratio, >4). Platelet expression of FcγRIIa can be used to identify patients at high and low risk of an event after MI or for patients with CAD.

[0049] The results disclosed herein demonstrate that activated platelets have phosphorylated FcγRIIa associated with membrane cytoskeletal proteins, fibrinogen, and coagulation factor XIII. These results indicate that platelet activation leads to plasma membrane cytoskeletal rearrangement, FcγRIIa clustering, and cross-linking of FcγRIIa with fibrinogen and factor XIII associated with lipid raft proteins. Lipid raft proteins (SRC kinases) phosphorylate FcγRIIa, which leads to downstream signal transduction and functions to amplify platelet activation.

[0050] Based on the results reported herein, it has been discovered that increased levels of FcγRIIa on platelets cause and therefore indicate increased platelet reactivity. Furthermore, the use of FcγRIIa is superior to other measures of platelet reactivity, as FcγRIIa levels are not affected by commonly used therapies and medications. Thus, the use of platelet FcγRIIa as a marker of platelet reactivity is not affected by antiplatelet treatment.

[0051] diagnosis The present disclosure features a diagnostic assay for identifying subjects with elevated levels of FcγRIIa, which indicates high platelet reactivity and an increased risk of thrombotic disease. In one embodiment, the level of platelet FcγRIIa is measured in a subject sample and used to characterize platelet reactivity in the subject. Any suitable method can be used to detect platelet FcγRIIa in a subject sample and to characterize platelet reactivity in blood from the subject. Biological samples include body fluids (e.g., blood, blood serum, plasma, and saliva). Successful implementation of the present disclosure can be achieved by one or a combination of methods that can detect and / or quantitate platelet FcγRIIa. Immunoassays in various formats (e.g., flow cytometry, ELISA) are common methods for detecting analytes captured on a solid phase. Such methods typically involve the use of FcγRIIa-specific antibodies.

[0052] Virtually any method known in the art can be used to detect FcγRIIa. For example, the level of platelet FcγRIIa can be compared by techniques well known in the art, such as flow cytometry, immunoassay, ELISA, Western blotting, radioimmunoassay, immunocytochemistry, binding to magnetic beads and / or antibody-coated beads, in situ hybridization, fluorescence in situ hybridization (FISH), flow chamber adhesion assay, microarray analysis, or colorimetric assay. The method may further include one or more of electrospray ionization mass spectrometry (ESI-MS), ESI-MS / MS, ESI-MS / (MS)n, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), surface-enhanced laser desorption / ionization time-of-flight mass spectrometry (SELDI-TOF-MS), desorption / ionization on silicon (DIOS), secondary ion mass spectrometry (SIMS), quadrupole time-of-flight (Q-TOF), atmospheric pressure chemical ionization mass spectrometry (APCI-MS), APCI-MS / MS, APCI-(MS)n, atmospheric pressure photoionization mass spectrometry (APPI-MS), APPI-MS / MS, and APPI-(MS)n, quadrupole mass spectrometry, Fourier transform mass spectrometry (FTMS), and ion trap mass spectrometry, where n is an integer greater than 0.

[0053] The detection method can include the use of biochip array.The biochip array useful in the present disclosure includes protein array and polynucleotide array.One or more markers are captured on the biochip array and are subjected to analysis to detect the level of the marker in the sample.

[0054] Platelet FcγRIIa can be captured using a capture reagent immobilized on a solid support, such as a biochip, a multi-well microtiter plate, a resin, or a nitrocellulose membrane, which is then probed for the presence or level of the marker. Capture can be on a chromatographic surface or a biospecific surface. For example, a sample containing the marker, such as serum, can be used to contact the activated surface of the biochip for a sufficient time to allow binding. Unbound molecules are washed away from the surface using a suitable eluent, such as phosphate-buffered saline. Generally, the more stringent the eluent, the more tightly the protein must be bound to be retained after washing.

[0055] Once captured on the biochip, the analytes can be detected by a variety of detection methods, such as gas-phase ion spectroscopy, optical methods, electrochemical methods, atomic force microscopy, and radio frequency methods. In one embodiment, mass spectrometry, particularly SELDI, is used. Optical methods include, for example, fluorescence, luminescence, chemiluminescence, absorbance, reflectance, transmittance, birefringence, or refractive index detection (e.g., surface plasmon resonance, ellipsometry, resonant mirror methods, grating coupler waveguide methods, or interferometry). Optical methods include microscopy (both confocal and non-confocal), imaging, and non-imaging methods. Electrochemical methods include voltammetry and amperometry. Radio frequency methods include multipolar resonance spectroscopy.

[0056] Mass spectrometry (MS) is a well-known tool for analyzing chemical compounds. Thus, in one embodiment, the method of the present disclosure comprises performing quantitative MS to measure serum peptide markers. The method can be performed in an automated format (Villanueva, et al., Nature Protocols (2006) 1(2):880-891) or semi-automated format. This can be achieved, for example, using an MS operatively linked to a liquid chromatography device (LC-MS / MS or LC-MS) or a gas chromatography device (GC-MS or GC-MS / MS). Methods for performing MS are known in the art and are disclosed, for example, in U.S. Patent Application Publication Nos. 20050023454; 20050035286; U.S. Patent No. 5,800,979, and the references disclosed therein.

[0057] Protein fragments, whether peptides derived from the protein backbone or side chain residues, are collected on a collection layer. They can then be analyzed by spectroscopic methods based on matrix-assisted laser desorption / ionization (MALDI) or electrospray ionization (ESI). A preferred technique is MALDI with time-of-flight (TOF) analysis, known as MALDI-TOF MS. This involves forming a matrix on a membrane using an agent that strongly absorbs incident light at the specific wavelength employed, as described, for example, in the literature. In a MALDI mass spectrometer, the sample is excited into the gas phase by UV or IR laser light. Ions are generated by evaporation, forming an ion plume. The ions are accelerated in an electric field and separated according to their travel time along a given distance, yielding highly accurate and sensitive mass-to-charge (m / z) readings. MALDI spectrometers are commercially available from PerSeptive Biosystems, Inc. (Frazingham, Mass., USA) and are described, for example, in the above-cited references by M. Kussmann and P. Roepstorff.

[0058] In other embodiments, the level of FcγRIIa is detected in combination with one or more additional markers.Although each individual marker is a useful diagnostic marker, in some cases, a combination of markers provides greater predictive value than a single marker alone.Detecting multiple markers in sample (or their absence in some cases) can increase the percentage of true positive and true negative diagnoses, and can reduce the percentage of false positive or false negative diagnoses.Therefore, the method disclosed in the present invention provides for the measurement of more than one marker or clinical parameter.

[0059] The use of multiple markers increases the predictive value of the test and provides greater utility in diagnosis, toxicology, patient stratification, and patient monitoring. A process called "pattern recognition" detects the patterns formed by multiple markers. The inclusion of additional markers can improve the sensitivity and specificity of determining the patient's risk of developing thrombotic diseases or disorders associated with undesirable increases in platelet reactivity. Subtle variations in data from clinical samples indicate that certain patterns of protein levels or expression (e.g., FcγRIIa levels) can predict phenotypes such as increased platelet reactivity, or identify patients who may benefit from more aggressive drug treatment (e.g., treatment with more potent antiplatelet agents such as clopidogrel, prasugrel, ticagrelor, or vorapaxar).

[0060] The expression level of platelet FcγRIIa correlates with platelet reactivity and is therefore useful for diagnosis. Antibodies that specifically bind to FcγRIIa, or any other method known in the art, can be used to monitor platelet FcγRIIa expression. Detection of changes compared to a normal reference sample can be used as a diagnostic indicator of platelet reactivity. In certain embodiments, a 1.3- to 2-, 3-, 4-, 5-, or 6-fold difference in platelet FcγRIIa levels or MFI measurements is indicative of platelet reactivity.

[0061] In one embodiment, platelet FcγRIIa levels are measured on at least two separate occasions, and changes in levels compared to normal reference levels over time are used as an indicator of platelet reactivity or propensity to develop thrombosis. Generally, platelet FcγRIIa levels are present at low levels (about 1,500 copies per platelet) in low-risk subjects (e.g., subjects without reactive platelets). In one embodiment, increased levels of platelet FcγRIIa (about 1,600, 1,700, 1,800, 1,900, 2,000, 2,100, 2,200, 2,300, 2,400, or 2,500 copies of FcγRIIa per platelet, or more, are indicative of platelet reactivity. Increased levels include ranges of 1,600-2500, 1,700-2,400, 1,800-2,300, 1,900-2,200, and all of the various combinations and points within these ranges. Alternatively, the FcγRIIa level may be any level, such as 1,600, 1,700, 1,800, 1,900, 2,000, 2,100, 2,200, 2,300, 2,400, or 2,500 copies of FcγRIIa per platelet, or greater than 1,600, 1,700, 1,800, 1,900, 2,000, 2,100, 2,200, 2,300, 2,400, or 2,500 copies of FcγRIIa per platelet, or greater. Preferably, the copies of FcγRIIa per platelet are measured using FACS analysis.

[0062] In one embodiment, the reference value is at or about 1,500 copies of FcγRIIa per platelet, and the increased level is at or about 1,600, 1,700, 1,800, 1,900, 2,000, 2,100, 2,200, 2,300, 2,400, or 2,500 copies of FcγRIIa per platelet, or at or above 1,600, 1,700, 1,800, 1,900, 2,000, 2,100, 2,200, 2,300, 2,400, or 2,500 copies of FcγRIIa per platelet. The increased level can be 1,600, 1,700, 1,800, 1,900, 2,000, 2,100, 2,200, 2,300, 2,400, or 2,500 copies of FcγRIIa per platelet, or any level greater than 1,600, 1,700, 1,800, 1,900, 2,000, 2,100, 2,200, 2,300, 2,400, or 2,500 copies of FcγRIIa per platelet, or higher.

[0063] The diagnostic methods described herein can be used individually or in combination with any other diagnostic method described herein for more accurate diagnosis of the presence or severity of thrombotic disease.

[0064] The correlation may take into account the amount of platelet FcγRIIa in the sample compared to a control amount of platelet FcγRIIa (e.g., in a low-risk subject or a subject with undetectable platelet reactivity). The control may be, for example, the average or median amount of platelet FcγRIIa present in comparable samples from normal subjects. The control amount is measured under the same or substantially similar experimental conditions as those used to measure the test amount. As a result, a control with a known normal phenotype can be used as a reference standard, and each result can be compared to that standard rather than rerunning the control.

[0065] Therefore, marker profile can be obtained from subject sample and compared with reference value obtained from reference population, so that subject can be classified as belonging to or not belonging to reference population.Correlation can take into account the presence or absence of marker in test sample and the detection frequency of the same marker in control.Correlation can take into account both of these factors to help determine the current state of cancer.

[0066] In certain embodiments, the method further comprises selecting an antithrombotic therapy. For example, if platelet reactivity indicated by measuring the MFI or actual copy number of FcγRIIa is increased by 1.3 to 5 times, 5 to 10 times, or 10 to 25 times compared to the reference, the patient is identified as likely to benefit from more aggressive drug treatment (e.g., treatment with a more potent antiplatelet agent such as clopidogrel, prasugrel, ticagrelor, or vorapaxar). The present disclosure also provides such a method, in which platelet FcγRIIa is measured again after antithrombotic therapy. In these cases, the method is used to monitor the current state of platelet reactivity.

[0067] antibody Antibodies are glycoproteins. The basic functional unit of each antibody is an immunoglobulin (Ig) monomer (containing only one Ig unit). An Ig monomer is a "Y"-shaped molecule consisting of four polypeptide chains: two identical heavy chains and two identical light chains connected by disulfide bonds. Each chain is composed of structural domains called immunoglobulin domains, which consist of seven (constant domains) to nine (variable domains). The type of heavy chain present defines the antibody class; these chains are found in IgA, IgD, IgE, IgG, and IgM antibodies, respectively. Each heavy chain has two regions: the constant region and the variable region. The constant region is identical in all antibodies of the same isotype but differs in antibodies of different isotypes. The variable region of the heavy chain differs in antibodies produced by different B cells but is the same in all antibodies produced by a single B cell or B cell clone. Mammals have two types of immunoglobulin light chains: lambda and kappa. The light chain has two consecutive domains: one constant domain and one variable domain.

[0068] The variable domain, also called the Fv region, is the most important region for antigen binding. More specifically, the variable loops (three in the light (VL) chain and three in the heavy (VH) chain) are responsible for antigen binding. These loops are called complementarity-determining regions (CDRs).

[0069] By "antigen-binding fragment thereof" or "fragment thereof," we refer to a portion of a polypeptide that retains its ability to specifically and selectively bind to an antigen or target. In the present application, the antigen or target is FcγRIIa. Preferably, the antigen-binding fragment contains the antigen-binding region of a VLR so as to retain its ability to selectively and specifically bind to FcγRIIa. Those skilled in the art will be able to easily determine a suitable antigen-binding fragment capable of binding to FcγRIIa using the methods described in the Examples below.

[0070] As reported herein, antibodies that specifically bind to FcγRIIa are useful in diagnostic methods as well as therapeutic methods. For example, antibodies that act as platelet FcγRIIa antagonists (e.g., IV.3 Fab) are particularly useful in the methods of the present disclosure. In certain aspects, the present disclosure provides a method of using an anti-platelet FcγRIIa antibody to inhibit platelet reactivity. IV.3 is a monoclonal anti-FcγRIIa antibody that inhibits the phosphorylation of platelet FcγRIIa during platelet activation.

[0071] Other antibodies useful in the present disclosure are antibodies that attenuate platelet FcγRIIa signaling. Methods for preparing antibodies are well known to those skilled in the scientific field of immunology. As used herein, the term "antibody" refers not only to intact antibody molecules but also to fragments of antibody molecules that retain immunogen-binding ability. Such fragments are also well known in the art and are routinely employed both in vitro and in vivo. Thus, as used herein, the term "antibody" refers not only to intact immunoglobulin molecules but also to the well-known active fragments F(ab')2 and Fab. F(ab')2 and Fab fragments, which lack the Fc fragment of an intact antibody, are cleared from the circulation more rapidly and may have less nonspecific tissue binding than an intact antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983)). Antibodies of the present disclosure include natural whole antibodies, bispecific antibodies; chimeric antibodies; Fab, Fab', single chain V region fragments (scFv), fusion polypeptides, and non-conventional antibodies.

[0072] Non-conventional antibodies include, but are not limited to, nanobodies, linear antibodies (Zapata et al., Protein Eng. 8(10): 1057-1062, 1995), single-domain antibodies, single-chain antibodies, and antibodies with multiple valencies (e.g., diabodies, tribodies, tetrabodies, and pentabodies). Nanobodies are the smallest fragments of naturally occurring heavy-chain antibodies that have evolved to be fully functional in the absence of light chains. Nanobodies possess the affinity and specificity of conventional antibodies, despite being only half the size of a single-chain Fv fragment. A consequence of this unique structure, coupled with their extreme stability and high degree of homology to human antibody frameworks, is that nanobodies can bind to therapeutic targets inaccessible to conventional antibodies. Multivalent recombinant antibody fragments offer high binding avidity and unique target specificity to cancer cells. These multimeric scFvs (e.g., diabodies, tetrabodies) offer an improvement over parental antibodies because small molecules, approximately 60-100 kDa in size, offer faster blood clearance and rapid tissue uptake. See Power et al. (Generation of recombinant multimeric antibody fragments for tumor diagnosis and therapy. Methods Mol Biol, 207, 335-50, 2003); and Wu et al. (Anti-carcinoembryonic antigen (CEA) diabody for rapid tumor targeting and imaging. Tumor Targeting, 4, 47-58, 1999).

[0073] Various techniques for making and using non-conventional antibodies have been described. Bispecific antibodies generated using leucine zippers have been described by Kostelny et al. (J. Immunol. 148(5):1547-1553, 1992). Diabody technology has been described by Hollinger et al. (Proc. Natl. Acad. Sci. USA 90:6444-6448, 1993). Another strategy for making bispecific antibody fragments by using single-chain Fv (sFv) diners has been described by Gruber et al. (J. Immunol. 152:5368, 1994). Trispecific antibodies have been described by Tutt et al. (J. Immunol. 147:60, 1991).

[0074] Single-chain Fv polypeptide antibodies include covalently linked VH::VL heterodimers, which can be expressed from nucleic acids containing VH- and VL-encoding sequences either directly joined or joined via a peptide-encoding linker as described by Huston et al. (Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988). See also U.S. Patent Nos. 5,091,513, 5,132,405, and 4,956,778; and U.S. Patent Application Publication Nos. 20050196754 and 20050196754.

[0075] In one embodiment, the antibody that binds to platelet FcγRIIa is monoclonal. Alternatively, the anti-platelet FcγRIIa antibody is a polyclonal antibody. The preparation and use of polyclonal antibodies are also known to those skilled in the art. The present disclosure also encompasses hybrid antibodies in which a pair of heavy and light chains is obtained from a first antibody, while the other pair of heavy and light chains is obtained from a different second antibody. Such hybrids can be formed using humanized heavy chains and humanized light chains. Such antibodies are often referred to as "chimeric" antibodies.

[0076] Intact antibodies are generally said to contain an "Fc" region and a "Fab" region. The Fc region is involved in complement activation but not antigen binding. Antibodies from which the Fc' region has been enzymatically cleaved or which have been produced without the Fc' region are designated "F(ab')2" fragments and retain both of the intact antibody's antigen-binding sites. Similarly, antibodies from which the Fc region has been enzymatically cleaved or which have been produced without the Fc region are designated "Fab'" fragments and retain one of the intact antibody's antigen-binding sites. Fab fragments consist of a covalently linked antibody light chain and a portion of the antibody heavy chain, designated "Fd." The Fd fragment is the primary determinant of antibody specificity (a single Fd fragment can associate with up to 10 different light chains without altering antibody specificity). Isolated Fd fragments retain the ability to specifically bind to immunogenic epitopes.

