Method for diagnosing predisposition to the development of thrombocytopenia in an organism

Abstract

The present invention relates to a method for diagnosing a predisposition to the development of thrombocytopenia in an organism and uses relating to this method.

Description

[Technical Field]

[0001] The present invention relates to a method for diagnosing a predisposition to developing or suffering from thrombocytopenia in an organism, and uses relating to this method.

[0002] The present invention relates to the field of molecular medicine, more particularly to the field of molecular diagnostics, preferably to the diagnosis of molecular blood markers associated with thrombocytopenia. [Background technology]

[0003] Thrombocytopenia is a condition characterized by an abnormally low number of platelets (also known as thrombocytes) in the blood. It is the most common clotting disorder in intensive care patients, affecting 20% ​​of medical patients and one-third of surgical patients.

[0004] A normal human platelet count is between 150,000 and 450,000 platelets per microliter of blood. Values ​​outside this range do not necessarily indicate disease. One common definition of emergency thrombocytopenia is a platelet count of fewer than 50,000 platelets per microliter of blood. Thrombocytopenia can be contrasted with conditions in which the number of platelets in the blood is abnormally high, such as thrombocythemia (when the cause is unknown) and thrombocytosis (when the cause is known).

[0005] Heparin-induced thrombocytopenia (HIT) is a type of thrombocytopenia caused by the administration of various forms of heparin, an anticoagulant. HIT predisposes to thrombosis, the abnormal formation of blood clots inside blood vessels, due to the release of microparticles from platelets that activate thrombin. If thrombosis is identified, the disorder is called heparin-induced thrombocytopenia-thrombosis (HITT). HIT is caused by the formation of abnormal antibodies that activate platelets. Certain blood tests can confirm HIT in people receiving heparin if they develop a new thrombosis, if an existing thrombosis worsens, or if their platelet count drops.

[0006] HIT is suspected when blood tests confirm a decrease in platelet counts in individuals receiving heparin, or even when blood tests confirm a decrease in platelet counts after heparin administration has already been discontinued. Professional guidelines recommend that individuals receiving heparin undergo periodic complete blood counts, including platelet counts, while on heparin. However, not all individuals who experience a decrease in platelet count while on heparin have developed HIT. The timing of the onset of thrombocytopenia, its severity, the occurrence of new thrombosis, and the presence of other causes all determine the likelihood of HIT.

[0007] The "4Ts" score, introduced in 2003, is commonly used to predict the likelihood of HIT. The 4Ts score ranges from 0 to 8; a score of 0 to 3 indicates a low probability of HIT. A 4Ts score of 4 to 5 indicates a moderate probability, while a 4Ts score of 6 to 8 indicates a high probability. Patients with high scores may require treatment with alternative drugs and additional, more sensitive and specific HIT testing. Patients with low scores are highly unlikely to develop HIT and can safely continue to receive heparin. Because calculating the 4Ts score is laborious, it is difficult to implement as a daily diagnostic routine, and its reliability as a prognostic value is very low.

[0008] Vaccine-induced immune thrombocytopenia (VITT), also known as vaccine-induced prothrombotic immune thrombocytopenia (VIPIT), is a rare type of blood clotting event first observed in a small number of people vaccinated with the COVID-19 vaccine (called AZD1222 or ChAdOx1) co-developed by the University of Oxford and AstraZeneca during the COVID-19 pandemic. VITT was subsequently reported with the Johnson & Johnson COVID-19 vaccine, leading to its suspension until safety was reassessed. In April 2021, AstraZeneca and the European Medicines Agency (EMA) updated information for healthcare professionals regarding the ChAdOx1 vaccine, stating that a causal relationship between the development of thrombocytopenia and vaccination is "considered plausible" and that "such adverse events are very rare," but reported a higher incidence than in the general population. Furthermore, on April 7, 2021, the European Medicines Agency (EMA) stated that one "plausible explanation" for the concomitant blood clotting and thrombocytopenia is an immune response causing a condition similar to that occasionally seen in patients treated with heparin, known as heparin-induced thrombocytopenia (HIT).

[0009] There is currently no way for a treating physician to proactively identify patients who should immediately receive a vaccine (e.g., a vector vaccine) who are prone to developing thrombosis or thrombocytopenia and therefore need to be switched to an alternative vaccine if necessary.

[0010] In this situation, the present invention aims to provide a new method for diagnosing a predisposition to developing thrombocytopenia in an organism and / or diagnosing the presence of thrombocytopenia in an organism. This method can avoid the drawbacks of the methods in the art, or at least has fewer drawbacks than the methods in the art. More specifically, a method is provided that is not too cumbersome and can provide results in a reliable and reproducible manner.

[0011] The present invention fulfills these and other needs. Summary of the Invention [Means for solving the problem]

[0012] The present invention provides a method for diagnosing a predisposition to developing thrombocytopenia in an organism, comprising the steps of: 1. Providing serum from an organism to be diagnosed; 2. Providing platelets of a healthy reference organism; 3. Among the platelets from step (2), the percentage (%) of platelets expressing P-selectin and phosphatidylserine on their surface is measured to determine M pre The process of determining the value; 4. Incubating the serum aliquot of step (1) with the platelet aliquot of step (2); 5. After the incubation in step (4), the percentage (%) of platelets expressing P-selectin and phosphatidylserine on their surface is measured to determine M post determining the value; and 6.M post >M pre diagnosing the organism as being predisposed to developing thrombocytopenia when The present invention provides a method comprising:

[0013] The present invention further provides a method for diagnosing thrombocytopenia in an organism, comprising the steps of: 1. Providing serum from an organism to be diagnosed; 2. Providing platelets of a healthy reference organism; 3. Among the platelets from step (2), the percentage (%) of platelets expressing P-selectin and phosphatidylserine on their surface is measured to determine M pre The process of determining the value; 4. Incubating the serum aliquot of step (1) with the platelet aliquot of step (2); 5. After the incubation in step (4), the percentage (%) of platelets expressing P-selectin and phosphatidylserine on their surface is measured to determine M post determining the value; and 6.Mpost >M pre diagnosing the organism as being predisposed to developing thrombocytopenia when The present invention provides a method comprising: DETAILED DESCRIPTION OF THE INVENTION

[0014] Surprisingly, the present inventors have found that the colocalization or coexpression of P-selectin and phosphatidylserine on the surface of platelets is a reliable diagnostic marker indicating a predisposition to and / or the presence of thrombocytopenia in an organism. In particular, the expression of both P-selectin and phosphatidylserine on the surface of platelets discovered in the present invention was found to be stimulated by serum obtained from individuals predisposed to or suffering from thrombocytopenia. This surprising finding led to the development of the method of the present invention.

[0015] Such a phenomenon was unknown in the art and was unexpected by those skilled in the art.

[0016] The method according to the present invention, unlike methods known in the art, is easy to perform and provides reliable results. The method according to the present invention not only allows for the diagnosis of a predisposition to developing thrombocytopenia or the presence of thrombocytopenia in an organism, but also allows for a good assessment of the risks associated with the administration of a vaccine suspected of inducing thrombocytopenia in rare cases. If such a predisposition is diagnosed, the treating physician can avoid the administration of this vaccine and select another vaccine, such as an mRNA vaccine, that is unlikely to cause thrombocytopenia or at least has a reduced risk profile.

