Thrombrogenase compound as well as detection method and application thereof
By rebuilding the coordinase primary complex on the plasma platelet on the plasma plates after washing, the problem that it is difficult to monitor FV and FXA activity in existing technologies has been solved, and the effectiveness of the effectiveness of AIFVD cases and platelet infusion products is realized. Concrete.
Patent Information
- Application Number
- CN202411530702.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-30
- Publication Date
- 2025-01-21
AI Technical Summary
The prior art is difficult to effectively monitor the activity of FV and FXa in the prothrombinase complex, affecting the confirmation of AiFVD cases and the evaluation of the effectiveness of platelet infusion products.
By rebuilding coagalase primer on plasma platelets on the plasma platelets after washing, including human coagulation factor FXA and human coagal enzymes, or using Platelin LS, human coagulation factor FXA, human coagulation factor FVA, and human coagozyrase, combined with hair color The substrate and anticoagulation drugs are monitored to the production of coordinase.
Effective monitoring of FV and FXA activity can be achieved, which can be used to confirm AIFVD cases, monitor patients' hemostasis after treatment, and evaluate the effectiveness of platelet infusion products, and use it to screen anticoagulant drugs.
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Figure CN119331858A_ABST
Abstract
Description
Technical Field
[0001] The present application relates to the field of biomedicine technology, and in particular to a prothrombinase complex and a detection method and application thereof. Background Art
[0002] In the human coagulation cascade, when tissue is damaged, tissue coagulation factor (TF) is released; on phospholipids (PL), coagulation factor VIIa and TF form exogenous Xase enzyme. At the same time, high molecular weight kininogen (HKa), prekallikrein and coagulation factor XII form a primary complex on collagen, which converts prekallikrein into kallikrein and converts coagulation factor XII into coagulation factor XIIa. Coagulation factor XIIa converts coagulation factor XI into coagulation factor XIa, which in turn activates coagulation factor IX into coagulation factor IXa. Coagulation factor IXa also comes from exogenous Xase enzyme. On PL, IXa and VIIIa together form endogenous Xase enzyme. Both of the above complexes can activate coagulation factor X into coagulation factor Xa. Afterwards, prothrombin, Xa and Va coagulation factors form a prothrombinase complex on PL, and thrombin (IIa) is generated from the prothrombinase complex. The prothrombinase complex is the central point of both the intrinsic and extrinsic coagulation pathways, e.g. Figure 1 (where C1 INH represents C1 inhibitor), which is composed of prothrombin, Xa coagulation factor, Va coagulation factor and phospholipids. In the prothrombinase complex, Xa coagulation factor acts as an enzyme, which can convert prothrombin into thrombin on the phospholipid surface with its co-coagulation factor Va coagulation factor. Thrombin not only cleaves fibrinopeptide A (FPA) and fibrinopeptide B (FPB) from fibrinogen, but also eventually polymerizes the fibrin monomers produced.
[0003] Prothrombin is a circulating zymogen precursor protein that belongs to the family of vitamin K-dependent blood coagulation proteins; it is the most abundant coagulation protein circulating in plasma at a concentration of 1 μM to 2 μM, with a molecular weight of 72,000 Da. The prothrombin molecule is mainly synthesized in the liver, and the mature protein molecule consists of 579 amino acids, contains ten γ-carboxyglutamic acid (Gla) residues at the NH2 end of 40 amino acids, and has three aspartate-linked glycosylation sites. Human FV (prothrombin) is a single-chain glycoprotein present in plasma at a concentration of 20 nM, with a molecular weight of approximately 330,000 Da. An additional plasma pool is sequestered in the α-granules of platelets, accounting for 18%-25% of the total FV in whole blood. Thrombin-activated V coagulation factor is a calcium-dependent heterodimer that acts as a coagulant factor in the prothrombinase complex and can accelerate the conversion of prothrombin to thrombin in the presence of calcium ions and procoagulant phospholipid surfaces. In the presence of FVa, the maximum rate of conversion of prothrombin to thrombin can be increased by 3000-fold. It is currently difficult to effectively monitor the activity of FV and FXa (coagulation factor Xa). Summary of the invention
[0004] The purpose of the present application is to provide a prothrombinase complex to monitor the activity of FV and FXa. The specific technical solution is as follows:
[0005] The first aspect of the present application provides a prothrombinase complex, comprising: washed human plasma platelets, human coagulation factor FXa and human prothrombin; or, the prothrombinase complex comprises: Platelin LS, human coagulation factor FXa, human coagulation factor FVa and human prothrombin.