[0077] Antibodies can be produced by any method known in the art using a soluble polypeptide or its immunogenic fragment as an immunogen. One method for obtaining antibodies is to immunize a suitable host animal with the immunogen and follow standard procedures for generating polyclonal or monoclonal antibodies. The immunogen promotes the presentation of the immunogen on the cell surface. Immunization of a suitable host can be carried out in several ways. A nucleic acid sequence encoding human FcγRIIa or an immunogenic fragment thereof can be provided to the host in a delivery vehicle that is taken up by the host's immune cells. The cells then express human FcγRIIa, thereby generating an immunogenic response in the host. Alternatively, a nucleic acid sequence encoding human FcγRIIa or an immunogenic fragment thereof can be expressed in cells in vitro, followed by isolating human FcγRIIa and administering it to a suitable host to produce antibodies.

[0078] Alternatively, antibodies against platelet FcγRIIa can be obtained from an antibody phage display library, if desired. Bacteriophage are capable of infecting and replicating within bacteria, and can be engineered to display human antibody proteins when combined with human antibody genes. Phage display is the process by which phages are engineered to "display" human antibody proteins on their surface. Genes from a human antibody gene library are inserted into a population of phages. Each phage carries the genes for a different antibody and thus displays a different antibody on its surface.

[0079] The produced antibodies can then be purified from the host by any method known in the art. Antibody purification methods can include salt precipitation (e.g., with ammonium sulfate), ion exchange chromatography (e.g., on a cation or anion exchange column, preferably run at neutral pH and eluted with a step gradient of increasing ionic strength), gel filtration chromatography (including gel filtration HPLC), and chromatography on affinity resins such as Protein A, Protein G, hydroxyapatite, and anti-immunoglobulin.

[0080] Antibodies can be conveniently produced from hybridoma cells engineered to express the antibody. Methods for producing hybridomas are well known in the art. Hybridoma cells can be cultured in a suitable medium, and the spent medium can be used as a source of antibody. Polynucleotides encoding the antibody of interest can then be obtained from the antibody-producing hybridoma, and antibodies can then be produced synthetically or recombinantly from these DNA sequences. To produce large amounts of antibody, it is generally more convenient to obtain ascites fluid. Methods for producing ascites fluid generally involve injecting hybridoma cells into immunologically naive, histocompatible, or immunotolerant mammals, particularly mice. The mammal can be primed for ascites production by prior administration of a suitable composition (e.g., pristane).

[0081] Monoclonal antibodies (Mabs) produced by the methods of the present disclosure can be "humanized" by methods known in the art. A "humanized" antibody is one in which at least a portion of its sequence has been altered from its original form so that it more closely resembles a human immunoglobulin. Techniques for humanizing antibodies are particularly useful when producing non-human animal (e.g., murine) antibodies. Examples of methods for humanizing murine antibodies are provided in U.S. Patent Nos. 4,816,567, 5,530,101, 5,225,539, 5,585,089, 5,693,762, and 5,859,205.

[0082] Antibody FcYRIIa-5G1 In one embodiment, an antibody designated FcYRIIa-5G1 is provided. The heavy chain of antibody FcYRIIa-5G1 has the following DNA sequence: (SEQ ID NO 3): Encoded by TIFF2025528831000004.tif25159.

[0083] The predicted protein sequence based on this DNA sequence encoding the heavy chain of antibody FcYRIIa-5G1 is provided below with the complementarity determining regions (CDRs) underlined. (SEQ ID NO 4) TIFF2025528831000005.tif11158

[0084] In particular, the CDRs of the heavy chain of antibody FcYRIIa-5G1 are: TIFF2025528831000006.tif22128

[0085] The light chain of antibody FcYRIIa-5G1 has the following DNA sequence: (SEQ ID NO 8) Encoded by TIFF2025528831000007.tif25159.

[0086] The predicted protein sequence based on this DNA sequence encoding the light chain of antibody FcYRIIa-5G1 is provided below with the complementarity determining regions (CDRs) underlined. (SEQ ID NO 9) TIFF2025528831000008.tif11158

[0087] In particular, the CDRs of the light chain of antibody FcYRIIa-5G1 are: TIFF2025528831000009.tif22128

[0088] Antibody FcYRIIa-11F9 In another embodiment, an antibody designated FcYRIIa-11F9 is provided. The heavy chain of antibody FcYRIIa-11F9 has the following DNA sequence: (SEQ ID NO 13) Encoded by TIFF2025528831000010.tif25159.

[0089] The predicted protein sequence based on this DNA sequence encoding the heavy chain of antibody FcYRIIa-11F9 is provided below with the complementarity determining regions (CDRs) underlined. (SEQ ID NO 14) TIFF2025528831000011.tif11159

[0090] In particular, the CDRs of the heavy chain of antibody FcYRIIa-11F9 are: TIFF2025528831000012.tif22128

[0091] The light chain of antibody FcYRIIa-11F9 has the following DNA sequence: (SEQ ID NO 18) Encoded by TIFF2025528831000013.tif25160.

[0092] The predicted protein sequence based on this DNA sequence encoding the light chain of antibody FcYRIIa-11F9 is provided below with the complementarity determining regions (CDRs) underlined. (SEQ ID NO 19) TIFF2025528831000014.tif11158

[0093] In particular, the CDRs of the light chain of antibody FcYRIIa-11F9 are: TIFF2025528831000015.tif22128

[0094] The disclosed monoclonal antibodies FcYRIIa-5G1 and FcYRIIa-11F9 can bind to FcγRIIa or certain subregions thereof with high affinity, specificity, and selectivity.

[0095] Some embodiments include an FcYRIIa-5G1 antibody comprising CDRH1 (SEQ ID NO: 5), CDRH2 (SEQ ID NO: 6), CDRH3 (SEQ ID NO: 7), CDRL1 (SEQ ID NO: 10), CDRL2 (SEQ ID NO: 11), CDRL3 (SEQ ID NO: 12). Some embodiments include an FcYRIIa-5G1 antibody comprising a VH (SEQ ID NO: 4) and a VL (SEQ ID NO: 9) or SEQ ID NO: 3 and SEQ ID NO: 8.

[0096] Some embodiments include an FcYRIIa-11F9 antibody comprising CDRH1 (SEQ ID NO: 15), CDRH2 (SEQ ID NO: 16), CDRH3 (SEQ ID NO: 17), CDRL1 (SEQ ID NO: 20), CDRL2 (SEQ ID NO: 21), and CDRL3 (SEQ ID NO: 22). Some embodiments include an FcYRIIa-11F9 antibody comprising a VH (SEQ ID NO: 14) and a VL (SEQ ID NO: 19) or SEQ ID NO: 13 and SEQ ID NO: 18.

[0097] The sequences of antibodies FcYRIIa-5G1 and FcYRIIa-11F9 were determined as follows: Total RNA was isolated from hybridoma cell line cultures (2 × 10 in each case). 6RNA was treated to remove aberrant transcripts and reverse transcribed using oligo(dT) primers. The resulting cDNA samples were amplified in separate PCRs using pairs of framework 1 and constant region primers specific for either the heavy or light chain. Reaction products were separated on an agarose gel and assessed for size. PCR reactions were prepared for sequencing using a PCR cleanup kit and sequenced at GENEWIZ using an Illumina® NovSeq 6000.

[0098] The DNA sequence data from all constructs was analyzed to determine the consensus sequences of the heavy and light chains. To eliminate artifacts and / or process contamination, the consensus sequences were compared with all known variable region sequences. The consensus sequences were then analyzed using online tools to verify that they could encode viable immunoglobulins.

[0099] Antibody preparation Purified FcγRIIa (produced by mammalian cells) was exposed to formaldehyde before injection into mice. Mouse serum and cell culture medium from hybridomas were screened for antibodies that bound to formaldehyde-exposed FcγRIIa. Direct immunoassays using formaldehyde-treated FcγRIIa were used to screen for the presence of the target antibody. Competitive immunoassays were performed to identify clones that produced antibodies with high binding affinity. Clones with antibodies that effectively competed with commercially available antibodies were selected for further evaluation. In these immunoassays, the FcγRIIa used was not exposed to formaldehyde. This approach was used to identify clones that bound with high affinity, rather than simply binding to FcγRIIa on "fixed" platelets, demonstrating the suitably high sensitivity of the developed antibodies.

[0100] Candidate clones were evaluated in a series of additional tests. FcγRIIa was stably transfected into Chinese hamster ovary (CHO) cells to assess clone binding using fluorescence microscopy and flow cytometry. In addition, antibodies from candidate clones were used to perform immunoprecipitation using platelet lysates. These experiments confirmed that binding was specific to FcγRIIa (no other proteins were immunoprecipitated). Based on these screening tests, candidate clones with high binding potential were selected.

[0101] One of the goals was to identify antibodies capable of quantifying FcγRIIa on the surface of fixed platelets in a manner similar to that of unfixed platelets. To accomplish this, selected antibodies were fluorescently labeled. The goal was to identify clones capable of quantifying FcγRIIa on the surface of fixed platelets within 10% of the quantification of previously known antibodies on unfixed platelets. Based on these experiments, two clones were identified, which generated the antibodies FcYRIIa-5G1 and FcYRIIa-11F9 described above.

[0102] Antibodies from selected FcγRIIa clones were tested for sensitivity and specificity. Each antibody was selected not only for its ability to bind fixative-exposed FcγRIIa but also for its ability to competitively inhibit the binding of a commercially available FcγRIIa antibody to unfixed FcγRIIa. Specificity was assessed by performing immunoprecipitation experiments using platelet lysates. In these experiments, FcγRIIa was the only precipitated protein.

[0103] Antibodies may also be obtained in a conventional manner, including introducing FcγRIIa, a subregion thereof, a peptide, or an altered version thereof into a host animal, followed by isolation of antibody-producing splenocytes and formation of suitable hybridomas.

[0104] Antibodies such as and including those described above that are conjugated to a detectable label are also contemplated herein. In a preferred embodiment, the label is conjugated to the antibody in a 1:1 ratio, which allows for comparison of detection output measurements and, as described below, comparison to a curve comparing instrument output (e.g., fluorescence measurements) to known levels of detectable label.

[0105] inhibitory nucleic acid The inhibitory nucleic acid molecule is an oligonucleotide that inhibits the expression or activity of platelet FcγRIIa for the prevention of thrombosis and the treatment of thrombosis-related disorders. Such oligonucleotides include single-stranded and double-stranded nucleic acid molecules (e.g., DNA, RNA, and analogs thereof) that bind to nucleic acid molecules encoding FcγRIIa (e.g., antisense molecules, siRNA, shRNA), as well as nucleic acid molecules (e.g., aptamers) that directly bind to platelet FcγRIIa polypeptides and modulate its biological activity.

[0106] Ribozymes Catalytic RNA molecules, i.e., ribozymes, targeting the antisense FcγRIIa sequences disclosed herein can be used to inhibit expression of FcγRIIa nucleic acid molecules in vivo. The inclusion of ribozyme sequences within antisense RNAs confers RNA-cleaving activity, thereby increasing the activity of the construct. The design and use of target RNA-specific ribozymes are described in Haseloff et al., Nature 334:585-591, 1988, and U.S. Patent Application Publication No. 2003 / 0003469A1, each of which is incorporated by reference. Accordingly, the present disclosure also features catalytic RNA molecules comprising antisense RNAs with 8 to 19 consecutive nucleobases in the binding arms. In a preferred embodiment of the present disclosure, the catalytic nucleic acid molecules are formed into a hammerhead or hairpin motif. An example of such a hammerhead motif is described by Rossi et al., Aids Research and Human Retroviruses, 8:183, 1992. Examples of hairpin motifs are described by Hampel et al., "RNA Catalyst for Cleaving Specific RNA Sequences," filed September 20, 1989, Hampel and Tritz, Biochemistry, 28:4929, 1989, which is a continuation-in-part of U.S. patent application Ser. No. 07 / 247,100, filed September 20, 1988, and Hampel et al., Nucleic Acids Research, 18:299, 1990. These specific motifs are not limiting of the present disclosure; one of skill in the art will recognize that what is important in an enzymatic nucleic acid molecule of the present disclosure is only that it has a specific substrate binding site complementary to one or more of the RNA regions of the target gene, and that it has a nucleotide sequence within or surrounding that substrate binding site that confers RNA cleavage activity to the molecule.

[0107] Small hairpin RNAs consist of a stem-loop structure with an optional 3'UU overhang. Although variable, the stem can range from 21 to 31 bp (preferably 25 to 29 bp), and the loop can range from 4 to 30 bp (preferably 4 to 23 bp). For intracellular shRNA expression, plasmid vectors containing either the polymerase III H1-RNA promoter or the U6 promoter, a cloning site for stem-loop RNA inserts, and a transcription termination signal of four to five thymidines can be employed. Polymerase III promoters generally have clearly defined start and stop sites, and their transcripts lack a poly(A) tail. The termination signal for these promoters is defined by a polythymidine tract, and the transcript is typically cleaved after the second uridine. Cleavage at this position generates a 3'UU overhang in the expressed shRNA, which resembles the 3' overhang of synthetic siRNAs. Additional methods for expressing shRNA in mammalian cells are described in the references cited above.

[0108] siRNA Short double-stranded RNAs of 21 to 25 nucleotides are effective in downregulating gene expression (Zamore et al., Cell 101: 25-33; Elbashir et al., Nature 411: 494-498, 2001, incorporated herein by reference). The therapeutic efficacy of the sirNA approach in mammals was demonstrated in vivo by McCaffrey et al. (Nature 418: 38-39, 2002). Given the sequence of a target gene, siRNAs can be designed to inactivate the gene. Such siRNAs can be administered, for example, directly to affected tissues or systemically. The nucleic acid sequence of the Parl gene can be used to design small interfering RNAs (siRNAs). 21 to 25 nucleotide siRNAs can be used, for example, as therapeutic agents for treating lupus.

[0109] The inhibitory nucleic acid molecule of the present disclosure can be employed as double-stranded RNA for RNA interference (RNAi)-mediated knockdown of platelet FcγRIIa expression.In one embodiment, platelet FcγRIIa expression is reduced in megakaryocytes.RNAi is a method for reducing the cellular expression of a specific target protein (reviewed in Tuschl, Chembiochem 2:239-245, 2001; Sharp, Genes & Devel. 15:485-490, 2000; Hutvagner and Zamore, Curr. Opin. Genet. Devel. 12:225-232, 2002; and Hannon, Nature 418:244-251, 2002).The introduction of siRNA into cells, either by transfection of dsRNA or by expressing siRNA using a plasmid-based expression system, is increasingly used to create loss-of-function phenotypes in mammalian cells.

[0110] In one embodiment of the present disclosure, double-stranded RNA (dsRNA) molecules are generated containing 8 to 19 consecutive nucleobases of the nucleobase oligomer of the present disclosure. The dsRNA can be two separate RNA strands that are duplexed together, or a single RNA strand that is self-duplexed (small hairpin (sh)RNA). Typically, the dsRNA is about 21 or 22 base pairs long, but can be shorter or longer (up to about 29 nucleobases) if desired. dsRNA can be generated using standard techniques (e.g., chemical synthesis or in vitro transcription). Kits are available, for example, from Ambion (Austin, Tex.) and Epicentre (Madison, Wis.). Methods for expressing dsRNA in mammalian cells are described in Brummelkamp et al. Science 296:550-553, 2002; Paddison et al. Genes & Devel. 16:948-958, 2002; Paul et al. Nature Biotechnol. 20:505-508, 2002; Sui et al. Proc. Natl. Acad. Sci. USA 99:5515-5520, 2002; Yu et al. Proc. Natl. Acad. Sci. USA 99:6047-6052, 2002; Miyagishi et al. Nature Biotechnol. 20:497-500, 2002; and Lee et al. Nature Biotechnol. 20:500-505. 2002, each of which is incorporated herein by reference.

[0111] Small hairpin RNAs consist of a stem-loop structure with an optional 3'UU overhang. Although variable, the stem can range from 21 to 31 bp (preferably 25 to 29 bp), and the loop can range from 4 to 30 bp (preferably 4 to 23 bp). For intracellular shRNA expression, plasmid vectors containing either the polymerase III H1-RNA promoter or the U6 promoter, a cloning site for stem-loop RNA inserts, and a transcription termination signal of four to five thymidines can be employed. Polymerase III promoters generally have clearly defined start and stop sites, and their transcripts lack a poly(A) tail. The termination signal for these promoters is defined by a polythymidine tract, and the transcript is typically cleaved after the second uridine. Cleavage at this position generates a 3'UU overhang in the expressed shRNA, which resembles the 3' overhang of synthetic siRNAs. Additional methods for expressing shRNA in mammalian cells are described in the references cited above.

[0112] Delivery of nucleobase oligomers Naked inhibitory nucleic acid molecules or their analogs can enter mammalian cells and inhibit the expression of genes of interest. Nevertheless, it may be desirable to utilize formulations that aid in the delivery of oligonucleotides or other nucleobase oligomers to cells (see, e.g., U.S. Patent Nos. 5,656,611, 5,753,613, 5,785,992, 6,120,798, 6,221,959, 6,346,613, and 6,353,055, each of which is incorporated herein by reference).

[0113] therapeutic application Also disclosed are methods for treating animals or humans by administering an effective amount of an antibody against FcγRIIa. The antibody may include, or be provided in conjunction with, or linked to, a platelet-specific antibody, for example, in the form of a bispecific antibody. Such antibodies are also considered to be within the scope of the present disclosure. These antibodies may include FcYRIIa-5G1 and FcYRIIa-11F9 or portions thereof, for example, one or more CDRs, as disclosed herein. Antibodies that are 80%, 85%, 90%, 95%, or 98% homologous to all or a portion of FcYRIIa-5G1 and FcYRIIa-11F9 disclosed herein are also contemplated herein.

[0114] Also disclosed herein are molecules that are or comprise antigen-binding fragments and / or derivatives of FcYRIIa-5G1 and FcYRIIa-11F9 disclosed herein, which comprise any one or more of the CDRs disclosed herein, including CDRs from both FcYRIIa-5G1 and FcYRIIa-11F9 disclosed herein, in a single molecule.