[0017] According to the present invention, an "organism" includes any living organism, particularly mammals, preferably humans.

[0018] According to the present invention, "serum components in blood" or simply "serum" refers to blood fluid and solute components that do not contribute to clotting. Serum is the liquid portion of blood obtained as the supernatant when a clotted blood sample is centrifuged. Serum may be defined as plasma that does not contain fibrinogen. Serum contains all proteins not utilized in blood clotting, as well as all electrolytes, antibodies, antigens, and hormones, and may contain extraneous substances (e.g., drugs and microorganisms). Serum does not contain white blood cells, red blood cells, platelets, or clotting factors.

[0019] According to the present invention, "platelets" or "thrombocytes" are blood components that, together with coagulation factors, form clumps in response to bleeding due to vascular injury and initiate blood coagulation. Platelets lack a nucleus. They originate as cytoplasmic fragments derived from megakaryocytes in the bone marrow and then enter the blood circulation. Inactive platelets in the blood circulation are biconvex discoid (lenticular) structures with a maximum diameter of 2-3 μm.

[0020] According to the present invention, "P-selectin" or "CD62" is a type 1 transmembrane protein encoded in humans by the SELP gene. P-selectin functions as a cell adhesion molecule (CAM) on the surface of activated endothelial cells lining blood vessels and on the surface of activated platelets.

[0021] According to the present invention, "phosphatidylserine" (abbreviated as Ptd-L-Ser or PS) refers to a phospholipid that is a component of cell membranes. Phosphatidylserine plays an important role in cell cycle signaling, particularly in relation to apoptosis.

[0022] According to the present invention, "M pre " is an index showing the percentage of platelets in a healthy individual before incubation with serum obtained from the individual being tested, and "M post " is an index indicating the percentage of platelets from a healthy individual after incubation with serum from the individual being tested.

[0023] In one embodiment of the present invention, "thrombocytopenia" includes thrombosis, including cerebral venous sinus thrombosis, and the method according to the present invention can be used to determine the risk of developing thrombosis in an organism, or to determine whether an organism is actually suffering from thrombosis.

[0024] In a modified method of the present invention, in steps (3) and (5), instead of measuring the proportion of platelets expressing P-selectin and phosphatidylserine on their surface, the amounts of P-selectin and phosphatidylserine on the surface of the platelets are measured.

[0025] In one embodiment of the present invention, the thrombocytopenia is heparin-induced thrombocytopenia (HIT).

[0026] By this means, the present invention can be adapted to the diagnosis of one or more of the most important thrombocytopenias for which there is currently no reliable and convenient method for determining the predisposition.

[0027] In one embodiment of the present invention, the thrombocytopenia is vaccine-induced immune thrombocytopenia (VITT), preferably VITT caused by an anti-SARS-CoV-2 vaccine, and more preferably VITT caused by an adenovirus-based vector vaccine or an adeno-associated virus-based vector vaccine.

[0028] By this means, the invention can be adapted to a prognostic method that allows physicians to make better decisions regarding the risk of developing thrombocytopenia when administering vaccines such as the ChAdOx1 vaccine that are suspected of inducing VITT.

[0029] In another embodiment, the platelets in step (2) are provided as washed platelets.

[0030] This method has the advantage of further improving the sensitivity of the method of the present invention with respect to the detection sensitivity of pathogenic antibodies, etc.

[0031] According to the present invention, "washed platelets" is understood to include platelets from which the majority, preferably substantially all, of the plasma, red blood cells and white blood cells has been removed and replaced with saline or another type of storage solution.

[0032] In yet another embodiment of the present invention, the platelets in step (2) are provided as platelet-rich plasma (PRP).

[0033] This method has the advantage that it does not require the time-consuming and costly step of washing platelets, and the method of the present invention can be carried out by personnel with a certain level of skill, without requiring skilled laboratory technicians.

[0034] According to the present invention, "platelet-rich plasma (PRP)," also known as autologous conditioned plasma, is a concentrate of platelet-rich plasma proteins obtained from a patient's own whole blood by plasma exchange using an autotransfusion device or a dedicated benchtop device. The separation principle is based on centrifugal force, and because the individual blood components have different specific gravities, each blood component can be collected individually by centrifuging the blood in layers (plasma exchange). Whole blood is separated into red blood cells, platelet-poor plasma (PPP), and platelet-rich plasma (PRP).

[0035] In one embodiment of the present invention, platelet factor IV (PF4) is added to the incubation mixture of serum and platelets in step 4. Alternatively, or in addition, heparin, preferably low molecular weight heparin (LMWH), is added to the incubation mixture of serum and platelets in step 4.

[0036] This approach has the advantage that it can further improve the sensitivity and / or specificity of the method of the present invention.

[0037] Platelet factor IV (PF4), also known as chemokine (C-X-C motif) ligand 4 (CXCL4), is a small cytokine belonging to the C-X-C chemokine family. This chemokine is released from the α-granules of activated platelets during platelet aggregation and promotes blood coagulation by mitigating the effects of heparin-like molecules.

[0038] "Heparin" is a natural polysaccharide that inhibits clotting, which leads to thrombosis. Natural heparin consists of molecular chains of various lengths and has a variety of molecular weights. Pharmaceutical-grade heparin, which consists of a wide range of molecular weights, ranges from 5,000 daltons to over 40,000 daltons. In contrast, "low molecular weight heparin (LMWH)" consists exclusively of short-chain polysaccharides. Low molecular weight heparin is defined as a heparin salt with an average molecular weight less than 8,000 Da, where at least 60% of all chains have a molecular weight less than 8,000 Da. Low molecular weight heparin can be obtained by various fractionation or depolymerization methods of polymerized heparin.

[0039] In one embodiment of the present invention, in step 3 and / or step 5, the percentage (%) of platelets expressing P-selectin and phosphatidylserine on their surface is measured by flow cytometry (FC).

[0040] This approach has the advantage that well-established methods can be used to measure newly discovered cell surface markers and can be easily implemented in daily diagnostic routine.

[0041] In one embodiment of the present invention, in step 6, if 10% or more of the platelets after the incubation in step (4) express P-selectin and phosphatidylserine on their surface, the patient is diagnosed as having the predisposition to the disease or as actually suffering from the disease.

[0042] This approach has the advantage of providing a specific threshold at which a diagnosis can be made easily and reliably.

[0043] In one embodiment of the present invention, the percentage of platelets expressing P-selectin and phosphatidylserine on their surface may be 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, or 9% or more, but is preferably 10% or more. According to the present invention, "10% or more" includes 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, and 100% or more.

[0044] Another subject of the present invention relates to the use of the colocalization of P-selectin and phosphatidylserine on the surface of platelets as a diagnostic marker for predisposition to the development of and / or the prevalence of thrombocytopenia in an organism, preferably heparin-induced thrombocytopenia (HIT) and / or vaccine-induced immune thrombocytopenia (VITT).

[0045] The features, properties, advantages and embodiments of the method according to the invention also apply to the use according to the invention.

[0046] The features mentioned above and the features to be described below can be used not only in the combinations shown in the respective embodiments, but also in other combinations or alone without departing from the scope of the present invention.