[0006] In some embodiments of the present application, the concentration of platelets in the washed human plasma platelets is (0.5-1.5)×10 8 / mL; the solvent in the washed human plasma platelets includes a Tyrodes buffer containing adenosine triphosphatase; the concentration of the adenosine triphosphatase is 0.01U / mL-0.03U / mL.
[0007] In some embodiments of the present application, the method for preparing washed human plasma platelets comprises the steps of:
[0008] Step 1: Place freshly collected human blood in a sodium citrate anticoagulation tube and centrifuge to obtain platelet-rich plasma;
[0009] Step 2: adding the platelet-rich plasma to a G10 sephadex chromatography column pre-equilibrated with a Tyrodes buffer, washing the G10 sephadex chromatography column with the Tyrodes buffer, and collecting the washing solution;
[0010] Step 3: The washing solution is centrifuged and then resuspended in a Tyrodes buffer containing ATPase to obtain the washed human plasma platelets.
[0011] In some embodiments of the present application, step 2 further includes a step of confirming that platelets are active, and the step of confirming that platelets are active includes: using a platelet aggregometer to test a turbid platelet-rich tube containing ADP and the washing solution; preferably, the concentration of ADP is 150 μM-250 μM.
[0012] The second aspect of the present application provides a kit, comprising the prothrombinase complex described in the first aspect of the present application.
[0013] In some embodiments of the present application, the kit further comprises a chromogenic substrate; preferably, the chromogenic substrate comprises at least one of the chromogenic substrate S2238 and the chromogenic substrate S2765.
[0014] The third aspect of the present application provides the use of the prothrombinase complex described in the first aspect of the present application or the kit described in the second aspect of the present application in screening anticoagulant drugs or monitoring the effectiveness of platelet transfusion products.
[0015] The fourth aspect of the present application provides a method for detecting a prothrombinase complex, which comprises the steps of:
[0016] BSA is added to the detection buffer for pre-blocking, and then the washed human plasma platelets, human coagulation factor FXa, human prothrombin, as well as a chromogenic substrate and an anticoagulant drug described in the first aspect of the present application are added to obtain a first reaction system; the first reaction system is monitored at a wavelength of 405 nm using a multi-well plate reader at room temperature for 20 min-40 min; or,
[0017] BSA is added to the detection buffer for pre-blocking, and then the PlatelinLS, human coagulation factor FXa, human coagulation factor FVa, human prothrombin, as well as chromogenic substrate and anticoagulant drugs described in the first aspect of the present application are added to obtain a second reaction system; the second reaction system is monitored at a wavelength of 405nm using a multiwell plate reader at room temperature for 20min-40min.
[0018] In some embodiments of the present application, the detection buffer comprises: 15mM-24mM HEPES, 140mM-160mM NaCl and 1.5mM-2.5mM Ca 2+ ; Preferably, the amount of BSA used is 0.2 vol%-0.8 vol% of the detection buffer.
[0019] In some embodiments of the present application, in the first reaction system: the concentration of platelets is (0.5-1.5)×10 7 / mL; the concentration of the chromogenic substrate is 0.2mM-0.4mM; the concentration of human coagulation factor FXa is 2nM-8nM; the concentration of human prothrombin is 0.5M-1.5M;
[0020] In the second reaction system: the concentration of the chromogenic substrate is 0.2mM-0.4mM; the concentration of the human coagulation factor FXa is 2nM-8nM; the concentration of the human coagulation factor FVa is 20pM-60nM; the concentration of the human prothrombin is 0.5M-1.5M; the amount of Platelin LS added to every 100μL of the second reaction system is 5μL-15μL.