[0115] Also disclosed are antigen binding proteins, such as antibodies or antigen binding fragments thereof, that specifically bind to FcYRIIa and comprise CDRs that are variants of the sequences set forth in CDR SEQ ID Nos. 5, 6, 7, 10, 11, 12, 15, 16, 17, 20, 21, 22.

[0116] Variants include partial changes to the amino acid sequences of one or more CDRs, heavy chains, and / or light chains by deletion or substitution of one to several amino acids from the CDRs, heavy chains, and / or light chains, and / or addition or insertion of one to several amino acids into the CDRs, heavy chains, and / or light chains, and / or combinations thereof. Variants may contain 1, 2, 3, 4, 5, or 6 amino acid substitutions, additions, and / or deletions (1 to 6 of each) in the amino acid sequences of the CDRs, heavy chains, and / or light chains. Substitutions of amino acid residues may be conservative, for example, substituting one hydrophobic amino acid for another.

[0117] Antigen-binding proteins that are variants of one or more CDRs, heavy chains, and / or light chains have the same or similar functional properties as antigen-binding proteins comprising the CDRs, heavy chains, and / or light chains described herein. Thus, antigen-binding proteins comprising variant CDRs bind to the same target protein or epitope with the same or similar binding affinity as the CDRs, heavy chains, and / or light chains described herein.

[0118] The provided antibodies can also be formed into suitable pharmaceutical compositions for administration to human or animal patients to regulate FcYRIIa-mediated conditions. Pharmaceutical compositions containing the provided antibodies, their variants, and / or active fragments can be formulated in combination with any suitable pharmaceutical vehicle, excipient, or carrier commonly used in the art, including conventional materials for this purpose, such as saline, dextrose, water, glycerol, ethanol, other therapeutic compounds, and combinations thereof. The particular vehicle, excipient, or carrier used will vary depending on the patient and their condition, and those skilled in the art will recognize that a variety of administration modes are suitable for the compositions. Suitable methods of administration for any pharmaceutical composition disclosed in the present application include, but are not limited to, topical, oral, anal, vaginal, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal, and intradermal administration.

[0119] Thus, the provided antibody compositions are useful for disrupting, modulating, or inhibiting the binding interaction between the FcYRIIa protein and its ligand protein, prothrombin, in blood and tissues, and thus have particular applicability in developing compositions and methods for preventing or treating staphylococcal infections and in inhibiting the activation of prothrombin.

[0120] The antibody compositions can be multispecific, eg, bispecific or trispecific, including, eg, a portion that selectively binds to platelets.

[0121] Thus, administering the antibodies disclosed herein in any of the conventional ways described above (e.g., topically, parenterally, intramuscularly, etc.) can provide a highly useful method for treating or preventing certain conditions in human or animal patients. By effective amount, we mean a level of use, such as a level of antibody titer, that is sufficient to bind to FcYRIIa and / or inhibit the binding of other molecules to FcYRIIa, for example, through steric inhibition or direct blocking of the binding site of other molecules, and thus be useful for platelet regulation. As those skilled in the art will recognize, the level of antibody titer required to be effective in treating or preventing staphylococcal infections will vary depending on the nature and condition of the patient and / or the level of FcYRIIa expression.

[0122] In addition to the uses of the disclosed antibodies and their degenerative or homologous forms, as described above, the inventors contemplate the use of these antibodies in a variety of ways, including detecting the presence of FcYRIIa in diagnosis or in formulating treatment regimens and options. After isolation of a sample, diagnostic assays utilizing the disclosed antibodies can be performed to detect the presence of FcYRIIa; such assay techniques for determining such presence in a sample are well known to those skilled in the art and include methods such as flow cytometry, radioimmunoassay, Western blot analysis, and ELISA assays.

[0123] Thus, the disclosed antibodies can be used for the specific detection or diagnosis of FcYRIIa-mediated conditions, for the prevention or inhibition of conditions mediated by FcYRIIa, for diagnostics, or for use as research tools. As used herein, the term "antibody" includes monoclonal antibodies, polyclonal antibodies, chimeric antibodies, single-chain antibodies, bispecific antibodies, simianized antibodies, and humanized or primatized antibodies, as well as F(ab) fragments, such as fragments that retain the binding specificity of the antibody to the FcYRIIa protein, including the products of an F(ab) immunoglobulin expression library. Thus, the use of single chains, such as the variable heavy and variable light chains of the antibody, as described below, is also contemplated. The production of any of these types of antibodies or antibody fragments is well known to those skilled in the art.

[0124] In some embodiments, the antibody provided herein detects reactive platelets.Reactive platelets may constitute a determining factor of thrombosis.Without wishing to be bound by any theory, measuring FcYRIIa using the antibody provided herein can provide information about subsequent therapeutic treatment, including the treatment of thrombosis.For example, measuring FcYRIIa antibody can be used to provide information about the use of blood thinners, aspirin, anticoagulants, surgery, and / or other drugs.

[0125] Test Device Design Test devices can take any desired form that provides for the flow of a liquid test sample from the point of contact with the test sample through the test and / or control sites. Generally, the test devices of the present disclosure include an internal flow channel comprising one or more liquid-permeable materials. In a first section, the device includes a site for applying a liquid sample. This first section of the device also includes an analyte-binding conjugate, such as an antibody that specifically binds to an antigen of interest (e.g., FcγRIIa, a platelet surface protein). The analyte-binding conjugate typically binds to the analyte to form a complex. Complex formation (e.g., formation of an antigen / antibody conjugate complex) can occur at any point in the internal flow channel after the analyte contacts the analyte-binding conjugate. For example, complex formation can occur or continue as the sample flows from the first section to the second section of the device.

[0126] The second portion of the device has a variety of features that enhance its functionality. In one embodiment, the second portion is constructed of a material capable of filtering the sample to prevent particulate matter from flowing through the device. In another embodiment, the second portion promotes complex formation by increasing the time required for fluid to flow from the application site to the test site. Thus, the dimensions of the second portion can be varied (e.g., increased or decreased) to empirically determine for each application those dimensions that increase sensitivity, i.e., optimize the signal-to-noise ratio, while reducing false positives. In yet another embodiment, the second portion of the device can be used to deliver desired agents to the fluid as it flows through the device. For example, the second portion can be impregnated with a buffer (e.g., TRIS, sodium carbonate), a surfactant (e.g., Tween, Triton), a preservative (e.g., sodium azide, thimerosol), a salt, or other agent such that contact of the sample with the second portion of the device alters the sample. Exemplary changes include an increase or decrease in the pH, salt concentration, buffering capacity of the sample, or in the binding between the conjugate and the analyte.

[0127] The third section of the device contains a test site that serves as a read zone, providing for the detection of an analyte in a sample. Various means for detecting the presence of an analyte at a test site are known in the art. In a competitive assay, the analyte of interest competes with a labeled probe for binding to a detector at the test site. The more analyte present in the sample, the more effectively the analyte can compete with and / or displace the binding of the detector. A hallmark of most competitive assays is that an increase in the amount of analyte in the sample results in a decrease in signal at the read zone. In contrast, a "sandwich" format typically involves mixing a test sample with a detection probe conjugated to a specific binding member (e.g., an antibody). In this embodiment, the conjugate and analyte bind to form a complex. These complexes are then brought into contact with a receptive material (e.g., an antibody) immobilized in the test site. The analyte / conjugate complex binds to the immobilized receptive material, forming a "sandwich complex" (e.g., antibody conjugate / antigen / antibody). In this approach, detection of the "sandwich complex" indicates the presence of the analyte in the sample.

[0128] It may be desirable to include a positive control to indicate that the liquid sample has traveled through the internal flow path from the application site through the test site. In a competitive assay format, the first portion of the device further comprises a control conjugate, and the third portion of the device comprises a control site comprising a receptive material that binds to the control conjugate. The control site is located downstream from the test site in the third portion of the device. Detection of binding of the control conjugate at the control site indicates that the liquid sample has flowed from the application site through the test site to the control site. In a sandwich assay format, a control antibody that binds to the anti-antigen antibody is immobilized at the control site. Excess anti-antigen antibody is detected at the control site in the presence or absence of the antigen.

[0129] The device may also include a wicking pad in a fourth portion that contains a sorbent material capable of absorbing or adsorbing excess liquid present in the liquid sample.

[0130] In one embodiment, the test device contains a liquid permeable material that defines the following portions of the capillary communication: a) a first portion that is a site for application of a liquid sample, the first portion comprising a liquid-permeable medium, an anti-antigen-antibody conjugate, and a control antibody conjugate, wherein the first portion has a length of 5 mm to 20 mm; for example, the length of the first portion is equal to any integer between 5 and 20 (e.g., lengths of 5, 10, 15, 20 mm); b) a second portion comprising a liquid-permeable medium, wherein the second portion overlaps the first portion by at least 1, 2, 3, 4, or 5 mm; and the length of the second portion is between 10 mm and 40 mm: for example, the length of the second portion is any integer between 10 and 40 (e.g., 10, 15, 20, 25, 30, 35, 40); and c) a third portion that is a site for detecting binding of an anti-antigen-antibody conjugate at the test site and binding of a control antibody conjugate at the control site, the third portion comprising a liquid-permeable medium having an antigen immobilized thereon at the test site and an antibody that binds to a control antibody present at the control site, wherein the third portion is 15 to 40 mm in length; e.g., any integer between 15 and 40 (e.g., 15, 20, 25, 30, 35, 40); and the second portion overlaps with the third portion by at least 1, 2, 3, 4, or 5 mm.

[0131] In the fourth section, the device can contain a sorbent material. The sorbent material has a length of 25 to 75 mm. For example, the length is an integer between 25 and 75 (e.g., 25, 35, 50, 60, 70, 75). In one embodiment, the fourth section overlaps with the third section by at least 1, 2, 3, 4, or 5 mm.

[0132] Typically, the internal flow path is 1 mm to 10 mm wide; for example, the width of the test device (e.g., test strip) is any integer between 1 and 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10). In one embodiment, the strip width is 3.8 mm. Desirably, the test devices of the present disclosure have increased sensitivity compared to conventional test devices. The sensitivity of the test devices of the present disclosure is increased by at least 5%, 10%, 25%, 50%, 75%, 100%, 150%, or 200% compared to conventional test devices.

[0133] As described herein, the test device comprises a conjugate that binds to the analyte. In one approach, the conjugate is an antibody that can bind to FcγRIIa, either alone or when conjugated to another compound. Any antibody, antibody conjugate, or fragment thereof that binds to the target antigen can be used in the present disclosure. Such an antibody or antibody conjugate is present in the internal flow channel of the test device. Suitable antibodies include, but are not limited to, polyclonal antibodies, monoclonal antibodies, or fragments thereof.

[0134] To detect antibody / antigen complexes in a test device, a detection reagent or conjugate must be coupled to the antibody or antigen. Exemplary conjugates include colored reagents, fluorescent compounds, enzymes, and radioisotopes. Colored or fluorescent compounds include gold particles, colored or fluorescent latex particles, polystyrene beads, and dyes such as fluorescein isothiocyanate, BODIPY FL, Oregon Green, Alexa Fluor 488, phycoerythrin, and phycocyanin. Colloidal metals, metal sols, and other types of colored particles useful as marker substances in immunoassay procedures are known in the art. See, for example, U.S. Patent No. 4,313,734. Antibody conjugates are widely available from a variety of well-known commercial sources (e.g., Molecular Probes (e.g., Zenon® labeling technology), Nanoprobes (e.g., Nanogold® gold-antibody conjugates). A preferred label for use as a detection reagent is the fluorescent dye phycoerythrin ("PE").

[0135] Enzymes that can be coupled to antibodies include peroxidases (such as horseradish peroxidase), phosphatases (such as acid phosphatase or alkaline phosphatase), β-galactosidase, urease, glucose oxidase, carbonic anhydrase, acetylcholinesterase, glucoamylase, lysozyme, malate dehydrogenase, glucose-6-phosphate dehydrogenase, β-glucosidase, protease, pyruvate decarboxylase, esterase, luciferase, or any other enzyme known to those skilled in the art. Enzymes are not themselves detectable but must be combined with a substrate in order to catalyze a reaction in which the end product of the reaction is detectable.

[0136] Antibodies, antibody conjugates, protein-antigen conjugates, and protein-hapten conjugates are immobilized in the internal flow channels using standard methods known to those skilled in the art. Protein immobilization protocols are known to those skilled in the art. See, for example, Laboratory Techniques in Biochemistry and Molecular Biology, Tijssen, Vol. 15, Practice and Theory of Enzyme Immunoassays, Chapter 13, The Immobilization of Immunoreactants on Solid Phases, pp. 297-328, and the references cited therein. In one approach, antibodies are directly immobilized on a solid support by physical adsorption, or are bound covalently or through a bridging molecule such as protein A or polylysine, or are bound to a solid support.

[0137] Internal flow path A test device can be provided with an internal flow path that facilitates the flow of a liquid sample through the device. The internal flow path contains one or more liquid-permeable materials or membranes composed of any relatively inert material or combination of materials (e.g., glass fiber, polyester, nitrocellulose, cellulose or its derivative fibers, non-cellulose hydrocarbon materials, ceramics) suitable for transporting liquid from the contact site through the test site and / or control site, and optionally to the reservoir. Materials suitable for use in the internal flow path are wettable and exhibit low nonspecific binding. Materials with increased sorptive properties facilitate liquid flow. Different materials with different absorption characteristics or sorptive properties can be used in various portions of the flow path. If desired, the materials used can be screened for optimal pore size and density to facilitate controlled distribution of antibodies within the membrane, optimize reaction kinetics, or optimize the sensitivity, discrimination, or signal-to-noise ratio of the device.

[0138] solid support In most applications, the test device comprises an internal flow channel fixed to a solid support. While the physical form of the solid support is not critical, some forms may be more convenient than others for purposes of the present invention. Thus, the solid support may be in the form of a paper strip, a dipstick, a membrane (e.g., a nylon membrane or cellulose filter), a plate (e.g., a microtiter plate), or solid particles (e.g., latex beads). The solid support may be made from any suitable material, including, but not limited to, plastic (e.g., polyethylene, polypropylene, polystyrene, latex, polyvinyl chloride, polyurethane, polyacrylamide, polyvinyl alcohol, nylon, polyvinyl acetate, or any suitable copolymer thereof), cellulose (e.g., various types of paper, such as nitrocellulose paper), silicone polymers (e.g., siloxane), polysaccharides (e.g., agarose or dextran), or ion exchange resins (e.g., conventional anion or cation exchange resins).

[0139] sorbent reservoir The test device optionally includes a fourth section forming a reservoir made of an adsorbent or absorbent material. This reservoir absorbs excess liquid as it flows through the test device. In some applications, such as when the concentration of antigen in the test sample is particularly low, it may be desirable to apply a large volume of liquid test sample to the test device. In such cases, the presence of an adsorbent material may increase the sensitivity of antigen detection. Optionally, the region of the flow path in the test cell defining the test and control sites has a restricted cross-sectional area compared to other regions of the flow path. This feature creates a "bottleneck" effect, whereby antigen in the entire volume of the adsorbed sample must pass through the restricted flow region immediately above the test site. This "bottleneck" may promote the formation of a sandwich. Suitable sorbent materials include virtually any commercially available material (e.g., synthetic or natural materials such as cotton) capable of absorbing many times its weight in water. Such materials are widely available commercially.

[0140] How to use the test device The present disclosure provides methods of using the test devices of the present disclosure to detect an analyte (e.g., an antigen) in a test sample. In one example, the assay is performed by placing the leading edge (first portion) of the lateral flow device in contact with a liquid test sample. In another example, the sample is brought into contact with the device by applying the liquid test sample dropwise to the first portion of the lateral flow device.

[0141] Test sample The methods and compositions of the present disclosure are useful for identifying an analyte (FcγRIIa) in a test sample. In one embodiment, the methods of the present disclosure are suitable for detecting an analyte of biological origin. Test samples include, but are not limited to, any liquid containing a dissolved or dispersed analyte (FcγRIIa) of biological origin. Exemplary test samples include body fluids (e.g., blood, plasma, amniotic fluid, sputum, urine, cerebrospinal fluid, lymph, tears, feces, or gastric juice), tissue extracts, or any liquid or biological fluid containing platelets. Exemplary conjugates that specifically bind to platelets include, but are not limited to, antibodies against glycoprotein (GP) IIb (e.g., anti-CD41 or CD41a); antibodies against GP IIIa (e.g., anti-CD61); antibodies against GP V (e.g., anti-CD42d); antibodies against GP Ib (e.g., anti-CD42b); antibodies against GP IX, such as anti-CD42a; ​​antibodies against lysosomal membrane proteins (e.g., anti-CD63); and antibodies against PECAM (e.g., anti-CD31). In various embodiments, the test device of the present disclosure detects FcγRIIa peptide or FcγRIIa protein (e.g., on platelets). If the test sample is not sufficiently fluid by itself for the purposes of the present invention, it can be mixed with a suitable fluid to the desired fluidity, for example, by homogenization.

[0142] kit In another aspect, the present disclosure provides kits for aiding in assessing platelet reactivity (e.g., determining the level of FcγRIIa expressed on platelets in a sample, identifying subjects with or at risk of having thrombosis, selecting a treatment method for a subject with or at risk of having thrombosis, etc.), which kits are used to detect biomarkers according to the present disclosure. In one embodiment, the kit includes an agent that specifically recognizes FcγRIIa. In a specific embodiment, the agent is an antibody. Fluorescently labeled antibody levels are useful when flow cytometry is used to determine the level of FcγRIIa expressed on platelets in a sample. In a further embodiment, such a kit can include instructions for use in any of the methods described herein. In various embodiments, the instructions, in the form of a label or a separate package insert, provide suitable operating parameters. For example, the instructions can inform the consumer on how to collect the sample, how to wash the probe or the particular biomarker to be detected, or how to determine platelet reactivity based on the measured FcγRIIa level. In still other embodiments, the kit can include one or more containers containing controls (e.g., biomarker samples) used as standards for calibration. In still other embodiments, the kit can include one or more therapeutic agents for the treatment of thrombosis (e.g., ADP receptor antagonists, PAR antagonists, etc.).