[0047] The present invention will be further described in detail by reference to the following embodiments, which describe additional features, characteristics, and advantages of the present invention. Furthermore, the following embodiments are for illustrative purposes only and are not intended to limit the spirit or scope of the present invention. Features described in specific embodiments are general features of the present invention and are not only applicable to specific embodiments, but also applicable alone and to all embodiments of the present invention. [Brief explanation of the drawings]

[0048] The present invention will be described and explained in more detail with reference to the following examples and figures, but the present invention is not limited to these examples and figures. [Figure 1] Antibody-mediated platelet activation and the production of procoagulant platelets are shown. The results of a modified platelet activation assay (HIPA) in patients with vaccine-induced immune thrombotic thrombocytopenia (VITT) are shown. Each dot represents the median value across four donors. VITT patients showed significantly increased platelet activation by the PF4 / heparin complex in buffer-only conditions, but this platelet activation was suppressed by high doses of heparin (Panel A). In a separate experimental setting, procoagulant platelets (CD62P / phosphatidylserine (PS)-positive cells) were analyzed by Annexin V-FITC antibody staining and CD62p-APC antibody staining. Platelets were treated with PF4, 0.2 U / ml or 100 U / ml heparin, receptor-binding domain (RBD), or ChAdOx1 nCoV-19A vaccine, as indicated (Panel B). The results of the HIPA assay using serum from VITT patients with various titers are shown. Note that diluted serum (dilution factor 1:64 or greater) activated platelets only in the presence of PF4 (Panel C). Furthermore, the effect of various titers of serum from VITT patients on the production of procoagulant platelets is shown. Note that diluted serum (dilution factor 1:8 or greater) activated platelets only in the presence of PF4 (Panel C). Data are presented as the mean ± standard deviation (SD) of the fold increase measurements compared to the control (not significant, *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001). The number of sera tested is indicated on each graph. The dotted line indicates the cutoff value determined by testing serum from healthy donors. [Figure 2]The graphs show the correlation between serum anti-COVID IgG and IgA antibody levels and HIT-EIA. The graphs show the MFI of anti-COVID IgG and IgA antibody levels against spike trimer, RBD, S1, S2, and nucleocapsid in VITT patients (Panel A), vaccinated volunteers (Panel B), and COVID-19 patients (Panel C) quantified by a bead-based Luminex assay. No correlation was observed between anti-PF4 antibody levels and anti-COVID antibodies in VITT patients and vaccinated volunteers (Panel D). Each symbol represents an individual subject, and the graph shows the number of subjects tested. [Figure 3] IgG binding to healthy platelets is shown. Panel A shows representative flow cytometry histograms demonstrating anti-human globulin (AHG) binding. Anti-human globulin (AHG) binding was measured in the presence of buffer or high concentrations of heparin (100 IU / ml heparin) after incubation of platelets from healthy donors (HCs) with serum from VITT patients. Separate panels show IgG binding to healthy platelets after incubation with serum from vaccinated volunteers (Panel B) or serum from COVID-19 patients (Panel C), as assessed by flow cytometry and shown as fold increase corrected relative to control. Platelets were treated with PF4, 0.2 U / ml or 100 U / mL heparin, the receptor-binding domain (RBD), or the ChAdOx1 nCoV-19A vaccine, as indicated (abbreviations: ns: not significant; **p<0.01). IgG binding to the receptor-binding domain (RBD) of SARS-CoV-2 was tested in VITT patients and assessed by EIA (shown as fold increase over buffer control). A trend toward increased IgG binding to RBD was observed with increasing RBD concentrations (Panel D). No IgG binding to the SARS-CoV-2 S2 domain was observed in VITT patients (Panel E). Each symbol represents an individual subject, and the graph indicates the number of subjects tested. [Figure 4]Antibody-mediated platelet activation and the production of procoagulant platelets are shown. The results of a modified platelet activation assay (HIPA) in patients with vaccine-induced immune thrombotic thrombocytopenia (VITT) are shown. Each dot represents the median value across four donors. VITT patients showed significantly increased platelet activation by the PF4 / heparin complex in buffer-only conditions, but this platelet activation was suppressed by high doses of heparin (Panel A). In a separate experimental setting, procoagulant platelets (CD62P / phosphatidylserine (PS)-positive cells) were analyzed by Annexin V-FITC antibody staining and CD62p-APC antibody staining. Platelets were treated with PF4, 0.2 U / ml or 100 U / ml heparin, receptor-binding domain (RBD), or ChAdOx1 nCoV-19A vaccine, as indicated (Panel B). The results of the HIPA assay using serum from VITT patients with various titers are shown. Note that diluted serum (dilution factor 1:64 or greater) activated platelets only in the presence of PF4 (Panel C). Furthermore, the effect of various titers of serum from VITT patients on the production of procoagulant platelets is shown. Note that diluted serum (dilution factor 1:8 or greater) activated platelets only in the presence of PF4 (Panel C). Data are presented as the mean ± standard deviation (SD) of the fold increase measurements compared to the control (not significant, *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001). The number of sera tested is indicated on each graph. The dotted line indicates the cutoff value determined by testing serum from healthy donors. [Figure 5]Heparin-induced platelet activation assay (HIPA) results are shown. The graph shows the results of the modified platelet activation assay (HIPA) in the presence or absence of PF4, 0.2 U / ml or 100 U / ml heparin, RBD, or ChAdOx1 nCoV-19 vaccine. No platelet activation was observed in any subject under any condition, except for one vaccinated control in the presence of the receptor-binding domain (RBD) of the spike protein (Panel A). Similar results were observed in COVID-19 patients, except that one sample from two of four donors showed enhanced activation in the presence of 0.2 U / ml heparin (*p<0.05, Panel B). Each symbol represents an individual subject, and the graph indicates the number of subjects tested. [Figure 6] Representative dot plot histograms of procoagulant platelets are shown. Washed platelets (PLTs) were incubated with serum from a VITT patient. Panels I–VII show representative dot plots for: [I] a healthy control in the presence of buffer; [II] case No. 8 in the presence of buffer; [III] case No. 8 in the presence of 0.2 U / ml heparin; [IV] case No. 8 in the presence of 100 U / ml heparin; [V] case No. 8 in the presence of IV.3 antibody; [VI] case No. 8 in the presence of intravenous immunoglobulin (IVIG); [VII] case No. 8 in the presence of PF4; or [VIII] case No. 8 in the presence of IV.3 antibody and PF4. [Figure 7] Procoagulant platelets (CD62P / phosphatidylserine (PS)-positive cells) are shown in sera from vaccinated volunteers (Panel A) or from COVID-19 patients with anti-PF4 antibodies (Panel B) in various experimental settings. Data are presented as the mean ± standard deviation (SD) of fold-increase measurements compared to controls (not significant, and *p<0.05). The dotted line indicates the cutoff value as the mean fold-increase (FI) determined by testing sera from healthy donors. The number of sera tested is indicated on each graph. [Figure 8]In a separate experimental setup, procoagulant platelets (CD62P / phosphatidylserine (PS)-positive cells) were analyzed after incubation of washed platelets with serum. Platelets were analyzed by Annexin V-FITC antibody staining and CD62p-APC antibody staining. Platelets were treated with PF4, 0.2 U / ml or 100 U / ml heparin, or monoclonal antibody IV.3, as indicated on the graph. Data are presented as the mean ± standard error of the mean (SEM) of the fold increase measurements compared to the buffer control. [Figure 9] In a separate experimental setup, procoagulant platelets (CD62P / phosphatidylserine (PS)-positive cells) were analyzed after incubation of HIPA assay-negative serum with platelet-rich plasma (PRP). Platelets were analyzed by Annexin V-FITC antibody staining and CD62p-APC antibody staining. Platelets were treated with PF4 or 0.2 U / ml or 100 U / ml heparin, as indicated on the graph. Data are presented as the mean ± standard error of the mean (SEM) of the fold increase measurements compared to the buffer control. [Figure 10] Procoagulant platelet formation due to serum HIT antibodies is heparin-dependent. Each graph shows the number of patient sera tested. The dashed line indicates the preliminary cutoff value for determining the presence or absence of procoagulant platelet formation. Comparisons between groups for unpaired data sets were performed using the Mann-Whitney U test, and comparisons between groups for paired data sets were performed using the paired Wilcoxon test. *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001. ns (not significant); CD62p: P-selectin; N: number of healthy controls or patients; PS: phosphatidylserine. [Figure 11]Procoagulant platelets are induced only in sera from patients with confirmed heparin-induced thrombocytopenia (HIT). The number of patient sera tested is shown in each graph. The dashed line indicates the preliminary cutoff value for determining procoagulant platelet formation. Comparisons between groups were performed using paired Wilcoxon tests. *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001. ns (not significant); CD62p: P-selectin; N: number of healthy controls or patients; PS: phosphatidylserine. [Figure 12] Procoagulant platelet formation occurs through the binding of HIT IgG antibodies to platelet FcγRIIA-(A). Each graph shows the number of patient sera tested. The dashed line indicates the preliminary cutoff value for determining the presence or absence of procoagulant platelet formation. Comparisons between groups for unpaired data sets were performed using the Mann-Whitney U test, and comparisons between groups for paired data sets were performed using the paired Wilcoxon test. *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001. ns (not significant); CD62p: P-selectin; N: number of healthy controls or patients; PS: phosphatidylserine. [Example]