[0143] In a further aspect, the present disclosure provides a kit comprising a test device for detecting an analyte in a sample. In one aspect, the kit comprises a lateral flow device as described herein. In some aspects, the kit comprises a container containing the lateral flow device; such a container can be a box, an ampoule, a bottle, a vial, a tube, a bag, a pouch, a blister pack, or other suitable container form known in the art. In one aspect, such a container can be sterile. Such a container can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for containing pharmaceuticals.

[0144] If desired, the device is provided with instructions for using it to identify the presence or absence of FcγRIIa in a sample. The instructions generally include information about using the device to identify a specific analyte, such as FcγRIIa. The instructions can be printed directly on the container (if present), or as a label attached to the container, or as a separate sheet, pamphlet, card, or folder provided in or with the container. If desired, the kit can also include a standard measuring pipet, test vials, and / or a liquid used to extract the sample (e.g., ethanol, methanol, an organic solvent, a suitable buffer such as phosphate-buffered saline, or water).

[0145] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are well within the skill of those in the art. Such techniques are fully explained in such references as "Molecular Cloning: A Laboratory Manual," second edition (Sambrook, 1989); "Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987); "Methods in Enzymology" "Handbook of Experimental Immunology" (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Current Protocols in Molecular Biology" (Ausubel, 1987); "PCR: The Polymerase Chain Reaction" (Mullis, 1994); "Current Protocols in Immunology" (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the present disclosure and, as such, may be considered when making and practicing the present disclosure. Techniques particularly useful for particular embodiments are discussed in the sections that follow.

[0146] Assay methodology In another aspect, the present disclosure provides an in vitro assay for the use of antibodies such as those disclosed herein. In this assay, 1) blood is anticoagulated with citrate or EDTA and fixed immediately after collection (which advantageously stabilizes platelets and allows them to be sent to a laboratory for analysis up to and including approximately 1-2 weeks after blood collection); 2) the fixed platelets are separated and washed; 3) the platelets are exposed to (i) an antibody such as those described herein for quantifying FcγRIIa, and (ii) preferably also an antibody for platelet identification; and 4) after dilution, FcγRIIa can be quantified using flow cytometry, and the output can be standardized. Testing has demonstrated that this assay maintains excellent precision (coefficient of variation for replicates of <5%). The assay may be useful in assessing and treating acute coronary syndromes, stroke, and cancer.

[0147] Bangs Beads (Bang Laboratories, Inc.), Quantibrite™ (BD Biosciences), or other suitable fluorescent microspheres can be used to convert fluorescence intensity units into FcγRIIa molecules on the platelet surface. (Kay S, Herishanu Y, Pick M, et al. Quantitative flow cytometry of ZAP-70 levels in chronic lymphocytic leukemia using molecules of equivalent soluble fluorochrome. Cytometry B Clin Cytom. 2006;70(4):218-226; Quadrini KJ, Hegelund AC, Cortes KE, et al. Validation of a flow cytometry-based assay to assess C5aR receptor occupancy on neutrophils and monocytes for use in drug development. Cytometry B Clin Cytom. 2016;90(2):177-190) In this way, FcγRIIa expression on platelets can be measured as the number of FcγRIIa molecules per platelet.

[0148] A more detailed description of an exemplary assay methodology follows. A blood sample is drawn from a patient, e.g., at a clinical site, into a standard citrate-anticoagulated tube (with sodium citrate solution in the tube). An aliquot of blood may then be added to a fixative tube containing formaldehyde or other suitable fixative. Optionally, the sample tube may contain only the anticoagulant and formaldehyde solution. This tube may be stored and shipped for analysis.

[0149] Sample processing may include the following steps. Dilute the sample with approximately 1,400 μl of HT buffer. Centrifuge at 1,200 g for 2 minutes. Decant the liquid. Resuspend the platelet pellet in 1,000 μl HT buffer. Centrifuge at 1,200 g for 2 minutes. Decant the liquid. Resuspend the platelet pellet in 1,000 μl HT buffer. Centrifuge at 1,200 g for 2 minutes. Resuspend the platelet pellet in 500 μl HT buffer. The final volume of the assay tube is typically approximately 30 μl and contains: 2 μl resuspended platelets 2 μg of 5G1 antibody or 11F9 antibody or other suitable antibody 2 μl CD42bPECy5 (Becton Dickinson) QS (quantity sufficient) with HT buffer. The antibody and platelets are incubated for 2 hours at room temperature. Dilute. Analyze by flow cytometry.

[0150] Another method for preparing a blood sample is provided, which includes the following steps.

[0151] The blood sample is treated with a suitable anticoagulant. Examples of suitable anticoagulants include those used in specimen collection, devices, transfusions, and equipment, as well as medical and surgical instruments. Examples include ethylenediaminetetraacetic acid (EDTA), and citrate can be provided in liquid form in the sample tube, e.g., pre-filled in the tube, or in a transfusion bag or other device. Forms of citrate can include sodium citrate or acid-citrate-dextrose, as well as certain oxalates (ethanedioates), e.g., as salts or other suitable forms.

[0152] A fixative, preferably formaldehyde, can be added to a concentration of approximately 0.5% formaldehyde. Other suitable fixatives, such as other aldehydes such as glutaraldehyde, can be used. Certain alcohols, such as ethanol and methanol, can function as fixatives, similar to other known fixatives.

[0153] The blood sample may be incubated, for example, for 15 minutes, followed by dilution of the sample. In one embodiment, the sample is diluted to a fixative concentration such as 0.1%. In some embodiments, the fixative is diluted with a buffer. Typical buffers include phosphate-buffered saline (PBS), Dulbecco's phosphate-buffered saline (DPBS), Hank's balanced salt solution (HBSS), HEPES, and MOPSO. The buffer may contain additional factors to aid in sample stability. In some embodiments, the buffer is combined with glycerol. Glycerol may be combined with PBS to form a solution, creating a glycerol-buffer solution. The glycerol-buffer solution may contain approximately 10% glycerol, 9% glycerol, 8% glycerol, 7% glycerol, 6% glycerol, 5% glycerol, 4% glycerol, 3% glycerol, 2% glycerol, 1% glycerol, 0.5% glycerol, 0.1% glycerol, or any range therebetween.

[0154] In some embodiments, a glycerol-buffer solution can be used to dilute a fixative. The fixative can be diluted with the glycerol-buffer solution at approximately 1 (fixative):1 (glycerol-buffer) solution, 1:2, 1:4, 1:8, 1:10, 10:1, 8:1, 4:1, 2:1, or any ratio therebetween.

[0155] In another embodiment, 100 μl of 1.5% formaldehyde fixative is added to a 2 ml screw-cap tube. 200 μl of citrate-anticoagulated whole blood is added to achieve a final formaldehyde concentration of approximately 0.5%. The mixture of fixative and citrate-anticoagulated whole blood is gently mixed, e.g., by flicking the bottom of the tube several times, and then incubated at room temperature for 15 minutes. 1.2 ml of phosphate-buffered saline or other suitable diluent is then added to dilute the sample, substantially reducing the formaldehyde concentration to approximately 0.1%. The resulting mixture can then be mixed, e.g., by inverting the tube, and then stored, e.g., at approximately +4°C, for later analysis and, optionally, for shipping. Additional steps can be added.

[0156] Another method for preparing a blood sample is provided, which includes the following steps.

[0157] The first step is performed (e.g., at a blood collection site). Blood is placed in a standard blue-capped tube, preferably containing 3.2% sodium citrate anticoagulant, which may be a vacutainer tube. The sample is then mixed to ensure the effectiveness of the anticoagulant, and 200 μl of blood is added to a collection tube containing 100 μl of fixative, e.g., formaldehyde fixative, to achieve a final formaldehyde concentration of approximately 0.5%. In some embodiments, the fixative is diluted with a buffer or buffer-glycerol solution. The contents of the collection tube may then be mixed, e.g., by tapping or flicking the bottom of the tube, as described above. The blood and fixative may then be incubated at room temperature for 15 minutes, and the sample may be diluted to achieve a final formaldehyde concentration of approximately 0.1%. After mixing, the sample may be stored, e.g., at approximately +4°C.

[0158] In some embodiments, blood can be fixed according to the methods provided herein for 1 day, 2 days, or up to 3 days after collection. Figure 30 shows the stability of anticoagulated blood when stored at room temperature for up to 3 days.

[0159] Figures 31 and 32 show the difference in stability over time after fixation with glycerol in diluent.

[0160] After storage, and if necessary, shipping (optionally on wet ice), analysis of the samples can be performed, even at a facility remote from the blood collection site. The tubes can be spun down in a centrifuge at 1,200 g for 2 minutes, the liquid can be decanted, the resulting pellet can be resuspended in phosphate-buffered saline (PBS), and the entire contents can be transferred to an Eppendorf tube. Further steps include: Add 100 μl of "perm" solution (ThermoFisher), which, along with standard reagents containing either ammonium chloride or acetic acid, induces lysis of red blood cells. Dissolution in ammonium chloride (10x concentration) 8.02 grams of NH4Cl (ammonium chloride) 0.84 grams of NaHCO3 (sodium bicarbonate) EDTA (disodium) 0.37g QS to 100ml with Millipore water.

[0161] Lysis of red blood cells can also be performed by adding 3% acetic acid (120 μl) to 5 μl of blood.

[0162] Incubate for 15 minutes. Add 800 μl of PBS. Centrifuge at 1,200 g for 2 minutes. Decant the liquid. Resuspend the platelet pellet in 1,000 μl of PBS. Centrifuge at 1,200 g for 2 minutes. Decant the liquid. Resuspend the platelet pellet in 1,000 μl of PBS. Centrifuge at 1,200 g for 2 minutes. The platelet pellet is resuspended in 800 μl of PBS containing 0.2% BSA and the platelets are stored at room temperature for at least 1 hour before the assay (blocking step). Final volume in assay tube = 35 μl, add the following: 5 μl resuspended platelets 0.5 μg of 5G1-PE antibody 2 μl CD42bPECy5 (Becton Dickinson) QS with PBS / 0.2% BSA. The antibody and platelets are incubated for 1 hour at room temperature. Dilute with 500 μl of PBS. Analyze by flow cytometry.

[0163] Flow cytometry Analyze platelet samples using template 032522 FCquant 5G1R-PE 375V_T. Set acquisition volume to 200ul and run samples at 200ul / min. Count 10,000 events (CD42b positive events) in the platelet gate. Platelets are first gated on an FSC vs. SSC plot. A second gate is created for CD42b PECY5 positive events. A histogram plot gated on CD42b-positive events is generated. The gate is set to the right of the non-immune IgG control. The x-mean of the histogram gate positive for 5G1R-PE binding is reported. IgG control assay tube, add the following: 5 μl resuspended platelets 2 μl IgG-PE 2 μl CD42bPECy5 (Becton Dickinson) 26 μl PBS / 0.2% BSA

[0164] Standardization of flow cytometry output By standardizing flow cytometry output measurements, samples can be measured using different instruments in different laboratories, on different days or times, and even under different conditions. Standardization allows for quantification of the number of FcγRIIa molecules per platelet, rather than relying on measurements of mean fluorescence. Problems with relying on mean fluorescence alone include the lack of reproducibility of results due to differences, for example, in individual instruments or reaction conditions.

[0165] Normalizing the mean fluorescence measurements from a flow cytometry instrument allows the flow cytometry output measurements to be converted into reliable and reproducible data, e.g., the number of antibody molecules bound per cell. The normalization process uses beads with a known ratio of phycoerythrin ("PE") molecules to beads, allowing the instrument or user to generate a curve, histogram, or other tool or metric that allows the number of PE molecules to be determined relative to the measured fluorescence output. By binding an antibody conjugated with PE and running the assay sample through the flow cytometry instrument using the same settings, the measured fluorescence output can be converted into the number of antibody bound. When antibodies bind to PE at a 1:1 ratio, the number of PE molecules per platelet is the number of FcγRIIa copies per platelet.

[0166] A detailed method using Quantibrite™ is described below.

[0167] Remove one Quantibrite™ tube (lyophilized pellet of beads conjugated with four levels of phycoerythrin (PE) available from Beckton Dickinson™) in a foil packet from the refrigerator. Add 500ul of PBS / 0.2% BSA to the Quantibrite™ tube. Mix gently. The beads are analyzed on a flow cytometer using the same voltage settings as for the platelet analysis, using a template Quantibrite™ BD375V_T. The beads are run at 200 ul / min and 20,000 events are counted. In this way, measurements of samples with known levels of fluorescent marker (here PE) can be obtained and used to normalize output measurements of samples with unknown levels of fluorescent marker.

[0168] In particular, measuring fluorescence using PE-labeled beads with known levels of PE allows for the establishment of a curve useful for converting mean fluorescence measurements into the actual number of FcγRIIa molecules per platelet.

[0169] The flow cytometry output (mean fluorescence intensity) can also be normalized by the use of Bang's Beads, allowing specific quantification of the number of FcγRIIa molecules per platelet.

[0170] One goal of the research described herein was to extend the time between blood collection and assay performance without adversely affecting the assay. This means that the antibody still binds to FcγRIIa on platelets, and a reasonably accurate measurement of FcγRIIa per platelet can still be achieved. Preferably, after a delay period before running the assay, the resulting FcγRIIa per platelet measurement remains relatively close to the measurement achieved after a short delay or no delay before running the assay. In preferred embodiments, the variability between measurements of FcγRIIa per platelet is 15% or less, or 10% or less, or 5% or less of measurements including a 7, 10, 14, or 21-day delay before the assay compared to measurements after only a short delay or no delay between blood collection and assay performance. Increasing the time between blood collection and assay performance allows for useful flexibility in performing the assay. For example, samples can be fixed and then stored or shipped before the assay is performed.

[0171] In another aspect, the present disclosure reflects the discovery that certain steps in preparing platelet samples impact sample stability and the ability to store the sample before performing an assay. Figure 23 shows the results of varying the time period between blood collection and performing an assay for quantification of platelet FcγRIIa in a study in which blood was stored in citrate anticoagulant. As shown, the measured levels of platelet FcγRIIa decreased with each day of storage. The line indicates the 10% mark.

[0172] Notably, it was discovered that starting with a low concentration of fixative, e.g., formaldehyde (0.5%), waiting 15 minutes, and then diluting the concentration in the sample to 0.1% formaldehyde allowed accurate quantification of platelet FcγRIIa even when samples were stored for up to two weeks after blood collection and platelet fixation, while maintaining less than 10% variation in measured platelet FcγRIIa compared to platelet FcγRIIa measured by an assay performed immediately after blood collection (see Figure 24). The line indicates the 10% marker and shows that even 10–15 days after fixation, measured platelet FcγRIIa levels did not fall below 10% of the earlier measurements on days 2–5.

[0173] Preferably, the blood sample is treated with fixative two hours or less after being drawn from the human patient into a blood sample tube.

[0174] In a preferred variation, certain steps of a method for preparing a blood sample are performed, including fixing the blood sample with a fixative at a concentration of 1.0 to 2.5%, incubating to allow fixation, then reducing the fixative concentration, for example to 0.5%, then optionally storing the blood sample for 1 to 14 days, and further processing the sample.

[0175] A suitable method may include: Adding a fixative to the blood sample at a concentration of about 0.1-2.5%, preferably about 0.2-2.0% or about 1.0-1.5%, more preferably about 0.5%, and preferably 0.5% by weight. Suitable concentrations include about 2.5%, 2.0%, 1.5%, and 1.0%. A preferred fixative is formaldehyde or a formaldehyde-based fixative. Incubating with the fixative for 5 to 70 minutes, preferably 5 to 60 minutes, 10 to 30 minutes, 10 to 20 minutes, and most preferably 15 minutes. Diluting the sample with an appropriate buffer to a fixative concentration of, for example, about 0.2% to 0.02%, or about 0.5% to 0.05%, preferably about 0.1% or 0.1%. Optionally, the sample can be washed to reduce the fixative concentration below 0.02%.

[0176] Optionally, the sample may be stored, eg, at 4° C. or room temperature, for a period of about 1 to about 21 days, preferably 1 to about 14 days, most preferably 1 to 14 days.

[0177] After optional storage, and if necessary after shipping, antibody assays can then be performed, optionally including removal of formaldehyde (centrifugation to pellet platelets and washing twice with PBS).

[0178] Figure 25 relates to test results using approximately 5% formaldehyde fixative. Tests were performed on blood from human subjects who are generally low-risk (not shown). Such subjects generally have lower platelet FcγRIIa expression. In these subjects, fixation with approximately 5% formaldehyde within 2 hours showed less than 10% variation in results (not shown). Subsequent analytic variation was performed using platelets from subjects with higher expression (shown). In patients with higher platelet FcγRIIa expression, a significant decay in mean fluorescence intensity was observed after 2 days of storage.

[0179] Figure 26 shows the results of a test based on varying the duration of time during which platelets were fixed. In this test, platelets in citrate-anticoagulated in vitro blood samples were fixed by introducing a low concentration of fixative, here 0.5% formaldehyde, into the sample and incubating for the time intervals in minutes indicated on the chart (5, 15, 30, or 60), and then diluting the fixative concentration in the sample to 0.1%. The mean fluorescence intensity based on FcγRIIa expression was measured according to the procedure described herein. As shown, fixative incubation periods of 15 minutes and longer resulted in a significant reduction in the standard deviation across the test results, as indicated by the error bars.

[0180] Figure 27 shows the results of a study to determine the impact of diluting fixative before sample storage. In this study, platelets in citrate-anticoagulated in vitro blood samples were fixed by introducing a low concentration of fixative, here 0.5% formaldehyde, to the sample and incubating for 15 minutes. The samples were then diluted with PBS to the concentration of formaldehyde fixative indicated on the chart (0.25%, 0.1%, 0.05%, or 0.01%) and stored for 4 days before testing for FcγRIIa expression. FcγRIIa was quantified 4 days after fixation / dilution. The resulting measurements of FcγRIIa expression (indicated by mean fluorescence intensity) showed a correlation between mean fluorescence intensity and increasing dilution of formaldehyde fixative. In particular, significantly lower mean fluorescence intensity measurements were observed for samples in which the formaldehyde fixative concentration was reduced to only 0.25%, compared to higher mean fluorescence intensity measurements for samples in which the formaldehyde fixative concentration was more diluted, e.g., to 0.1%, 0.05%, or 0.01%. These results demonstrate that fixative dilution, particularly to 0.1%, 0.05%, or 0.01%, impacts the ability to accurately measure FcγRIIa expression.

[0181] Figure 28 shows the results of a study to determine the impact of formaldehyde fixative concentration on quantification of FcγRIIa expression. In this study, platelets in citrate-anticoagulated in vitro blood samples were fixed within 2 hours of sample collection by introducing formaldehyde fixative to the concentration level indicated in the chart (2.5%, 2%, 1.5%, 1%, or 0.5%) for a 15-minute period. Each sample was then diluted to a final formaldehyde concentration of 0.1%. FcγRIIa expression assays were performed 4 days after fixation. Results showed an inverse correlation between the concentration of formaldehyde fixative used during the 15-minute formaldehyde fixation step and the resulting FcγRIIa expression measurements (as indicated by mean fluorescence intensity). Results for 0.5% showed a significant improvement in the resulting FcγRIIa expression measurements.

[0182] Previously known tests of platelet function were often affected by platelet count. In contrast, the innovative method described herein limits the impact of platelet count. Flow cytometry evaluates each particle that falls within a selected gate. In the context of the present invention, platelets are identified by their size and expression of universal platelet markers. The mean fluorescence intensity is the average of all counted platelets (typically a group of 10,000 or approximately 10,000 platelets). Because each individual platelet is evaluated, the measured expression is not affected by platelet count.

[0183] Figure 29 shows the results of a test on two different subjects using a commercially available FcγRIIa antibody compared to the use of the 5G1 antibody described herein. The commercially available antibody is the FLI8.26 monoclonal antibody from BD Pharmingen™. The test shows that when using unfixed platelets, the 5G1 antibody binds to a lower extent than the commercially available antibody. However, the 5G1 antibody binds to fixed platelets at a higher level. Notably, the results show that the 5G1 antibody disclosed herein binds to a greater extent to FcγRIIa on fixed platelets than to FcγRIIa on unfixed platelets.

[0184] It is known that when FcγRIIa expression is above 25,000 per platelet, the accuracy and variability of measurements increase. In contrast, the innovative method described herein ensures that the amount of antibody present is saturated. The greater variability associated with high expression suggests that the antibody is not saturated under high expression. This is addressed by increasing the concentration of antibody during incubation. Additional steps in the assay may include repeating the assay after additional dilution of platelets (effectively reducing the total amount of FcγRIIa and ensuring that the antibody is saturated again).

[0185] The innovative method described herein provides the ability to accurately quantify FcγRIIa expression across a wide range of patient expression levels, e.g., 1,500 to 30,000 molecules per platelet. Tests using microsphere beads have demonstrated that the method can detect 500 to 42,000 molecules per bead. The in vitro tests described herein can be applied to blood samples collected from humans or animals. As will be appreciated by those skilled in the art, when performing the tests described herein, the blood sample to be tested is preferably isolated from a human or animal patient. Furthermore, platelets can be isolated from the blood sample.

[0186] Calibrating Flow Cytometry Output - Mean Fluorescence Intensity Calibration of the flow cytometer can be, and preferably is, performed when the mean fluorescence intensity (MFI) of the mid-low beads, Quantibrite™ beads or Bang's MESF beads, deviates from ±5% of the nominal expression level.

[0187] Each flow cytometer device has individual characteristics. Moreover, results from a given flow cytometer device can vary, even from day to day. To ensure that results between devices are directly comparable, outputs (MFI) can be synchronized. Standardization beads (Quantibrite™, MESF™) can be used to assess MFI. Mid-low beads generally exhibit an MFI within the range of FcγRIIa expression on platelets. If the MFI of the beads is outside the nominal range of ±5%, preferably, calibration of the device is performed before analysis of clinical samples. To ensure that the measured signal remains stable, each lot of beads can be compared to the previous lot. Similarly, each lot of Rainbow beads is compared to the previous lot to ensure a consistent signal.

[0188] material BD Rainbow Calibration Beads: BD catalog #556286 (beads dyed for eight different fluorescent intensities and excited over a range of wavelengths) BD Quantibrite™; Fisher catalog #: 50-620-179 (standardized beads) or Bang's MESF Beads, Bang's catalog #827

[0189] procedure Create a new experiment on the flow cytometer instrument. · Set up a forward scatter / side scatter (FSC / SSC) linear plot. Set up two histogram plots, one for the BL2 (phycoerythrin - PE) channel and one for the RL1 (PECy5) channel. In the instrument settings, set the voltages that have been used to evaluate the Quantibrite™ Beads (as well as the control and platelet samples). A typical starting point is a voltage of 400 for BL2 and 350 for RL1. Set the threshold to 25. Add 3 drops of vortexed Rainbow Calibration beads to 1 ml of phosphate-buffered saline (PBS) and vortex before running on the flow cytometer. Set the acquisition volume to 100 μl and the flow rate to 25 μl / min. Adjust the FSC / SSC voltage until the beads are visible in FSC / SSC (approximately 350V for BL2 and 350V for RL1). Draw or name a gate around the single bead population (R1). · Set the histogram based on R1. Re-run the beads and collect 10,000 beads in R1. Draw five gates in each histogram plot and record the MFI. ·Adjust the voltages of the BL2 and RL1 channels until they fall within the ranges defined below. ○PE channel ○R2: 10,500~11,500 ○R3: 33,000~36,000 ○R4: 105,000~115,000 ○R5: 315,000 to 345,000 ○R6: 760,000 to 840,000 ○PECy5 channel ○R7: 3,000~3,600 ○R8: 11,000~12,000 ○R9: 36,000~39,000 ○R10: 95,000~105,000 ○R11: 160,000~176,000 Save the voltage settings in the FcγRIIa platelet assay template used for Quantibrite™, control beads, and platelet samples.

[0190] Preparation of high, low, and negative FcγRIIa control beads In another method, FcγRIIa control beads are prepared for use in flow cytometry.

[0191] material: Bang's Polystyrene Microspheres, 2.36 μm diameter, Catalog #: PC05002 Bang's PolyLink Protein Coupling Kit. Catalog #PL01N

[0192] Preferably, control beads with three different levels of FcγRIIa are prepared. Negative control beads (BSA control): FcγRIIa zero Low FcγRIIa beads: FcγRIIa 0.5μg: 125μl of bead solution High FcγRIIa beads: FcγRIIa 4μg: 125μl of bead solution

[0193] Example of a procedure for preparing low and high control beads: Warm the microparticles, coupling buffer (available from Bang's), and wash / storage buffer (available from Bang's) to room temperature. The coupling buffer is designed to activate the beads and prepare them for covalent coupling. Common buffers include MES (2-(N-morpholino)ethanesulfonic acid) buffer, (MES buffer), pH range 5.7-7.2. Dissolve 19.2 g of MES free acid (MW 195.2) in approximately 900 mL of purified water. Titrate to the desired pH with 1 N HCl or 1 N NaOH and adjust the final volume to 1000 mL with purified water. Vortex the beads at high speed four times for approximately 5 seconds each time, then pipette 125 μl into a microcentrifuge tube. Pellet the beads in a microcentrifuge (1200g for 3 minutes). Aspirate the supernatant and wash the beads by adding 400 μl of coupling buffer (CB). Pellet the beads again (centrifuge at 1200g for 3 minutes). Aspirate the supernatant and resuspend the beads in 170 μl of CB and transfer to a 2 ml microcentrifuge tube. Sonicate the beads for 60 seconds (maximum setting). Prepare EDAC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) solution: EDAC is preferably stored at -20°C and should be prepared fresh just before adding to the beads. Weigh out approximately 10 mg of EDAC and QS to 200 mg / ml with CB. Add 20 μl to 170 μl of microparticle suspension while vortexing at medium speed for approximately 5 seconds and allow the activation step to proceed for 15 minutes. · Sonicate the sample for 1 minute (maximum setting). Dilute FcγRIIa protein in 100 μl of CB and add to the activated beads while vortexing at medium speed. Mix by end-over-end for 30 minutes. Sonicate the sample (at maximum setting) for 1 minute after 15 minutes and for 60 seconds after 30 minutes. Add 50 μl of 1% BSA / PBS to the tube and continue incubation for 15 minutes. Vortex at high speed for approximately 5 seconds. Pellet the beads (centrifuge at 1200g for 5 minutes). Aspirate the supernatant and resuspend the beads in 400 μl of Wash / Storage Buffer (WSB) and vortex for approximately 10 seconds. Pellet the beads (centrifuge at 1200g for 5 minutes). Aspirate the supernatant and resuspend the beads in 400 μl of W / SB. Vortex at high speed for approximately 10 seconds. Add 100 μl of 1% BSA to W / SB for a final BSA concentration of 0.2% in a final volume of 500 μl. Analyze 1 μl on a flow cytometer to assess the single bead suspension. If significant aggregates are evident, sonicate the beads for 2 minutes.

[0194] BSA Negative Control Bead Procedure: The BSA negative control beads were saturated with BSA by absorption to prevent nonspecific binding of the FcγRIIa antibody. Pipette 125 μl of vortexed beads into a microcentrifuge tube. Pellet the beads in a microcentrifuge (1200g for 3 minutes). Aspirate the supernatant and wash the beads by adding 400 μl of coupling buffer (CB). Pellet the beads (centrifuge at 1200g for 3 minutes). Aspirate the supernatant and resuspend the beads in 400 μl of 1% BSA W / SB. Vortex the beads at high speed for approximately 5 seconds and mix by end-over-end mixing for 1 hour. After 30 minutes and at the end of the incubation, sonicate the beads for 1 minute (maximum setting). Pellet the beads (centrifuge at 1200g for 5 minutes). Aspirate the supernatant and resuspend the beads in 400 μl of W / SB. Vortex at high speed for approximately 10 seconds. Add 100 μl of 1% BSA to the W / SB for a final BSA concentration of 0.2%. Analyze 1 μl on a flow cytometer to assess the single bead suspension. If significant aggregates are evident, sonicate the beads for 2 minutes.

[0195] Assay for quantification of platelet FcγRIIa expression Main commercially available reagents ThermoFisher Fix Medium A; ThermoFisher catalog #GAS001S100 ThermoFisher Permeabilization Medium B; ThermoFisher catalog #GAS002S5 Phosphate-buffered saline (PBS); Fisher catalog #: 21031CM 10% bovine serum albumin (BSA) in PBS; Fisher catalog #: PI37525 BD Quantibrite; Fisher catalog #: 50-620-179 or Bang's MESF Beads, Bang's catalog #827 CD42b PECY5; Fisher Catalog #: BDB551141 Control IgG PE; Fisher Catalog #: BDB340013

[0196] Processes performed in clinical settings Collect in vitro blood samples in standard blue-capped (citrate) tubes or other suitable containers. Mix the blood in the blue-capped tube and then add 200 μl of blood to a collection tube (e.g., a screw-cap tube with a colored cap) containing 100 μl of Fix diluted 1:4 in PBS. Mix the contents of the collection tube by gently tapping the bottom of the tube. Incubate the blood with the Fix for 15 minutes at room temperature. Using the provided transfer pipette, transfer the entire contents (1.2 ml) of the diluent (phosphate buffered saline) into a collection tube. Mix contents by inverting. Store collection tubes at 4°C until shipping. Ship the tubes on ice.

[0197] Processes carried out in the laboratory Step 1: Calibration of the flow cytometer: preferably performed each day the assay is performed Each flow cytometer has its own unique characteristics. To ensure that results between instruments are directly comparable, outputs (mean fluorescence intensity - MFI) are preferably synchronized. Standardization beads (Quantibrite™) are used to assess MFI. Mid-low beads have an MFI that is within the range of FcγRIIa expression on platelets. To ensure that the signal remains stable, each lot of Quantibrite™ beads is preferably compared to the previous lot. If the MFI of the mid-low beads is outside the range of 27,000 to 30,000, instrument calibration (according to the calibration protocol) is preferably performed before analysis of clinical samples.

[0198] Preparation of Quantibrite™ standard curve Remove one Quantibrite™ tube from the foil packet. Add 500 μl of PBS / 0.2% BSA to the Quantibrite™ tube. Mix gently. Analyze the beads on a flow cytometer using the same voltage settings used for platelet analysis. Run the beads at 200 μl / min and count 20,000 events.

[0199] Preparation of Bang's™ MESF R-PE Standard Curve (Alternative to Quantibrite™) Label five Eppendorf tubes B, 1, 2, 3, and 4. Add 500 μl of PBS to each tube. Remove the MESF beads from the refrigerator and mix them gently by inverting and tapping the bottom of the tube. Add 20 μl of Bang's MESF beads to 500 ul of PBS. Vortex the tube. Analyze the beads on a flow cytometer using the same voltage settings used for platelet analysis.

[0200] Step 2: Analyze control samples: performed on each day the assay is performed High and low control beads are analyzed in duplicate. Assay Procedure: Add 50 μl of 1% BSA / PBS to the assay tube. Add 0.5 μg of phycoerythrin-conjugated 5G1 antibody (5G1PE). Add 1 μl of FcγRIIa control beads. Incubate the beads with the antibody for 30 minutes. Add 1 ml of PBS and spin at 1200 g for 3 minutes. Aspirate the supernatant and resuspend in 1000 μl of PBS. Flow cytometry analysis should be performed 1 hour after washing.

[0201] Flow cytometry analysis of control beads The control beads should be analyzed under the same settings as defined for the Quantibrite™ and platelet assays. Generate a log-forward scatter / side scatter (FSC / SSC) plot and gate around the single bead population. Generate a histogram plot based on 5G1PE gated on the single bead population gate. Run the labeled beads, set a gate to exclude the BSA peak (non-specific binding), and record the MFI of the low and high controls. The MFI of the low and high controls is converted to FcγRIIa molecules per bead using the calculation template (below). If the high and low control values ​​(FcγRIIa molecules per bead) are outside the predetermined range (provided with the beads), a calibration (Quantibrite™ and flow cytometry output) should be performed.

[0202] Step 3: Platelet FcγRIIa assay Centrifuge the collection tube at 1,200 g for 2 minutes. Decant the liquid. · Resuspend the platelet pellet in 100 μl of PBS and transfer the entire contents to an Eppendorf tube. Add 100 μl of "perm" solution. Incubate for 15 minutes. Add 800 μl of PBS. Centrifuge at 1,200g for 2 minutes. Decant the liquid. Resuspend the platelet pellet in 1,000 μl of PBS. Centrifuge at 1,200g for 2 minutes. Decant the liquid. Resuspend the platelet pellet in 1,000 μl of PBS. Centrifuge at 1,200g for 2 minutes. · Resuspend the platelet pellet in 800 μl of PBS containing 0.2% BSA and store the platelets at room temperature for at least 1 hour before the assay (blocking step). Final volume in assay tube = 35 μl 5 μl of resuspended platelets 0.5 μg of 5G1-PE 2 μl of CD42bPECy5 (Becton Dickinson) QS with PBS / 0.2% BSA. Incubate the antibody and platelets for 1 hour at room temperature. Add 50 μl of Medium A fixative and incubate at room temperature for 15 minutes. Dilute with 500 μl of PBS. Analyze by flow cytometry.

[0203] Flow cytometry Analyze the platelet sample using the template. Set the acquisition volume to 200 μl and run the sample at 200 μl / min. Count 10,000 events (CD42b positive events) in the platelet gate. Platelets are first gated by size (FSC / SSC plot). A second gate is created for CD42b PECY5 positive events. A histogram plot gated on CD42b-positive events is generated. The gate is set to the right of the non-immune IgG control (≤1% of platelets are in the gate). The MFI of the histogram gate binding to 5G1PE is reported. IgG control assay tube 5 μl of resuspended platelets 2 μl IgG-PE 2 μl of CD42bPECy5 (Becton Dickinson) 26 μl PBS / 0.2% BSA

[0204] Calculation of molecules FcγRIIa per platelet BD Quantibrite™ beads contain four MFI peaks with a specified number of PE molecules per bead. These results are used to generate a calibration curve that converts MFI to FcγRIIa molecules per platelet. A calculation template is provided. A calculation template (Excel file) is used to input the MFI from the histogram statistics. A lot-specific value of Quantibrite™ PE molecules per bead is used. A linear regression equation is generated relating log PE molecules per bead to log MFI. The slope and intercept of the standard curve are calculated. The formula converts MFI to FcγRIIa molecules per platelet (or FcγRIIa molecules per control bead).

[0205] Additional definitions "Adenosine diphosphate (ADP) receptor" refers to a purinergic G protein-coupled receptor stimulated by the nucleotide adenosine diphosphate (ADP). ADP receptors include P2Y12, which regulates thrombosis. Adenosine diphosphate (ADP) receptor antagonists are agents that inhibit adenosine diphosphate receptors. P2Y12 is the target of antiplatelet drugs, including prasugrel, clopidogrel, and other thienopyridines.

[0206] As used herein, the term "about" means ±5% of the stated value unless otherwise stated and always explicitly includes the stated value.

[0207] "Clopidogrel" means (+)-(S)-methyl 2-(2-chlorophenyl)-2-(6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)acetate, a potent platelet aggregation inhibitor.

[0208] "Prasugrel" means (RS)-5-[2-cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl]-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl acetate, a potent platelet aggregation inhibitor.

[0209] "Ticagrelor" refers to the potent platelet aggregation inhibitor (1S,2S,3R,5S)-3-[7-[(1R,2S)-2-(3,4-difluorophenyl)cyclopropylamino]-5-(propylthio)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl]-5-(2-hydroxyethoxy)cyclopentane-1,2-diol.