[0049] 1. Introduction Over the past 15 months, COVID-19 infection has caused considerable morbidity and mortality. In a very short period of time, several SARS-CoV-2 vaccines have been licensed and administered worldwide. However, safety signals have been observed. In early March 2021, the U.S. Centers for Disease Control and Prevention (CDC) reported 26 cases of venous thromboembolism, 20 cases of thrombosis, and 41 cases of ischemic stroke among people vaccinated with mRNA vaccines in the United States. EudraVigilance, a European database of suspected adverse events, reported over 200 cases of thrombosis per 34 million people among people vaccinated with the ChAdOx1 nCoV-19 vaccine. The European Medicines Agency (EMA) investigated the reported cases and found an association between the ChAdOx1 nCoV-19 vaccine and thrombotic events and associated thrombocytopenia. Although the WHO and EMA concluded that the benefits of ChAdOx1 nCoV-19 vaccination outweigh the risks associated with thrombosis and thrombocytopenia, several countries have imposed restrictions on the use of ChAdOx1 nCoV-19 vaccine. An unusual group of cases presenting with cerebral venous sinus thrombosis (CVST) and thrombocytopenia is termed vaccine-induced immune thrombotic thrombocytopenia (VITT). To better understand the pathophysiology of VITT, we investigated eight cases of thrombocytopenia primarily suspected to be CVST but also presenting with other thromboembolic complications. This study identified antibody-mediated procoagulant platelets as a novel mechanism associated with VITT.

[0050] 2. Method Evaluation of study cohort and clinical data Blood samples were collected and analyzed for coagulation parameters to exclude heparin-induced thrombocytopenia (HIT). Blood samples from unvaccinated healthy donors (n = 24) served as healthy controls (17 women, mean age 36.1 ± 13.7 years). Blood samples were also collected from colleagues at the Tübingen Blood Donation Center and Ulm University Hospital before and after the first dose of ChAdOx1 nCoV-19 vaccine and served as vaccinated controls (n = 41, 29 women, mean age 37.3 ± 10.9 years). None of the colleagues from whom blood samples were collected developed hematological abnormalities. All study subjects received the ChAdOx1 nCoV-19 vaccine (Bakiszebria, AstraZeneca, London, UK). Additionally, sera from 29 COVID-19 patients (7 women, mean age 65.3 ± 14.1 years) who underwent multiple serial HIT antibody EIA assays during their hospitalization were included in this study. Clinical data from 21 of these COVID-19 patients admitted to the ICU ward have been reported in a previous study.

[0051] Patients and sera Experiments were performed using remaining serum samples from HIT patients received by our laboratory between March 2019 and December 2021. The diagnosis of HIT was independently confirmed by two hematology specialists according to current guidelines (e.g., a 4T's score of >3) and based on laboratory findings in an IgG enzyme immunoassay and a heparin-induced platelet activation (HIPA) test. Additionally, serum samples were collected from healthy blood donors who provided written consent at the Tübingen Blood Donation Center. Serum samples were stored at -80°C and then thawed at 4°C before experimental manipulations. To eliminate nonspecific effects of serum components other than antibodies, all sera were heat-inactivated at 56°C for 30 minutes and centrifuged at 5,000 × g for 5 minutes. The resulting supernatant was used in this study.

[0052] A bead-based multiplex assay for detecting COVID-19 antibodies COVID-19 antibodies were measured using a multiplex assay (NMI, Reutlingen, Germany) with the FLEXMAP 3D® system (Luminex, Austin, USA). Each test was performed at the Tübingen blood donation center. Four types of bound antibodies were detected in a single run using the Luminex FLEXMAP 3D® system and Luminex xPONENT software 4.3 (settings: 50 events, gates: 7,500–15,000, reporter gain: standard PMT).

[0053] Anti-PF4 / heparin antibody test A commercially available IgG enzyme immunoassay (EIA) was used (Hyphen Biomed, Neuville-sur-Oise, France) according to the manufacturer's instructions. Samples were considered reactive if their optical density (OD) was ≥ 0.500, as recommended by the manufacturer. The platelet-activating capacity of serum was tested using a functional assay, the heparin-induced platelet aggregation assay (HIPA), as previously reported. Briefly, serum was tested using washed platelets (wPLTs) obtained from four healthy donors in the absence (buffer only) or presence of unfractionated heparin (0.2 IU / mL or 100 IU / mL [Ratiopharm, Ulm, Germany]). Reactions were performed in microtiter wells equipped with a spherical stir bar and agitated at approximately 500 revolutions per minute (rpm). Each well was optically monitored every 5 minutes (min) to observe a decrease in turbidity. Serum was considered reactive (positive) if the platelet suspension in at least two wells changed from turbid to clear within 30 minutes. The observation period was 45 minutes. In each test, diluted serum from a patient with HIT was used as a weak positive control, collagen (5 μg / mL) was used as a strong positive control, and serum from a healthy donor was used as a negative control.