[0210] "Vorapaxar" means ethyl N-[(3R,3aS,4S,4aR,7R,8aR,9aR)-4-[(E)-2-[5-(3-fluorophenyl)-2-pyridyl]vinyl]-3-methyl-1-oxo-3a,4,4a,5,6,7,8,8a,9,9a-decahydro-3H-benzo[f]isobenzofuran-7-yl]carbamate, a potent platelet aggregation inhibitor.

[0211] "Antithrombotic therapy" means any treatment used to inhibit or reduce thrombosis or to inhibit or reduce platelet aggregation in a subject.

[0212] By "agent" is meant any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragment thereof.

[0213] By "ameliorate" is meant to lessen, inhibit, attenuate, reduce, arrest, or stabilize the onset or progression of a disease.

[0214] By "alteration" is meant a change (increase or decrease) in the expression level or activity of a gene or polypeptide as detected by standard known methods, such as those described herein. As used herein, alteration includes a 10% change in expression level, preferably a 25% change, more preferably a 40% change, and most preferably a 50% or greater change in expression level.

[0215] "Analog" refers to a molecule that is not identical but has similar functional or structural features. For example, a polypeptide analog retains the biological activity of the corresponding naturally occurring polypeptide while possessing certain biochemical modifications that enhance the analog's function compared to the naturally occurring polypeptide. Such biochemical modifications may, for example, increase the analog's protease resistance, membrane permeability, or half-life without altering ligand binding. Analogs may include unnatural amino acids.

[0216] "Analyte" means any compound under investigation using an analytical method.

[0217] By "analyte-binding conjugate" is meant a detectable molecule that binds to the compound under investigation.

[0218] "Capillary communication" means promoting the flow of liquid between liquid-permeable materials.

[0219] "Capture reagent" refers to a reagent that specifically binds to a polypeptide or nucleic acid molecule for the purpose of selecting or isolating the polypeptide or nucleic acid molecule. In various embodiments, a capture reagent for an FcγRIIa polypeptide is an anti-FcγRIIa antibody. In other embodiments, the platelet capture reagent specifically binds to a platelet cell surface polypeptide (e.g., useful for binding platelets to a solid phase). Exemplary platelet capture reagents include, but are not limited to, antibodies against glycoprotein (GP) IIb (e.g., anti-CD41 or CD41a); antibodies against GP IIIa (e.g., anti-CD61); antibodies against GP V (e.g., anti-CD42d); antibodies against GP Ib (e.g., anti-CD42b); antibodies against GP IX, such as anti-CD42a; ​​antibodies against lysosomal membrane proteins (e.g., anti-CD63); and antibodies against PECAM (e.g., anti-CD31).

[0220] In this disclosure, "comprises," "comprising," "containing," and "having" and the like may have the meaning ascribed to them in U.S. patent law and may mean "includes," "including," and the like; "consisting essentially of" or "consisting essentially of" likewise have the meaning ascribed to them in U.S. patent law and the terms are open-ended, permitting the presence of more than what is recited, but excluding prior art aspects, so long as the basic or novel characteristics of the recited items are not altered by the presence of more than what is recited.

[0221] As used herein, "contacting," such as in "contacting a sample," refers to directly or indirectly contacting a sample in vitro, ex vivo, or in vivo (i.e., within a subject as defined herein). Contacting a sample can include adding an antibody to the sample, adding a compound to the sample, or administering it to a subject. Contacting encompasses administration to a solution, cell, tissue, mammal, subject, patient, or human. Additionally, contacting a cell includes adding an agent to a cell culture.

[0222] By "control conjugate" is meant a detectable molecule that does not substantially bind to the compound under investigation.

[0223] "Detection" refers to determining the presence, absence, or amount of the analyte being detected.

[0224] "Detectable label" refers to a composition that, when attached to a molecule of interest, renders the molecule of interest detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioisotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (e.g., those commonly used in ELISA), biotin, digoxigenin, or haptens, including, for example, phycoerythrin.

[0225] "Disease" means any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. Examples of diseases include thrombotic disorders, which involve an undesirable increase in platelet reactivity and / or the formation of blood clots, such as thrombi that result in ischemic events.

[0226] "Effective amount" refers to the amount of agent required to alleviate the symptoms of a disease compared to an untreated patient. The effective amount of an active compound or composition used to practice the present disclosure for the therapeutic treatment of a disease varies depending on the method of administration, the age, weight, and general health of the subject. Ultimately, the attending physician or veterinarian will determine the appropriate amount and dosage regimen. Such an amount is referred to as an "effective" amount.

[0227] "Flow cytometry" means a technique for counting and examining microparticles that allows for multiparameter analysis of the physical and / or chemical characteristics of the microparticles.

[0228] As used herein, the terms fixed, fixative, fixative, and their various forms refer to the treatment of living cells to preserve their properties or to prevent or at least slow decay or other deterioration. In certain embodiments, fixation is intended to terminate or slow biochemical responses and may increase the mechanical strength or stability of the treated cells. Formaldehyde fixation is preferred and useful in flow cytometry. Glutaraldehyde, a suitable alternative, exhibits autofluorescence. In the embodiments described herein, formaldehyde was used to fix platelets unless otherwise noted.

[0229] The present disclosure provides a number of targets useful for the development of highly specific drugs for treating thrombotic diseases or disorders (e.g., characterized by undesirable increases in platelet reactivity) characterized by the methods described herein.In addition, the methods of the present disclosure provide a convenient means for identifying therapies that are safe for use in subjects.In addition, the methods of the present disclosure provide a route for analyzing virtually any number of compounds for their effects on the thrombotic diseases described herein with high volume throughput, high sensitivity, and low complexity.

[0230] By "fragment" is meant a portion of a polypeptide or nucleic acid molecule. The portion preferably contains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

[0231] "Hybridization" means hydrogen bonding, which may be Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds.

[0232] "Inhibitory nucleic acid" refers to a double-stranded RNA, siRNA, shRNA, or antisense RNA, or a portion thereof, or a mimetic thereof, that, when administered to a mammalian cell, causes a reduction (e.g., 10%, 25%, 50%, 75%, or even 90-100%) in expression of a target gene. Typically, a nucleic acid inhibitor comprises at least a portion of a target nucleic acid molecule or its ortholog, or comprises at least a portion of the complementary strand of a target nucleic acid molecule. For example, an inhibitory nucleic acid molecule comprises at least a portion of any or all of the nucleic acids delineated herein.

[0233] "Isolated polynucleotide" refers to a nucleic acid (e.g., DNA) that is free of the genes adjacent to it in the naturally occurring genome of the organism from which the nucleic acid molecule of the present disclosure is derived.Thus, this term includes, for example, recombinant DNA incorporated into a vector; recombinant DNA incorporated into an autonomously replicating plasmid or virus; or recombinant DNA incorporated into the genomic DNA of a prokaryotic or eukaryotic organism; or recombinant DNA that exists as a separate molecule independent of other sequences (e.g., cDNA or genomic fragments or cDNA fragments generated by PCR or restriction endonuclease digestion).In addition, this term also includes RNA molecules transcribed from DNA molecules, as well as recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequences.

[0234] By "isolated polypeptide" is meant a polypeptide of the present disclosure that has been separated from components that naturally accompany it. Typically, a polypeptide is isolated when at least 60% by weight is free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a polypeptide of the present disclosure. Isolated polypeptides of the present disclosure can be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

[0235] "Lateral flow device" means a test device that relies on the flow of liquid via capillary action, wicking, or wetting of a liquid-permeable medium present in the device.

[0236] "Liquid-permeable material" means a material that is susceptible to wetting, wicking, or transmission of liquids by capillary action.

[0237] By "marker" is meant any protein or polynucleotide having an altered expression level or activity that is associated with a disease or disorder. For example, increased levels, activity, phosphorylation, or expression of FcγRIIa is associated with increased platelet reactivity and / or an increased propensity to develop a thrombotic disease or disorder.

[0238] Percentages, when used herein to refer to the amount of an ingredient in a composition, refer to percentages by weight unless otherwise indicated.

[0239] By "portion" is meant a fraction of a whole. A portion of a test device can be, for example, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 of the length of the internal flow path of the device.

[0240] "Protease-activated receptor (PAR)" refers to a G protein-coupled receptor that is activated by cleavage of a portion of its extracellular domain. PARs are highly expressed in platelets and include the thrombin receptors PAR1, PAR3, and PAR4. PARs are activated by the action of serine proteases, such as thrombin (which activates PAR1, PAR3, and PAR4). Cleavage of the N-terminus of the receptor generates a tethered ligand (SFLLRN) that acts as an agonist, triggering physiological responses. The cellular effects of thrombin are mediated by protease-activated receptors (PARs). Thrombin signaling in platelets contributes to hemostasis and thrombosis. Thrombin receptor antagonists include vorapaxal (SCH 530348), a PAR1 antagonist.

[0241] As used herein, "obtaining," as in "obtaining an agent," includes synthesizing, purchasing, or otherwise acquiring the agent.

[0242] "Primer set" refers to a set of oligonucleotides that can be used, for example, in PCR. A primer set consists of at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 30, 40, 50, 60, 80, 100, 200, 250, 300, 400, 500, 600, or more primers.

[0243] By "reduce" is meant at least a 10%, 25%, 50%, 75%, or 100% negative change in a parameter.

[0244] "Reference" means a standard or control condition.

[0245] " Reference sequence " is a defined sequence that is used as a basis for sequence comparison. Reference sequence can be a subset or the entirety of a specified sequence; for example, a segment of a full-length cDNA or a segment of a gene sequence, or a complete cDNA or complete gene sequence. For polypeptides, the length of a reference polypeptide sequence is generally at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of a reference nucleic acid sequence is generally at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, even more preferably about 100 nucleotides or about 300 nucleotides, or any integer number close thereto or therebetween.

[0246] " siRNA " refers to double-stranded RNA.Optimally, siRNA is 18, 19, 20, 21, 22, 23 or 24 nucleotides in length and has a 2-base overhang at its 3' end.These dsRNAs can be introduced into individual cells or whole animals; for example, they can be introduced into the whole body through bloodstream.Such siRNAs are used to down-regulate mRNA level or promoter activity.

[0247] By "specifically binds" is meant a compound or antibody that recognizes and binds to a polypeptide of the present disclosure, but does not substantially recognize and bind to other molecules in a sample, e.g., a biological sample, that naturally contains the polypeptide of the invention.

[0248] Nucleic acid molecules useful in the disclosed methods include any nucleic acid molecule encoding a polypeptide of the present invention or a fragment thereof. Such nucleic acid molecules do not need to be 100% identical to the endogenous nucleic acid sequence, but typically exhibit substantial identity. "Percentage of sequence identity" or "percent similarity" is determined by comparing two optimally aligned sequences over a comparison window, where the portion of the polynucleotide or peptide sequence in the comparison window may contain additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions) due to optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where the same nucleic acid base or amino acid residue appears in both sequences to determine the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to determine the percentage of sequence identity.

[0249] The term "substantial identity" or "substantial similarity" of a polynucleotide or peptide sequence means that the polynucleotide or peptide contains a sequence with at least 75% sequence identity. Alternatively, the percent identity can be any integer between 75% and 100%. More preferred embodiments include at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity compared to a reference sequence using a program described herein, preferably BLAST, with standard parameters as described. These values ​​can be adjusted appropriately to determine the corresponding identity of proteins encoded by two nucleotide sequences, taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like.

[0250] For purposes of this invention, "substantial identity" of amino acid sequences typically means at least 75% polypeptide sequence identity. Preferred percent polypeptide identity can be any integer between 75% and 100%. More preferred embodiments include at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.7%, or 99%.

[0251] " Percentage of sequence identity " and " percentage of homology " are used interchangeably herein to refer to the comparison between polynucleotides and polypeptides, and are determined by comparing two optimally aligned sequences across a comparison window, where the portion of the polynucleotide sequence or polypeptide sequence in the comparison window may contain additions or deletions (i.e., gaps) compared to the reference sequence due to the optimal alignment of the two sequences.Percentage can be calculated by determining the number of positions where the same nucleic acid base or amino acid residue appears in both sequences to determine the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to determine the percentage of sequence identity.Alternatively, percentage can be calculated by determining the number of positions where either the same nucleic acid base or amino acid residue appears in both sequences, or the number of positions where nucleic acid base or amino acid residue is aligned with gaps, to determine the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to determine the percentage of sequence identity. Those skilled in the art appreciate that there are many established algorithms available to align two sequences.Optimal alignment of sequences for comparison can be accomplished, for example, by the local homology algorithm of Smith and Waterman, (1981) Adv. Appl. Math. 2:482, by the homology alignment algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. 48:443, by the similarity search method of Pearson and Lipman, (1988) Proc. Natl. Acad. Sci. USA 85:2444, by computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA), or by visual inspection (see generally Current Protocols in Molecular Biology, F.M. Ausubel et al., eds., Current Protocols, Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995 Supplement)).

[0252] Examples of suitable algorithms for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., (1990), J. Mol. Biol. 215: 403-410 and Altschul et al., (1977) Nucleic Acids Res. 3389-3402, respectively. Software for performing BLAST analyses is publicly available through the website of the National Center for Biotechnology Information.

[0253] Similar DNA and protein sequences Two DNA sequences are "substantially similar" when approximately 70% or more (e.g., at least about 80%, at least about 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of the nucleotides match over a defined length of the DNA sequence. Substantially homologous sequences can be identified by comparing sequences using standard software available in sequence data banks or in Southern hybridization experiments, e.g., under stringent conditions defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Maniatis et al., Molecular Cloning: A Laboratory Manual, 1982; DNA Cloning, Vols. I & II, supra; Nucleic Acid Hybridization, [BD Hames & SJ Higgins eds. (1985)].

[0254] "Substantially similar" also means a DNA sequence that is not identical to that set forth in any disclosed sequence due to the degeneracy of the genetic code, but still encodes the same amino acid sequence; or a DNA sequence that encodes a different amino acid sequence that retains the activity of the protein either because an amino acid has been replaced by a similar amino acid, or because the change (whether a substitution, deletion, or insertion) does not affect the active site of the protein.

[0255] Two amino acid sequences or two nucleic acid sequences are "substantially similar" when approximately 70% or more (e.g., at least about 80%, at least about 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of the amino acids match over a defined length of the sequences.

[0256] As evidenced herein, modifications and changes can be made to the structure of peptides and the DNA segments that encode them and still result in functional molecules that encode proteins or peptides with desirable characteristics. Amino acid changes in proteins can be used to create equivalent or even improved second-generation molecules.

[0257] For example, certain amino acids can be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with the structure, such as with the antigen-binding region of an antibody or a binding site on a substrate molecule. Because it is the interactive capacity and properties of a protein that define its biological functional activity, certain amino acid sequence substitutions can be made in the protein sequence, and of course in the underlying DNA coding sequence, and still obtain a protein with similar properties. Thus, it is contemplated by the inventors that various changes can be made in the peptide sequences of the disclosed compositions, or the corresponding DNA sequences encoding the peptides, without appreciable loss of their biological utility and activity.

[0258] In making such changes, the hydropathic amino acid index may be considered. The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte and Doolittle, J. Mol. Biol., 157(1):105-132, 1982).

[0259] It is known in the art that certain amino acids can be substituted with other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., a biologically functionally equivalent protein. When making such changes, there is a general tendency to substitute amino acids with hydropathic indices within a range of about ±0.5 to about ±2. It is also understood in the art that substitution of similar amino acids can be effectively made based on hydrophilicity. For example, the local average hydrophilicity of a protein, which is influenced by the hydrophilicity of its neighboring amino acids, can be correlated with the biological properties of the protein.

[0260] It is understood that an amino acid can be substituted for another amino acid having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent, protein.

[0261] Thus, as outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, e.g., their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions that take into consideration the various aforementioned characteristics are well known to those of skill in the art and include, but are not limited to, arginine for lysine; glutamic acid for aspartic acid; serine for threonine; glutamine for asparagine; and valine for leucine and isoleucine.

[0262] Polypeptides can be chemically synthesized. Synthetic polypeptides are prepared using well-known techniques, such as, but not limited to, solid-phase techniques, liquid-phase techniques, or peptide condensation techniques, or any combination thereof, and can include natural and unnatural amino acids.

[0263] As used herein, the recitation of a list of elements in any definition of a variable includes definitions of that variable as any single element or combination (or subcombination) of the listed elements. As used herein, the recitation of an embodiment includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0264] For example, a stringent salt concentration is typically less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be achieved in the absence of organic solvents, such as formamide, while high stringency hybridization can be achieved in the presence of at least about 35% formamide, and more preferably at least about 50% formamide. Stringent temperature conditions typically include a temperature of at least about 30°C, more preferably at least about 37°C, and most preferably at least about 42°C. Various additional parameters, such as hybridization time, the concentration of detergents, such as sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency can be achieved by combining these various conditions as needed. In a preferred embodiment, hybridization is carried out at 30° C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization is carried out at 37° C. in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg / ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization is carried out at 42° C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg / ml ssDNA. Useful modifications to these conditions will be readily apparent to those of skill in the art.

[0265] In most applications, the stringency of the washing step following hybridization also varies. The stringency of the washing can be defined by salt concentration and temperature. As mentioned above, the stringency of the washing can be increased by decreasing the salt concentration or increasing the temperature. For example, stringent salt concentrations for the washing step are preferably less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the washing step typically include a temperature of at least about 25°C, more preferably at least about 42°C, and even more preferably at least about 68°C. In a preferred embodiment, the washing step is performed at 25°C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, the washing step is performed at 42°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, the wash step is carried out at 68°C in 15mM NaCl, 1.5mM trisodium citrate, and 0.1% SDS. Additional modifications to these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

[0266] "Subject" means a mammal, including, but not limited to, a human or a non-human mammal, such as a cow, horse, dog, sheep, or cat. In some embodiments, the subject is at increased risk of thrombosis. In some embodiments, the subject is suspected of having an increased risk of thrombosis or platelet reactivity, or any condition related to thrombosis or reactive platelets. The subject may also have or be suspected of having coronary artery disease, renal disease, or end stage genital disease, one or more myocardial infarctions, cardiovascular disease, stroke, diabetes, or atherosclerosis.

[0267] Ranges provided herein are understood to be shorthand for all values ​​within the range. For example, the range 1 to 50 is understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0268] As used herein, the terms "treat," "treating," "treatment," and the like refer to reducing or alleviating a disorder and / or its associated symptoms. It will be understood that treating a disorder or symptom does not require, but does not exclude, the complete abolition of the disorder, condition, or its associated symptoms.

[0269] "Test device" means a device used in the detection of an analyte in a sample.

[0270] "Wick" means to absorb and adsorb liquid.

[0271] Unless specifically stated or clear from context, as used herein, the term "or" is understood to be inclusive. Unless specifically stated or clear from context, as used herein, the terms "a," "an," and "the" are understood to be singular or plural.

[0272] Unless otherwise specified or clear from the context, the term "about" as used herein is understood to mean within the normal tolerance range in the art, for example, within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values ​​provided herein are modified by the term about.

[0273] Herein, the recitation of a list of chemical groups in any definition of a variable includes definitions of that variable as any single group or combination of listed groups. Herein, the recitation of an embodiment for a variable or aspect includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0274] Any composition or method provided herein can be combined with one or more of any other compositions and methods provided herein.

[0275] Other Aspects In certain aspects, the present disclosure relates to any of the following examples:

[0276] 1. At least one heavy chain complementarity determining region (CDRH) and at least one light chain complementarity determining region (CDRL) Including, wherein the CDRH is selected from SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, and the CDRL is selected from SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, wherein SEQ ID NO: 11 encodes the amino acid sequence DTS; or wherein the CDRH is selected from SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, and the CDRL is selected from SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, wherein SEQ ID NO: 21 encodes the amino acid sequence WAS; An isolated antibody or antigen-binding fragment thereof that specifically binds to FcγRIIa. 2. At least two heavy chain complementarity determining regions (CDRHs) and at least two light chain complementarity determining regions (CDRLs) Including, wherein the CDRH is selected from SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, and the CDRL is selected from SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12; or wherein the CDRH is selected from SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, and the CDRL is selected from SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22. The isolated antibody or antigen-binding fragment thereof of Example 1. 3. a. a CDRH1 region comprising SEQ ID NO: 5, a CDRH2 region comprising SEQ ID NO: 6, and a CDHR3 region comprising SEQ ID NO: 7; and a CDRL1 region comprising SEQ ID NO: 10, a CDRL2 region comprising SEQ ID NO: 11, and a CDRL3 region comprising SEQ ID NO: 12, and a sequence at least 90% identical to SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, or b. a CDRH1 region comprising SEQ ID NO: 15, a CDRH2 region comprising SEQ ID NO: 16, and a CDHR3 region comprising SEQ ID NO: 17; and a CDRL1 region comprising SEQ ID NO: 20, a CDRL2 region comprising SEQ ID NO: 21, and a CDRL3 region comprising SEQ ID NO: 22, as well as sequences at least 90% identical to SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22. The antibody or antigen-binding fragment thereof of Example 1 or 2, comprising: 4. The heavy chain complementarity determining region is encoded by SEQ ID NO: 4 and the light chain complementarity determining region is encoded by SEQ ID NO: 9, and the sequences are 90% identical to SEQ ID NO: 4 and SEQ ID NO: 9; or the heavy chain complementarity determining region is encoded by SEQ ID NO: 14 and the light chain complementarity determining region is encoded by SEQ ID NO: 19, and the sequences are 90% identical to SEQ ID NO: 14 and SEQ ID NO: 19. The antibody or antigen-binding fragment thereof of any one of Examples 1 to 3. 5. The antibody of any one of the preceding examples, wherein FcγRIIa is present on platelets. 6. The antibody or antigen-binding fragment of any one of Examples 1 to 5, wherein the antibody is linked to a detectable label. 7. The antibody or antigen-binding fragment of any one of Examples 1 to 6, wherein FcγRIIa is present on a platelet and the platelet has been fixed with a fixative prior to binding of the antibody to the FcγRIIa. 8. The antibody or antigen-binding fragment of any one of the preceding examples, wherein said antibody is an isolated monoclonal antibody. 9. The antibody or antigen-binding fragment of any one of the preceding examples, wherein said antibody is a monoclonal antibody. 10. The antibody or antigen-binding fragment of any one of the preceding examples, wherein the antibody is, or is generated using, recombinant antibody technology, nucleic acid aptamer technology, or non-immunoglobulin protein scaffold technology. 11. A conjugate of an antibody that binds to FcγRIIa, wherein the antibody comprises any antibody or antigen-binding fragment of any one of the preceding examples. 12. A method for detecting FcγRIIa, comprising the step of binding FcγRIIa to an antibody, wherein the antibody comprises the antibody or antigen-binding fragment of any one of the preceding examples. 13. A method for detecting the presence of FcγRIIa in a blood sample, comprising the step of binding an antibody to FcγRIIa, wherein the antibody comprises an antibody or antigen-binding fragment of any one of the preceding examples. 14. The method of Example 13, wherein the platelets in the blood sample are treated with a fixative solution before or at the same time as the antibody is introduced into the blood sample. 15. The method of Example 13 or 14, wherein the fixative is combined with the blood sample for up to 2 days after collection of the blood sample. 16. The method of any one of Examples 13-15, wherein the fixative is diluted with a solution comprising a buffer and glycerol before being combined with the blood sample. 17. The method of any one of Examples 13-16, wherein the buffer is phosphate buffered saline (PBS) and the glycerol comprises approximately in the range of 2% to 7% of the total volume of the buffer-glycerol solution. 18. The method of Example 16 or 17, wherein the glycerol is 5% of the total volume of the buffer-glycerol solution. 19. The method of any one of Examples 12-18, wherein the bound FcγRIIa and antibody complexes are detected via flow cytometry to measure the level of FcγRIIa in the sample. 20. The method of Example 19, wherein the measured level of FcγRIIa in the sample is normalized based on a comparison of the measured level of fluorescence in the sample to a known level of a fluorescent marker. 21. A method for detecting the presence of FcγRIIa in a blood sample, comprising: treating the blood sample with an anticoagulant; adding a fixative to the blood sample; separating and washing platelets from said blood sample; incubating the platelets with an antibody comprising any one of the antibodies or antigen-binding fragments of any one of Examples 1 to 11; and performing an analysis to quantify FcγRIIa in the sample. The method comprising: 22. The method of Example 21, wherein said analysis is selected from the group consisting of flow cytometry, immunoassay, ELISA, Western blotting, and radioimmunoassay. 23. The method of Example 22, wherein the analysis is flow cytometry. 24. Reducing the concentration of fixative in the blood sample; and storing the blood sample for a period of up to and including 1 to 14 days before incubating the platelets with the antibody. The method of any one of Examples 21-23, further comprising: 25. A method for preparing a blood sample, comprising: treating the blood sample with an anticoagulant; adding a fixative to the blood sample; subsequently diluting the concentration of the fixative in the blood sample; and storing the blood sample for a period of time up to and including 1 to 14 days. The method comprising: 26. The method of Example 25, in which fixative is added to a concentration of 5% and subsequently diluted to 1.25%. 27. A method for detecting FcγRIIa, comprising the step of binding FcγRIIa to an antibody, the antibody comprises at least one heavy chain complementarity determining region (CDRH) and at least one light chain complementarity determining region (CDRL); wherein the CDRH is selected from SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, and the CDRL is selected from SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, wherein SEQ ID NO: 11 encodes the amino acid sequence DTS; or wherein the CDRH is selected from SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, and the CDRL is selected from SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, wherein SEQ ID NO: 21 encodes the amino acid sequence WAS; The method. 28. The method of Example 27, wherein FcγRIIa is detected on platelets from a blood sample. 29. The method of Example 27 or 28, further comprising comparing the level of detection of FcγRIIa between a blood sample from a human having or suspected of having heart disease, thrombosis, coronary artery disease, kidney disease, or myocardial infarction and a blood sample from a control human who does not have heart disease, thrombosis, coronary artery disease, kidney disease, or myocardial infarction. 31. The antibody or antigen-binding fragment of any one of Examples 1 to 11 for use in a method for detecting platelet reactivity in a subject.

[0277] All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each individual patent and publication was specifically and individually indicated to be incorporated by reference.

[0278] From the foregoing description, it will be apparent that variations and modifications may be made to the subject matter described herein to adapt it to various uses and conditions. Such embodiments also fall within the scope of the following claims.

[0279] The following examples are put forward so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assays, screening methods, and treatments of the present disclosure, and are not intended to limit the scope of what the inventors regard as their disclosure. [Example]

[0280] Example 1: FcγRIIa is phosphorylated after platelet activation We quantified the phosphorylation of FcγRIIa immunoprecipitated from platelets isolated from low-risk subjects after activation with thrombin, convulxin, ADP, and PAF (Figures 1A–1E). Although a band corresponding to the expected molecular weight of FcγRIIa was evident at 45 kDa, additional bands at 55 kDa, 75 kDa, and 90 kDa demonstrated phosphorylation. In subsequent experiments, immunoprecipitation was performed using an anti-phosphotyrosine antibody (4G10). These gels confirmed that phosphorylated FcγRIIa was associated with each of the four bands originally identified (Figure 2A). Maximal phosphorylation of FcγRIIa was observed early (1–2 min) after exposure to thrombin, convulxin, and PAF, whereas the maximal effect of ADP was not evident until later (3–5 min).

[0281] Mass spectrometry was used to identify proteins or protein fragments that co-immunoprecipitated with FcγRIIa after platelet activation. As expected, approximately 45% of the protein content of the 55 kDa band was IgG (used for immunoprecipitation). Fibrinogen (mainly the β chain, 33%), actin (3%), and the SRC kinase Lyn (1%) were co-immunoprecipitated proteins in the 55 kDa band. Without being bound by theory, the minor phosphorylation of FcγRIIa is associated with the significant phosphorylation of Lyn. Proteins constituting the 75 kDa band included coagulation factor XIII (A subunit, 38%) and coagulation factor V (4%), fibrinogen (α, β, and γ chains, 24%), nexilin (3%), actin (1%), and myosin (1%). Proteins constituting the 90-kDa band included fibrinogen (γ chain, 80%), gelsolin (6%), filamin (1%), and coagulation factor XIII (1%). Thus, upon platelet activation, FcγRIIa primarily associated with fibrinogen, coagulation factor XIII, and components of the platelet cytoskeleton (actin, myosin, nexilin, gelsolin, and filamin).

[0282] Example 2: FcγRIIa co-localizes with lipid rafts upon platelet activation Lipid rafts were isolated, and in extracts from nonactivated platelets, FcγRIIa was demonstrated in fractions 8–10, which are the membrane cytoskeleton (Figure 2B). After activation, FcγRIIa remained associated with the membrane cytoskeleton (fractions 8–10), phosphorylated FcγRIIa was found in fractions 8–10, and plasma membrane lipid rafts were found in fractions 9 and 10 (Figure 2B). FcγRIIa was not found associated with soluble lipid rafts (fractions 4–6).

[0283] Confocal microscopy demonstrated uniform surface expression of FcγRIIa in the absence of activation and clustering of FcγRIIa after activation (Figure 3). Consistent with previous results demonstrating that CD36 is restricted to the plasma membrane (Roper K, et al., Nat Cell Biol 2000; 2:582-592), non-activated platelets exhibited uniform surface expression of CD36, and activated platelets exhibited clustering of CD36. Colocalization of lipid rafts with FcγRIIa was more prevalent in activated platelets (Figure 3).

[0284] Example 3: Inhibition of FcγRIIa phosphorylation attenuates platelet activation Patients (n = 3) with coronary artery disease (CAD) whose platelet expression of FcγRIIa was two-fold greater than the average expression by platelets from low-risk subjects were selected for inclusion in this study. FcγRIIa expression was confirmed using flow cytometry and confirmed by Western blot analysis (Figure 4A). Extracts from platelets with greater FcγRIIa expression exhibited greater phosphorylation of FcγRIIa (approximately two-fold) after activation by thrombin (Figure 4B).

[0285] We identified concentrations of IV.3 Fab (100 μg / ml) and the SRC kinase antagonist PP2 (20 μM) that prevented FcγRIIa phosphorylation during platelet activation. Tirofiban was used to block fibrinogen binding to GP IIb-IIIa. Goat anti-FcγRIIa crosslinks FcγRIIa and was used as a direct-acting activator of FcγRIIa.

[0286] Inhibition of FcγRIIa phosphorylation by IV.3 attenuated platelet activation, particularly when activation was confirmed by PAC-1 binding (Figures 5A and 5B). The results were confirmed by the use of transmitted light platelet aggregometry (Figure 5C). When thrombin or TRAP were used as agonists, the degree of inhibition caused by IV.3 was more pronounced when lower concentrations of the agonist were used (Figure 5D). As expected, IV.3 effectively blocked most of the platelet activation induced by goat anti-FcγRIIa. In contrast, a concentration of tirofiban (0.5 μg / ml) that completely blocked PAC-1 binding to platelets did not attenuate platelet activation induced by either agonist, nor did it alter activation induced by goat anti-FcγRIIa. Confocal microscopy results suggested that IV.3 attenuated FcγRIIa clustering (Figure 3). Consistent with the results seen with the specific antagonist IV.3, inhibition of SRC kinase with PP2 attenuated platelet activation (Fig. 6).

[0287] Purified human fibrinogen was added to whole blood from low-risk subjects (to increase the concentration by 500 mg / dL), and platelet activation in response to 0.5 μM ADP was assessed by measuring P-selectin surface expression. The addition of fibrinogen increased ADP-induced platelet activation (fold induction -1.5 ± 0.2, n = 3). This effect was blocked by pretreatment with IV.3 (100 μg / ml, fold induction -0.9 ± 0.1, p < 0.05) and appeared to be attenuated by tirofiban (0.5 μg / ml, fold induction -1.2 ± 0.1, p = 0.11).

[0288] Blood from low-risk subjects was spiked with human coagulation factor XIII (to increase the concentration by 1 μM), and platelet activation in response to 0.5 μM ADP was assessed by measuring P-selectin surface expression. Addition of factor XIII increased ADP-induced platelet activation (fold induction - 3.9 ± 0.1, n = 3). This effect was blocked by pretreatment with factor XIII (100 μg / ml, fold induction - 0.7 ± 0.1, p < 0.001) and tirofiban (0.5 μg / ml, fold induction - 0.8 ± 0.2, p < 0.001).

[0289] Example 4: FcγRIIa and platelet activation in disease settings As shown in Figure 7, platelets from patients with end-stage renal disease (ESRD) expressing high levels of FcγRIIa demonstrated a higher percentage of activated platelets than platelets expressing lower levels of FcγRIIa.

[0290] As shown in Figure 8A, platelets from patients with coronary artery disease (CAD) and patients with end-stage renal disease (ESRD) express higher levels of FcγRIIa than platelets from low-risk controls. As shown in Figure 8B, platelets from patients with coronary artery disease (CAD) and one or more myocardial infarctions express higher levels of FcγRIIa than platelets from low-risk controls. Notably, patients with multiple myocardial infarctions have the highest FcγRIIa expression, and all patients with CAD have higher FcγRIIa expression.

[0291] As shown in Figure 9, several inflammation-related cytokines and growth factors affect the expression of FcγRIIa.

[0292] As shown in Figure 10, IFNγ treatment increases the expression of FcγRIIa by megakaryocytic cell lines.

[0293] As shown in Figure 11, IFNγ treatment increases the expression of FcγRIIa by human stem cell-derived megakaryocytes.

[0294] FIG. 12 shows the effect of IFNγ treatment on the expression of FcγRIIa on platelets.

[0295] FIG. 13 shows the effect of IFNγ treatment on FcγRIIa expression in monocytic and myeloid cell lines.

[0296] The relationship between FcγRIIa, platelet activation, and atherogenesis is illustrated in FIG.

[0297] As shown in Figure 15, antibodies specific for FcγRIIa can cause platelet activation.

[0298] As shown in Figures 16 and 17, FcγRIIa becomes phosphorylated in thrombin-activated and ADP-activated platelets, respectively.

[0299] The structure of lipid rafts is illustrated in FIG.

[0300] As shown in Figure 19, platelet activation entails cytoskeletal rearrangements.

[0301] FIG. 20 shows the effect of coagulation factor XIII on platelet activation.

[0302] The role of FcγRIIa in platelet activation is illustrated in FIG.

[0303] The examples presented in this application were carried out using the following materials and methods, unless otherwise indicated.

[0304] Blood sample collection In accordance with a protocol approved by the University of Vermont Institutional Review Board, blood was collected from low-risk subjects or patients with coronary artery disease after they provided written informed consent. Patients with coronary artery disease had a previous myocardial infarction or coronary revascularization procedure, elevated FcγRIIa expression on platelets, and were treated with aspirin but no other antiplatelet or anticoagulant medications. Blood was collected using a 21-gauge butterfly needle, a tourniquet was applied for less than 90 seconds, and the first 3 ml of blood was discarded. Blood (1 ml) for platelet function assays was anticoagulated with 32 μg / ml corn trypsin inhibitor (CTI, Haematologic Technologies Inc, Essex Junction, Vt.), a specific inhibitor of factor XIIa that does not alter platelet activation (Schneider DJ, et al., Circulation 96:2877-83, 1997) or have no effect on other coagulation factors (Rand MD, et al., Blood 1996;88:3432-45). Washed platelets were prepared from blood anticoagulated with acid citrate dextrose (ACD, trisodium citrate, 0.085 M; citric acid, 0.071 M; glucose, 0.1 M, pH 4.5, 1:10 v / v).