[0054] Preparation of washed platelets Fresh washed platelets (wPLTs) were prepared from venous blood samples as previously described. Briefly, fresh whole blood was collected from healthy donors by antecubital venipuncture into acid dextrose-containing Vacutainer tubes (Becton Dickinson, Plymouth, UK) and incubated at 37°C for 45 minutes. After centrifugation (120 × g, 20 minutes, room temperature [RT], no brake), platelet-rich plasma (PRP) was gently isolated and supplemented with apyrase (5 μL / mL, Sigma-Aldrich, St. Louis, USA) and prewarmed ACD-A (333 μL / mL, Sigma-Aldrich, St. Louis, USA). After further centrifugation (650 × g, 7 min, room temperature, no brake), the platelet pellet was resuspended in 5 mL of wash solution (modified Tyrode's buffer: 5 mL of bicarbonate buffer, 20% bovine serum albumin, 10% glucose solution [Braun, Melsungen, Germany], 2.5 U / mL apyrase, 1 U / mL hirudin [Pentapharm, Basel, Switzerland], pH 6.3) and incubated at 37 °C for 15 min. After a final centrifugation (650 × g, 7 min, room temperature, no brake), washed platelets (wPLTs) were resuspended in 2 mL of resuspension buffer (50 mL of modified Tyrode's buffer, 0.5 mL of 1 mM MgCl2, 1 mL of 2 mM CaCl2, pH 7.2) and counted on a Cell-Dyn Ruby hematology analyzer (Abbott, Wiesbaden, Germany). The number of washed platelets was 300 × 10 3 The concentration was adjusted to 1 / μL.

[0055] Preparation of immunoglobulin G A commercial IgG purification kit (Melon) was used according to the manufacturer's recommendations. TMIgG fractions were isolated from serum samples from HIT patients and controls using a Gel IgG Spin Purification Kit (Thermo Fisher Scientific, Waltham, USA). Briefly, heat-inactivated serum was diluted 1:10 in purification buffer and incubated with the kit's proprietary Gel IgG Purification Support for 10 min, followed by four cycles of incubation. After each 10-min incubation, the serum was centrifuged at 5,000 × g for 1 min through a 10 μm-pore filter tube. The flow-through was collected in a 100 kDa-pore centrifugal filter (Amicon Ultra 4, Merck Millipore, Cork, Ireland) and centrifuged (10–15 min, 2,000 × g, 4°C, brake on) to concentrate the serum sample to its original volume. IgG concentrations were measured using a NanoDrop One (VWR, Bruchsal, Germany) with excitation at 340 nm.

[0056] Serological evaluation of PF4 antibodies Antibody binding to PF4 and the receptor-binding domain of the spike protein (spike-RBD) was analyzed using an enzyme immunoassay (ELISA) developed in our laboratory. PF4 (25 μg / mL, ChromaTec, Greifswald, Germany) or the spike-RBD domain (0–100 μg / mL) was immobilized at various concentrations on microtiter plates (Nunc MaxiSorp, Thermo Fisher Scientific, Waltham, MA, USA).

[0057] Antibody-based evaluation of procoagulant platelets To exclude nonspecific effects such as platelet activation via complement or nonspecific immune complexes, all sera were heat-inactivated (56°C for 30 minutes) and centrifuged at 5,000 × g to collect the supernatant. For all experiments using patient sera, 5 μL of serum and washed platelets (7.5 × 10 6The platelets were incubated with 25 μL of PF4 (25 μg / mL), spike protein (0–100 μg / mL), or vaccine (1:75 (V:V)) where indicated. Next, the samples were washed once (7 min, 650 × g, room temperature, no brake) and gently resuspended in 75 μL of phosphate-buffered saline (PBS; Biochrom, Berlin, Germany). Platelets were then stained with Annexin V-FITC and CD62-APC (Immunotools, Friesoyte, Germany) and analyzed directly by flow cytometry (FC). Washed platelets incubated with ionomycin (5 μM, 15 min at room temperature) or TRAP-6 (10 μM, 30 min at room temperature) served as positive controls. Results were calculated as the fold increase in the proportion of PS / CD62p double-positive events in platelets after incubation with patient serum compared with platelets incubated with healthy donor serum tested in parallel.

[0058] Evaluation of procoagulant platelets using washed platelets and antibodies Before use, all sera were heat-inactivated at 56°C for 30 minutes and centrifuged at 5,000 × g. The supernatant was collected in a new tube. To measure procoagulant platelets, 5 μL of serum and washed platelets (7.5 × 10 6 Platelets were incubated with 25 μL of PS / CD62p double-positive platelets (PS / CD62p) for 1 hour at room temperature with orbital shaking. Where indicated, the cell suspension was preincubated with PF4 (10 μg / ml) or heparin (0.2 IU / ml or 100 IU / ml). The samples were then washed once (7 min, 650 × g, room temperature, no brake) and gently resuspended in 75 μL of phosphate-buffered saline (PBS; Biochrom, Berlin, Germany). Platelets were then stained with Annexin V-FITC and CD62-APC (Immunotools, Friesoyte, Germany) and analyzed directly by flow cytometry (FC). Results were calculated as the fold increase in the percentage of PS / CD62p double-positive events in platelets after incubation with patient serum compared to baseline.

[0059] Evaluation of procoagulant platelets in platelet-rich plasma (PRP) To measure procoagulant platelets in PRP, serum was prepared as described above. To prepare PRP, venous blood from healthy volunteers was collected in a Vacutainer blood collection tube (BD, Plymouth, UK) containing 0.105 M (3.2%) sodium citrate and allowed to stand at room temperature for 20 minutes. Next, PRP was prepared by centrifugation (20 minutes, 120 × g, room temperature). Autologous platelet-poor plasma (PPP [10 minutes, 2000 × g, room temperature]) was added to the PRP to obtain a 300 × 10 6 The platelet count was adjusted to 11.25 × 10 cells / mL. 6 37.5 μL of each sample was mixed and brought to a total volume of 50 μL with PBS and incubated for 1 hour at room temperature with orbital shaking. Where indicated, PRP was preincubated with PF4 (10 μg / ml) or heparin (0.2 IU / ml or 100 IU / ml). Samples were then processed and analyzed by flow cytometry (FC) as described for washed platelets. Results were calculated as the fold increase in the percentage of PS / CD62p double-positive events in platelets after incubation with patient serum compared to baseline.

[0060] Treatment of platelets with serum / IgG 37.5 μl of washed platelets (wPLT) / platelet-rich plasma (PRP) was mixed with 1 μl of 10 IU heparin (final concentration: 0.2 IU) or 1 μl of 5000 IU heparin (final concentration: 100 IU) and 5 μl of serum / IgG from HIT patients or control serum / IgG. PBS was added to each sample to a final volume of 50 μl, and the mixture was incubated at room temperature for 1 hour with gyroscopic shaking. Next, 5 μl of the platelet suspension was added to Hank's balanced salt solution (HBSS) containing 137 mM NaCl, 1.25 mM CaCl2, and 5.5 mM glucose (Carl-Roth, Karlsruhe, Germany) to a final volume of 100 μL. 1 μL of anti-CD62p-APC (BD, San Jose, USA), 1 μL of annexin-FITC (Immunotools, Friesoythe, Germany), or 2 μL of anti-CD42a-PerCP (BD, San Jose, USA) was added and incubated for 30 minutes at room temperature in the dark. Platelets treated with thrombin receptor-activating peptide (TRAP-6) (10 μM, 30 minutes at room temperature) or ionomycin (5 μM, 15 minutes at room temperature) (both Sigma-Aldrich, St. Louis, USA) served as positive controls. Platelets were then resuspended in HBSS to a final volume of 500 μL and immediately assessed by flow cytometry ([FC]) (Navios, Beckman Coulter, Brea, USA).

[0061] Analysis of the mechanism of HIT antibody-induced procoagulant platelet formation To investigate the mechanism of platelet changes induced by HIT antibodies, we used an FcγRIIA-blocking monoclonal antibody (moAb) anti-CD32 (moAb IV.3; stemcell TM Seventy-five microliters of washed platelets (wPLT) / platelet-rich plasma (PRP) was pretreated with either monoclonal isotype control ([SC-2025] Santa Cruz Biotechnology, Inc., Vancouver, Canada) or monoclonal isotype control ([SC-2025] Santa Cruz Biotechnology, Inc., Dallas, USA) for 30 minutes at room temperature before treatment with HIT serum / IgG.

[0062] ethics statement This study was conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from all volunteers, VITT patients, or their relatives before any procedures related to this study were performed. All tests were performed using remaining routine laboratory specimens. The study protocol was approved by the Institutional Review Board of the University of Tübingen (No. 236 / 2021BO1). The collection and analysis of serum from people vaccinated with the ChAdOx1 nCoV-19 vaccine was approved by the Ethics Committee of the University of Ulm (No. 99 / 21).

[0063] statistical analysis Statistical analysis was performed using GraphPad Prism (version 7.0, GraphPad, La Jolla, CA, USA). Because FC measurements can vary within a day, potentially introducing bias into data analysis, each test result was corrected for the results of two healthy donors tested in parallel at the same time point (raw data are provided in the Supplementary Data). Data herein are presented as median (range), mean ± standard deviation (SD), or n (%).

[0064] 3.Results IgG binding profile of sera from VITT patients High titers of PF4 / heparin antibodies were detected in all sera (8 of 8 samples, 100%) as measured by an IgG antibody enzyme immunoassay (ELISA) for the detection of PF4 / heparin complex antibodies. Interestingly, binding of all sera was inhibited in the presence of high concentrations of heparin (mean optical density [OD] of IgG antibodies to PF4 / heparin complex: 2.591 ± 0.642 (without heparin) vs. 0.176 ± 0.073 (with high-concentration heparin), p < 0.0001, Figure 1A). No correlation was observed between PF4 / heparin antibodies and detected COVID-19 antibodies in either VITT patients or vaccinated controls (Figures 2A–2D [I–IV]). In unvaccinated controls, PF4 / heparin antibodies were detected by EIA in only one subject (4%) (data not shown).

[0065] Next, we investigated PF4 seroconversion after ChAdOx1 nCoV-19 vaccination and during severe SARS-CoV-2 infection (Figure 1B). We found that 4 of 41 vaccinated healthy subjects (9.8%) and 4 of 41 severe COVID-19 patients (9.8%) seroconverted PF4 and produced IgG antibodies against the PF4 / heparin complex within 14 days (Figure 1B). Next, we tested IgG binding to platelets by flow cytometry (FC). Increased IgG binding to the test samples was observed (fold increase in mean fluorescence intensity [MFI fold increase (FI)] compared to healthy controls: 4.39 ± 1.15 vs. 1 ± 1.10 in healthy controls, p = 0.026, Figures 1C and 3A). IgG binding to platelets was inhibited by high concentrations of heparin (fold increase in MFI of IgG binding: 1.51 ± 0.66, p = 0.016), but not by low concentrations of heparin (fold increase in MFI of IgG binding: 3.60 ± 2.01, p = 0.688). Only one patient's serum showed increased binding to platelets in the presence of PF4 and the ChAdOx1 nCoV-19 vaccine (Case No. 4, Figure 1C). The receptor-binding domain of the spike protein (spike-RBD) did not induce significant changes in IgG binding in VITT patients (Figure 1C). Similar results were observed when S2 protein was added (Figure 1E). IgG binding was also observed when serum from a ChAdOx1 nCoV-19-vaccinated volunteer with PF4 IgG antibodies was tested (Figure 3B). On the other hand, in severe COVID-19 patients with PF4 IgG antibodies, no increase in IgG binding was observed (Figure 3C).

[0066] Effect of spike-RBD on anti-PF4 antibody binding Using an in-house developed EIA, sera from VITT patients showed stronger binding to PF4 compared with sera from healthy controls (OD of IgG antibodies against PF4: VITT patients 1.03 ± 0.04 vs. healthy controls 0.110 ± 0.002, p-value < 0.0001, Figure 1C). On the other hand, sera from VITT patients showed weak binding to spike-RBD, but the difference was not significant (Figure 3D). Most importantly, in the presence of PF4, IgG binding decreased when the RBD concentration was above 6.5 μg / mL (Figure 1D). However, sera from VITT patients did not show significant binding to S2 protein, either in the presence or absence of PF4 (Figure 1E and Figure 3E).

[0067] Platelet activation in the heparin-induced platelet aggregation (HIPA) assay. To examine the platelet-activating potential of patient sera, we used a heparin-induced platelet aggregation (HIPA) assay with some modifications. Patient sera and washed platelets were incubated in the presence of: I) buffer; II) 0.2 IU / mL low-molecular-weight heparin (LMWH); III) 100 IU / mL unfractionated heparin (UFH); IV) Fcγ receptor IIa (FcγRIIA)-blocking monoclonal antibody (mAb IV.3); VI) 30 mg / mL intravenous immunoglobulin (IVIG); VII) 25 μg / mL PF4; VIII) 50 μg / mL spike-RBD; IX) PF4 / spike-RBD complex; X) PF4 + RBD; or XI) ChAdOx1 nCoV-19 vaccine (XII). The PF4- and RBD-supplemented conditions were also replicated in the presence of high-concentration heparin (100 IU / mL UFH). In all eight VITT patients, platelet activation was observed in the presence of buffer (median time to platelet aggregation: 5 min, 5-10 min, Figure 4A). In contrast, platelet activation was not observed in the serum of a vaccinated volunteer with anti-PF4 antibodies who had no clinical signs of thromboembolic complications or side effects (Figure 5A). Furthermore, only one serum sample from a severe COVID-19 patient with a positive PF4 / heparin EIA showed platelet activation in the HIPA assay (Figure 5B). Interestingly, this response was reduced in the presence of low-molecular-weight heparin (median time to aggregation: 5 min, 5-10 min (no aggregation) vs. 30 min, prolonged from 5 to 45 min, Figure 4A). Both responses were suppressed with high doses of heparin (p-value = 0.008, Figure 4A). In the presence of PF4, sera from VITT patients demonstrated potent platelet activation (median time to aggregation: 5 min, 5-5 min, Figure 4A). Most importantly, platelet activation was completely inhibited by mAb IV.3, which blocks FcγRIIa, or high doses of IgG (>45 min, no aggregation, Figure 4A). Furthermore, addition of the PF4 / RBD complex did not significantly alter platelet activation. Antibody-mediated platelet activation was inhibited when the three sera were treated with low concentrations of low molecular weight heparin (LMWH).When serum from a VITT patient was diluted, specific binding to PF4 was observed, but no reaction was observed in the presence of buffer (Fig. 4C).