[0305] Assessment of platelet function: To assess platelet function, a 5 μl aliquot of whole blood was added to a tube containing HEPESTyrodes (HT) buffer (5 mM HEPES, 137 mM NaCl, 2.7 mM NaHCO, 0.36 mM NaHPO, 2 mM CaCl, 4 mM MgCl, and 5 mM glucose, pH 7.4) and a fluorescent dye-labeled ligand. The volume used minimized platelet aggregation during activation. Platelet activation was confirmed by anti-CD62-phycoerythrin (PE, which identifies P-selectin) and fluorescein isothiocyanate (FITC)-conjugated PAC-1 (which binds to activated GP IIb-IIIa) as described (Serrano FA, et al., Thromb J 2007; 5:7; Schneider DJ, et al., Circulation 96:2877-83, 1997; Kabbani SS, et al., Circulation 2001; 104:181-6; Aggarwal A, et al., Am J Kidney Dis 2002; 40:315-22; and Schneider DJ, et al., Diabetes Care, 32:944-9, 2009). Platelet FcγRIIa expression was quantified using anti-CD32-PE. For both assays (quantification of activation and FcγRIIa expression), PE-Cy5-anti-CD42b was used as an activation-independent marker of platelets. When thrombin or coagulation factor XIII was used, the peptide GPRP (Gly-Pro-Arg-Pro) was added to prevent fibrinogen polymerization (Achyuthan KE, et al., Biochim Biophys Acta 1986;872:261-8). Fluorochrome-conjugated antibodies were from Becton Dickinson (San Jose, Calif.).Human α-thrombin and coagulation factor XIII were from Haematologic Technologies Inc. (Essex Junction, Vt.), PAF from EMD Biosciences (Gibbstown, NJ), fibrinogen (purity >95%) from Sigma (St. Louis, Mo.), and ADP from BioData (Horsham, Pa.). Convulxin is a collagen-mimetic lectin that binds to GP VI (Clemetson JM, et al., J Biol Chem 1999;274:29019-24, Pentapharm, Basel, Switzerland). Fab fragments of the FcγRIIa antagonist (IV.3) were prepared from IgG produced by hybridoma cells (HB-217 cells, American Tissue Culture Center, Manassas, Va.) using the Pierce Fab Preparation Kit (Thermo Scientific, Rockford, Ill.) (Looney, RJ et al., J Exp Med 1986;163:826-836). PP2 (EMD Biosciences), an SRC kinase antagonist, was dissolved in dimethyl sulfoxide (DMSO, Sigma). Tirofiban was obtained from Merck Research Laboratories (Whitehouse Station, NJ).

[0306] The reaction mixture was incubated at room temperature for 15 minutes without stirring, after which the platelets were fixed and the red blood cells were lysed by adding Optilyse-C solution (Beckman Coulter). Flow cytometry analysis was performed using a Beckman Coulter FC500 (Miami, Fla.). Platelets were identified based on size (forward and side scatter) and binding of an activation-independent ligand (anti-CD42b). As described, non-immune IgG was used as a control sample to define the activation-dependent binding threshold (Serrano FA, et al., Thromb J 2007; 5:7; Schneider DJ, et al., Circulation 96:2877-83, 1997; Kabbani SS, et al., Circulation 2001; 104:181-6; Aggarwal A, et al., Am J Kidney Dis 2002; 40:315-22; and Schneider DJ, et al., Diabetes Care, 32:944-9, 2009). Platelet activation is reported as the percentage of platelets bound to activation-dependent ligands, and our results show a direct correlation with mean fluorescence intensity (Schneider DJ, et al., Thromb Haemost 2001; 85:309-13).

[0307] Turbidimetric platelet aggregation was performed using a PAP-4 aggregometer (BioData, Horsham, Pa.). Maximum aggregation was reported after 4 minutes. In the platelet aggregation assay, a thrombin receptor agonist peptide (TRAP, Bachem, Torrance, Calif.) was used to mimic the effects of thrombin.

[0308] Platelet protein assessment: Washed platelets (2 × 10 in 0.5 ml) isolated by gel filtration (Sepharose CL-2B, Sigma) from platelet-rich plasma (PRP centrifugation at 140 g × 15 min at room temperature) were used to demonstrate phosphorylation of FcγRIIa.8 Platelets (number of cells) were activated at selected intervals without stirring at room temperature. Whole platelet lysates were prepared by adding an equal volume of 2x lysis buffer (2% Nonidet P-401, 300 mM NaCl, 50 mM Tris, 2 mM Na3VO4, halt protease, and phosphatase inhibitor cocktail [Pierce Biochemicals], pH 7.3) for 30 min in an ice bath. Lysates used for immunoprecipitation were precleared of antibody by the addition of Protein G Dynabeads (Invitrogen, Carlsbad, Calif.).

[0309] Immunoprecipitation was performed overnight at 4°C using goat anti-CD32A / C (Santa Cruz Biotechnology, Santa Cruz, Calif.) or mouse anti-phosphotyrosine conjugated to magnetic beads (4G10, Millipore, Billerica, Mass.). When anti-CD32A / C was used, antigen-antibody complexes were isolated for 3 hours using protein G-coated magnetic beads (Dynabeads, Invitrogen, Carlsbad, Calif.). The beads containing the antigen-antibody complexes were washed once with 0.5x lysis buffer and twice with phosphate-buffered saline. Proteins were separated from the beads in sample buffer, followed by electrophoresis and wet transfer to an Immobilon FL membrane (Millipore, Billerica, Mass.). Phosphorylation was confirmed using mouse anti-phosphotyrosine (clone 4G10, Millipore) or goat anti-CD32A / C (Santa Cruz Biotechnology, Santa Cruz, Calif.). Bands were detected with the Li-Cor Odyssey Infrared Imaging System (Li-Cor Biosciences, Lincoln, Nebr.) or chemiluminescence (Amersham / General Electric Healthcare, Piscataway, NJ).

[0310] Washed platelets (4 x 10 in 0.5 ml) were activated for 90 seconds at room temperature without agitation as described (Lee FA, et al., J Biol Chem 2006; 281:39330-8) and then lysed by the addition of 2x lipid raft lysis buffer (20 mM Tris, 100 mM NaCl, 60 mM sodium pyrophosphate, 20 mM sodium glycerophosphate, 0.02 w / v% sodium azide, 0.025 Triton X-100, 2 mM sodium vanadate protease inhibitor tablet, pH 8.0). 8 Lipid rafts were isolated from 1000 cells / ml of 10 ...

[0311] Proteomic evaluation of proteins co-immunoprecipitated with FcγRIIa was performed by the University of Vermont Proteomics Core (co-director Dwight Matthews). After GelCode staining (Pierce Biotechnology, Rockford, Ill.), visible bands were excised, trypsin-digested (Protease MAX protocol, Promega, Madison, Wis.), and identified using liquid chromatography-mass spectrometry. Protein digests were separated on a 100 μm × 50 mm column packed with Michrom 3μ C18 AQ (Auburn, Calif.) using high-pressure liquid chromatography (Shimadzu, Columbia, Md.). Peptides eluted from the column were analyzed using a linear ion trap mass spectrometer (Thermo Scientific, San Jose, Calif.) equipped with a Michrom Advance electrospray source. Proteins were identified from peptide amino acid sequences determined using Thermo Scientific's SEQUEST algorithm.

[0312] Confocal microscopy Confocal microscopy was performed as previously described (Schneider DJ, et al., J Am Coll Cardiol 1999; 33:261-6). Platelets exposed to the selected conditions were fixed with Optilyse-C for 15 minutes and then pretreated with 1% bovine serum albumin (BSA) in HT buffer for 15 minutes before incubation with primary antibodies. Primary goat anti-FcγRIIa / CD32a was used to identify FcγRIIa, and anti-CD36 was used to identify lipid rafts (Gousset K, et al., J Cell Physiol 2002; 190:117-28). CD36 was chosen to identify lipid rafts because it differentially associates with lipid rafts located in the platelet plasma membrane (Gousset K, et al., J Cell Physiol 2002; 190:117-28). Platelets isolated by centrifugation (1,500 g × 10 min) were washed three times (HT) before adding secondary antibodies to identify CD36 (Alexa 488 anti-mouse IgG) and FcγRIIa (Alexa 555 anti-goat IgG). After centrifugation (1,500 g × 10 min), platelets were resuspended and applied to glass microscope slides for 30 min before being washed twice (HT). Coverslips were applied, and platelets were imaged using a Zeiss LSM 510 META confocal / scanning laser microscope (Zeiss Microimaging, Thornwood, NY). Control slides containing primary and secondary antibodies alone were used to verify autofluorescence and nonspecific association of the secondary antibodies.

[0313] statistical analysis Results are means ± standard deviations. The significance of differences was assessed using Student's t-test. Significance was established by p<0.05.

[0314] FcγRIIa as a biomarker for identifying high and low cardiovascular risk To evaluate the value of quantifying FcγRIIa expression on platelets, a single-center prospective trial was conducted. Quantification of FcγRIIa expression on platelets was performed using citrate-anticoagulated whole blood. A 2-μl aliquot of blood was added to a tube containing two antibodies: one to identify platelets and one to identify FcγRIIa. The sample was then fixed and red blood cells were lysed. After sufficient dilution to create a single-particle stream, the sample was analyzed using flow cytometry. The output (mean fluorescence intensity) was normalized to quantify FcγRIIa molecules per platelet.

[0315] This prospective study was conducted to determine whether platelet FcγRIIa expression identifies patients at high and low cardiovascular risk (Schneider DJ, McMahon SR, Chava S, et al. FcγRIIa: A New Cardiovascular Risk Marker. J Am Coll Cardiol. 2018;72(2):237-238). Patients (n=197) were enrolled immediately prior to discharge after hospitalization for a heart attack (including both ST-elevation and non-ST-elevation heart attacks). All patients were treated with ASA (81 mg), and treatment with clopidogrel (approximately 64%) and ticagrelor (approximately 36%) was balanced between patients with high and low platelet FcγRIIa expression. Clinical characteristics were well balanced, with the exception of older age, diabetes, and previous revascularization, which were more prevalent in the high-expression group. Referring to Figure 22, the primary endpoint, a composite of heart attack, stroke, death, and coronary revascularization, was lower in patients with platelet FcγRIIa expression <11,000.

[0316] Cox multivariate analysis for the combined risk of heart attack, stroke revascularization, and death demonstrated a hazard ratio of 3.0 (p=0.02) for platelet FcγRIIa expression >11,000 when age, diabetes, and previous revascularization were included as covariates. For the composite endpoint of heart attack, stroke, and death, Cox regression analysis demonstrated that platelet FcγRIIa expression was the only covariate associated with increased risk of MI, stroke, and death (hazard ratio 3.9, p=0.035).

[0317] This prospective study has demonstrated the power of this biomarker to improve care and fill critical gaps by providing clinicians with a precise tool that can effectively guide personalized care.To realize its potential, the test has been improved to significantly reduce potential sources of variation.In the original assay, platelets are exposed to antibody and then fixed.The main purpose of the improvement is to make it possible to fix platelets first and then expose them to antibody.Fixed platelets can be stored and separated from blood to improve analytical specificity.To achieve this goal, the antibody disclosed herein has been developed to bind to FcγRIIa on the surface of pre-fixed platelets.

Claims

1. at least one heavy chain complementarity determining region (CDRH) and at least one light chain complementarity determining region (CDRL) Including, wherein the CDRH is selected from SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, and the CDRL is selected from SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, wherein SEQ ID NO: 11 encodes the amino acid sequence DTS; or wherein the CDRH is selected from SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, and the CDRL is selected from SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, wherein SEQ ID NO: 21 encodes the amino acid sequence WAS; An isolated antibody or antigen-binding fragment thereof that specifically binds to FcγRIIa.

2. At least two heavy chain complementarity determining regions (CDRHs) and at least two light chain complementarity determining regions (CDRLs) Including, wherein the CDRH is selected from SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, and the CDRL is selected from SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12; or wherein the CDRH is selected from SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, and the CDRL is selected from SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO:

22.

2. The isolated antibody or antigen-binding fragment thereof of claim 1.

3. a. a CDRH1 region comprising SEQ ID NO: 5, a CDRH2 region comprising SEQ ID NO: 6, and a CDHR3 region comprising SEQ ID NO: 7; and a CDRL1 region comprising SEQ ID NO: 10, a CDRL2 region comprising SEQ ID NO: 11, and a CDRL3 region comprising SEQ ID NO: 12, and a sequence at least 90% identical to SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, or b. a CDRH1 region comprising SEQ ID NO: 15, a CDRH2 region comprising SEQ ID NO: 16, and a CDHR3 region comprising SEQ ID NO: 17; and a CDRL1 region comprising SEQ ID NO: 20, a CDRL2 region comprising SEQ ID NO: 21, and a CDRL3 region comprising SEQ ID NO: 22, as well as sequences at least 90% identical to SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO:

22. The antibody or antigen-binding fragment thereof of claim 2, comprising:

4. a. the heavy chain complementarity determining regions are encoded by SEQ ID NO:4 and the light chain complementarity determining regions are encoded by SEQ ID NO:9, and are 90% identical to SEQ ID NO:4 and SEQ ID NO:9; or b. the heavy chain complementarity determining region is encoded by SEQ ID NO: 14 and the light chain complementarity determining region is encoded by SEQ ID NO: 19, and the sequences are 90% identical to SEQ ID NO: 14 and SEQ ID NO: 19; The antibody or antigen-binding fragment thereof according to claim 3.

5. The antibody of claim 1, wherein FcγRIIa is on platelets.

6. 6. The antibody or antigen-binding fragment of claim 5, wherein the antibody is linked to a detectable label.

7. The antibody or antigen-binding fragment of claim 6, wherein FcγRIIa is present on a platelet and the platelet is fixed with a fixative before the antibody is allowed to bind to the FcγRIIa.

8. 8. The antibody or antigen-binding fragment of claim 7, wherein the antibody is an isolated monoclonal antibody.

9. The antibody or antigen-binding fragment of claim 8, wherein the antibody is a monoclonal antibody.

10. 10. The antibody or antigen-binding fragment of any one of the preceding claims, wherein the antibody is or is generated using recombinant antibody technology, nucleic acid aptamer technology, or non-immunoglobulin protein scaffold technology.

11. A conjugate of an antibody that binds to FcγRIIa, the antibody of claim 6.

12. A method for detecting FcγRIIa, comprising a step of binding FcγRIIa to an antibody, wherein the antibody comprises the antibody or antigen-binding fragment of claim 6.

13. A method for detecting the presence of FcγRIIa in a blood sample, comprising a step of binding an antibody to FcγRIIa, wherein the antibody comprises an antibody or antigen-binding fragment described in claim 6.

14. 14. The method of claim 13, wherein the platelets in the blood sample are treated with a fixative solution before or at the same time as the antibody is introduced into the blood sample.

15. 14. The method of claim 13, wherein the fixative is combined with the blood sample for up to two days after collection of the blood sample.

16. 15. The method of claim 14, wherein the fixative is diluted with a solution comprising a buffer and glycerol before being combined with the blood sample.

17. 17. The method of claim 16, wherein the buffer is phosphate buffered saline (PBS) and the glycerol comprises in the range of approximately 2% to 7% of the total volume of the buffer-glycerol solution.

18. 18. The method of claim 17, wherein the glycerol is 5% of the total volume of the buffer-glycerol solution.

19. The method of claim 17, wherein the bound FcγRIIa and antibody complex is detected via flow cytometry to measure the level of FcγRIIa in the sample.

20. The method of claim 19, wherein the measured level of FcγRIIa in the sample is normalized based on a comparison of the known level of a fluorescent marker with the measured level of fluorescence in the sample.

21. 1. A method for detecting the presence of FcγRIIa in a blood sample, comprising: treating the blood sample with an anticoagulant; adding a fixative to the blood sample; separating and washing platelets from said blood sample; incubating the platelets with an antibody comprising any one of the antibodies or antigen-binding fragments of claim 6; and performing an analysis to quantify FcγRIIa in the sample. The method comprising:

22. 22. The method of claim 21, wherein said analysis is selected from the group consisting of flow cytometry, immunoassay, ELISA, Western blotting, and radioimmunoassay.

23. 23. The method of claim 22, wherein the analysis is flow cytometry.

24. Reducing the concentration of fixative in the blood sample; and storing the blood sample for a period of up to and including 1 to 14 days before incubating the platelets with the antibody.

22. The method of claim 21, further comprising:

25. 1. A method for preparing a blood sample, comprising: treating the blood sample with an anticoagulant; adding a fixative to the blood sample; Subsequently, diluting the concentration of the fixative in the blood sample; and storing said blood sample for a period of time up to and including 1 to 14 days. The method comprising:

26. 26. The method of claim 25, wherein the fixative is added to a concentration of 5% and subsequently diluted to 1.25%.

27. A method for detecting FcγRIIa, comprising a step of binding FcγRIIa to an antibody, the antibody comprises at least one heavy chain complementarity determining region (CDRH) and at least one light chain complementarity determining region (CDRL); wherein the CDRH is selected from SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, and the CDRL is selected from SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, wherein SEQ ID NO: 11 encodes the amino acid sequence DTS; or wherein the CDRH is selected from SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, and the CDRL is selected from SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, wherein SEQ ID NO: 21 encodes the amino acid sequence WAS; The method.

28. The method of claim 27, wherein FcγRIIa is detected on platelets from a blood sample.

29. The method of claim 28, further comprising comparing the level of detection of FcγRIIa between a blood sample from a human having or suspected of having heart disease, thrombosis, coronary artery disease, kidney disease, or myocardial infarction and a control human blood sample that does not have heart disease, thrombosis, coronary artery disease, kidney disease, or myocardial infarction.

30. 1. A method for detecting platelet reactivity, comprising: where platelets are contacted with the antibody, the antibody comprises at least one heavy chain complementarity determining region (CDRH) and at least one light chain complementarity determining region (CDRL); wherein the CDRH is selected from SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, and the CDRL is selected from SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, wherein SEQ ID NO: 11 encodes the amino acid sequence DTS; or wherein the CDRH is selected from SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, and the CDRL is selected from SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, wherein SEQ ID NO: 21 encodes the amino acid sequence WAS; The method.

31. 10. The antibody or antigen-binding fragment of claim 1 for use in a method for detecting platelet reactivity in a subject.

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