[0068] Induction of a PF4-dependent procoagulant phenotype by VITT patient sera To investigate the mechanism of coagulation dysregulation in VITT, we incubated washed platelets prepared from healthy donors with VITT patient serum in the presence of buffer, heparin, mAb IV.3, intravenous immunoglobulin (IVIG), PF4, PF4 + IVIG, PF4 + RBD, spike-RBD protein, or the ChAdOx1 nCoV-19 vaccine. Flow cytometry analysis revealed that VITT patient serum significantly altered the distribution of CD62p / PS-positive platelets (fold increase (FI): 22.94 ± 6.14 from VITT patient serum vs. 0.90 ± 0.63 from control, p = 0.009, Figure 4B, Figure 6). In contrast, incubation of washed platelets with serum from vaccinated controls had little effect on the CD62p / PS-positive platelet population (Figure 7A). Interestingly, the production of procoagulant platelets in VITT patients was reduced by the addition of 0.2 IU / mL low molecular weight heparin (LMWH) (fold increase (FI) of CD62p / PS-positive platelets: 13.32 ± 11.50, p = 0.016, Figure 4B) and completely suppressed by the addition of high concentrations of unfractionated heparin (UFH) (fold increase (FI) of CD62p / PS-positive platelets: 1.92 ± 0.96, p = 0.008, Figure 4B, Figure 6). Furthermore, these responses were also suppressed by blockade of FcγRIIA by mAb IV.3 and by high concentrations of IgG (fold increase (FI) of CD62p / PS-positive platelets with mAb IV.3: 1.04 ± 0.22, p = 0.031; fold increase (FI) of CD62p / PS-positive platelets with high concentrations of IgG: 7.88 ± 5.56, p = 0.031, Figure 4B). No significant increase in procoagulant platelets was observed in the presence of PF4 (fold increase (FI) of CD62p / PS-positive platelets: 37.07 ± 23.73, p = 0.078) or spike-RBD alone (fold increase (FI) of CD62p / PS-positive platelets: 22.02 ± 17.09, p = 0.195).Although increased production of procoagulant platelets was observed after incubation with serum from severe COVID-19 patients, no significant changes were observed when tested with serum from vaccinated volunteers with anti-PF4 antibodies (Figure 7A-B).

[0069] To identify the target antigens of platelets that activate the antibodies, we performed the HIPA and FACS assays again using various titers of serum from VITT patients. Interestingly, only in the presence of PF4, diluted serum (at a dilution of 1:64 or greater) was able to activate platelets and induce a procoagulant phenotype (Figures 4C and 4D).

[0070] Procoagulant platelets in heparin-induced thrombocytopenia As shown in the graphs, patient serum was incubated with washed platelets (Figure 8) or PRP (Figure 9) prepared from healthy donors in the presence of buffer, heparin, PF4, or IV.3 antibody. While 0.2 IU / mL low molecular weight heparin (LMWH) potently produced procoagulant platelets, the addition of high concentrations of unfractionated heparin (UFH) completely suppressed procoagulant platelet production. An increase in procoagulant platelets was also observed in the presence of PF4, but this increase was suppressed by the Fcγ receptor IIA-blocking monoclonal antibody IV.3 (Figure 8). Furthermore, serum from a HIPA-negative patient was incubated with PRP from a healthy volunteer (Figure 8). A strong increase in the procoagulant phenotype was observed in the presence of PF4 when treated with 0.2 IU / mL heparin (low molecular weight heparin (LMWH)).

[0071] Therefore, heparin-induced thrombocytopenia (HIT) has been shown to be associated with procoagulant platelets. Furthermore, PF4 can be used as a predictive biomarker for HIT. The use of PF4 can improve the sensitivity of flow cytometry assays using PRP. Furthermore, the specificity of these assays can be improved by using high concentrations of heparin or monoclonal antibodies (IV.3).

[0072] Heparin-dependent procoagulant platelet formation due to serum HIT antibodies Washed platelets (wPLTs) from healthy donors were incubated with serum from well-characterized HIT patients or healthy control (HC) serum in the presence of therapeutic (0.2 IU) or supratherapeutic (100 IU) heparin (Figure 10). Sera from 15 of 35 patients (43%) induced a significant increase in the formation of CD62p / PS-positive platelets, whereas no such change was observed in platelets incubated with healthy control (HC) serum (mean percentage [mean %] ± SEM: patient serum 44.99 ± 6.26 vs. healthy control 1 ± 0, p = 0.0001). Most importantly, the procoagulant platelet phenotype induced by sera from HIT patients was completely inhibited in the presence of a supratherapeutic dose of heparin (100 IU heparin), indicating that the HIT-specific heparin / PF4 antibody complex was disrupted by increasing heparin concentrations (mean % ± SEM: procoagulant platelet phenotype induced by HIT patient sera 44.99 ± 6.26 vs. procoagulant platelet phenotype 1.18 ± 0.28 in the presence of high-dose heparin, p = 0.0001). Interestingly, sera from 5 of 36 patients (14%) induced significant amounts of procoagulant platelets even under buffer conditions. These patient sera were able to induce the formation of procoagulant platelets in the presence of therapeutic concentrations of heparin (0.2 IU), but procoagulant platelet formation was suppressed in the presence of supratherapeutic doses of heparin, suggesting that these patient serum samples may have been contaminated with platelet-activating factors (e.g., thrombin), which led to the formation of procoagulant platelets under buffer conditions.

[0073] Procoagulant platelets induced solely by serum from patients with confirmed HIT Interestingly, among the clinically significant observations, the procoagulant platelet formation was not detected in the subgroup of patients who were HIT-negative (HIT neg., [EIA-HIPA-]) or in the subgroup of patients who had only specific heparin / PF4 antibodies in their serum but a negative HIPA test (EIA-IgG pos., [EIA+HIPA-]), suggesting that the serum-induced procoagulant platelet formation effect was restricted to patients with confirmed HIT (HIT pos., [EIA+HIPA+]) (Fig. 11). This finding indicates that procoagulant platelet formation is induced only in patients with confirmed HIT by laboratory tests, i.e., patients who have specific antiheparin / PF4 antibodies and a positive HIPA test.

[0074] HIT IgG antibody and its binding to platelet FcγRIIA induce the formation of procoagulant platelets A) To confirm that the procoagulant platelet formation effect caused by HIT serum was induced by the specific heparin / PF4 HIT antibody and not by other nonspecific activation pathways, IgG fractions were prepared from selected HIT serum and incubated with platelet-rich plasma from healthy donors. Triple staining and subsequent FC analysis revealed that, similar to the results observed with serum, the HIT IgG fraction induced procoagulant platelets in the presence of a therapeutic dose (0.2 IU) of heparin. However, this change was not observed in platelets incubated with IgG from healthy controls (HCs) or in the presence of a supratherapeutic dose (100 IU) of heparin (Figure 12A). To confirm that HIT antibody-induced procoagulant platelet formation was induced by the specific platelet FcγRIIA signaling mechanism and not by other activation pathways, platelet-rich plasma was preincubated with the specific monoclonal inhibitor antibody IV.3 and then incubated with HIT IgG antibody (Figure 12B).

[0075] 4. Discussion Increasingly reported cases of rare thrombotic events after SARS-CoV-2 vaccination have attracted public attention and raised concerns about the safety of SARS-CoV-2 vaccines, given the unknown cause of these undesirable reactions. To understand the pathophysiology of this phenomenon, so-called vaccine-induced immune thrombotic thrombocytopenia (VITT), we analyzed serum samples from eight patients. Most of these patients were young and generally matched a cohort of patients with acute atypical thrombosis, primarily cerebral venous sinus thrombosis, an extremely rare manifestation of thrombosis in the general population (although some cases did not present with cerebral venous sinus thrombosis). All cases presented within 6–20 days after administration of the ChAdOx1 nCoV-19 vaccine, suggesting a temporal relationship between vaccination and symptoms. Key findings in these cases included thrombocytopenia, elevated D-dimer levels, and decreased fibrinogen levels, along with high titers of anti-PF4 IgG antibodies, which can induce a procoagulant platelet phenotype.

[0076] Intensive clinical laboratory investigation of VITT cases allowed us to identify a serological profile of pathogenic antibodies. Approximately 10% of a small cohort of vaccinated volunteers developed IgG antibodies against the PF4 / polyanion complex within 14 days after the first vaccination. These volunteers had not been exposed to heparin within the past 100 days. IgG binding to PF4 in serum from these volunteers and in serum from VITT patients was inhibited by heparin and by high concentrations of the spike-RBD protein. These data suggest that the antibodies are specific for a conformational change in PF4 that may be induced by a negatively charged structure. It should be noted that significant IgG binding to platelets was not observed in the presence of the ChAdOx1 nCoV-19 vaccine. Given these findings, it is highly unlikely that the vector (pCDNA4) is responsible for the high seroconversion rate of PF4 in vaccinated individuals. Similar data were previously reported in two recent studies during the preparation of this application. In addition to these two findings, our study demonstrated that serum from VITT patients directly induces procoagulant platelets, suggesting a potential mechanism for the thrombotic events observed in VITT patients.

[0077] Our data demonstrated that anti-PF4 IgG antibodies increased the production of procoagulant platelets in patients with VITT, but we could not exclude the possibility of other auxiliary factors inducing thromboembolic complications in vivo.

[0078] Our study also provides insight into potential therapeutic strategies. First, the increase in the proportion of procoagulant platelets (CD62p / PS positive) in response to VITT patient serum in vitro appears to represent a central pathological mechanism in VITT. Furthermore, our data also demonstrate that anticoagulation using non-heparin therapies, such as argatroban and danaparoid, is safe for the treatment or prevention of venous sinus thrombosis (CVST) in VITT.

[0079] In this study, we also report on VITT after vaccination with ChAdOx1 nCoV-19, the only SARS-CoV-2 vaccine containing a simian adenovirus. Intravenous administration of therapeutic vectors consisting of adenovirus genes has been reported to perturb platelets, but it is unclear how this relates to thrombocytopenia, a common adverse event of the vaccine.

[0080] Furthermore, the clinical and laboratory features observed in VITT are exceptional and extremely rare. Therefore, the benefits of COVID-19 vaccination, which confers crucial protective effects, should be considered to outweigh the significant health risks associated with COVID-19. Further understanding of this rare complication and the availability of effective treatments may further reconsider the risk-benefit ratio of the ChAdOx1 nCoV-19 vaccine.

[0081] 5. Conclusion Although the incidence of VITT after ChAdOx1 nCoV-19 vaccination is very low, its mortality rate is high (37% in the cases observed by the inventors). With the current global vaccination campaign, the number of people affected by this side effect is expected to increase as more people are vaccinated, highlighting the importance of a more detailed understanding of the pathophysiology of VITT. This study presents immunological and pathological findings in patients with VITT. Furthermore, it demonstrates the contribution of antibody-mediated platelet activation in the pathogenesis of VITT.

[0082] Based on the above, the inventors have developed, for the first time, a method that can easily and reliably determine a subject's predisposition to the development of thrombocytopenia and / or thrombosis, or whether the subject actually suffers from thrombocytopenia and / or thrombosis.

Claims

1. 1. A method for aiding in the diagnosis of a predisposition to developing vaccine-induced immune thrombocytopenia (VITT) in an organism, comprising:

1. The percentage of platelets expressing P-selectin and phosphatidylserine on their surface was measured among platelets from healthy reference organisms to determine M pre determining the value; 2. Incubating an aliquot of serum from the organism to be determined with an aliquot of platelets from said healthy reference organism; 3. After the incubation in step (2), the percentage of platelets expressing P-selectin and phosphatidylserine on their surface was measured to determine M post determining the value; and 4. Comparing the M pre and M post values Including, In step (2), platelet factor IV (PF4) is added to the incubation mixture consisting of the serum and the platelets; A method wherein, in step (4), if M post > M pre , the organism being determined is indicated to have a predisposition to developing vaccine-induced immune thrombocytopenia (VITT).

2. 2. The method of claim 1, wherein the VITT is caused by an anti-SARS-CoV-2 vaccine.

3. 3. The method of claim 2, wherein the anti-SARS-CoV-2 vaccine is a vector-based vaccine.

4. A method according to any one of claims 1 to 3, wherein the platelets of the healthy reference organism are washed platelets.

5. A method described in any one of claims 1 to 4, wherein the platelets of the healthy reference organism are platelet-rich plasma (PRP).

6. The method according to any one of claims 1 to 5, wherein in step (2), heparin is added to the incubation mixture consisting of the serum and the platelets.

7. The method of claim 6, wherein the heparin is a low molecular weight heparin (LMWH).

8. The method according to any one of claims 1 to 7, wherein in step (1) and / or step (3), the percentage (%) of platelets expressing P-selectin and phosphatidylserine on their surface is measured by flow cytometry (FC).

9. The method according to any one of claims 1 to 8, wherein in step (4), the presence of the predisposition is indicated when 10% or more of the platelets after the incubation in step (2) express P-selectin and phosphatidylserine on their surfaces.

10. 1. A method for aiding in the diagnosis of vaccine-induced immune thrombocytopenia (VITT) in an organism, comprising:

1. The percentage of platelets expressing P-selectin and phosphatidylserine on their surface was measured among platelets from healthy reference organisms to determine M pre determining the value; 2. Incubating an aliquot of serum from the organism to be determined with an aliquot of platelets from said healthy reference organism; 3. After the incubation in step (2), the percentage of platelets expressing P-selectin and phosphatidylserine on their surface was measured to determine M post determining the value; and 4. Comparing the M pre and M post values Including, In step (2), platelet factor IV (PF4) is added to the incubation mixture consisting of the serum and the platelets; A method wherein, in step (4), if M post > M pre , the organism to be determined is indicated to be suffering from vaccine-induced immune thrombocytopenia (VITT).

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