[0021] Beneficial effects of this application:
[0022] The prothrombinase complex provided in the present application is used to monitor the activity of FV and FXa, which can be used to confirm AiFVD cases and monitor the hemostasis status of patients after treatment; or to monitor the effectiveness of platelet transfusion products; or to screen anticoagulant drugs from candidates.
[0023] Of course, implementing any product or method of the present application does not necessarily require achieving all of the advantages described above at the same time. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application, and for ordinary technicians in this field, other embodiments can also be obtained based on these drawings.
[0025] Figure 1 The coagulation cascade in human hemostasis is shown;
[0026] Figure 2 The results of the progress curve of pNA (peptide nucleic acid) generation by the prothrombinase complex in Example 1 in the presence of different concentrations of PA11 are shown;
[0027] Figure 3 The results of the progress curve of pNA production in Example 2 on 10 μl of Platelin LS surface and in the presence of different concentrations of PA11 are shown. DETAILED DESCRIPTION
[0028] The following will be combined with the embodiments of the present application and the accompanying drawings to clearly and completely describe the technical solutions in the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. All other embodiments obtained by those skilled in the art based on the present application belong to the scope of protection of the present application.
[0029] The first aspect of the present application provides a prothrombinase complex, comprising: washed human plasma platelets, human coagulation factor FXa and human prothrombin; or comprising: Platelin LS, human coagulation factor FXa, human coagulation factor FVa and human prothrombin.
[0030] The human coagulation factor FXa, human coagulation factor FVa and human prothrombin described in the present application can all be purchased from commercial sources, for example, purified human coagulation factor FXa can be purchased from Calbiochem and Merck Millipore, human coagulation factor FVa and human prothrombin can be purchased from Haematologic Technologies Inc. USA; Platelin LS can be purchased from Organon Teknika Corporation LLC, Munich, Germany.
[0031] The present application reconstitutes the prothrombinase complex on human plasma platelets and can monitor the activities of FV and FXa.
[0032] In some embodiments of the present application, in the prothrombinase complex, the concentration of platelets in the washed human plasma platelets is (0.5-1.5)×10 8 / mL; the solvent in the washed human plasma platelets includes a Tyrodes buffer containing adenosine triphosphatase (apyrase); the concentration of the adenosine triphosphatase is 0.01U / mL-0.03U / mL.
[0033] The pH of the Tyrodes buffer described in the present application may be 7.4, and the components may include: 10 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 2.9 mM NaH2PO4, 5.6 mM glucose and 0.35 vol% BSA.
[0034] In some embodiments of the present application, the method for preparing washed human plasma platelets comprises the steps of:
[0035] Step 1: Place freshly collected human blood in a sodium citrate anticoagulation tube and centrifuge to obtain platelet-rich plasma (PRP);
[0036] Step 2: adding the platelet-rich plasma to a G10 sephadex chromatography column pre-equilibrated with a Tyrodes buffer, washing the G10 sephadex chromatography column with the Tyrodes buffer, and collecting the washing solution;
[0037] Step 3: The washing solution is centrifuged and then resuspended in a Tyrodes buffer containing ATPase to obtain the washed human plasma platelets.
[0038] The present application has no particular limitation on the concentration of the sodium citrate solution contained in the sodium citrate anticoagulation blood vessel, as long as the purpose of the invention of the present application can be achieved, for example, it can be 3.5wt%-4.0wt%.
[0039] The present application has no particular limitation on the volume ratio of the sodium citrate solution and freshly collected human blood in the sodium citrate anticoagulation blood vessel, as long as the purpose of the invention of the present application can be achieved, for example, it can be 1:(8-10).
[0040] The present application has no particular limitation on the speed and time of centrifugation, as long as the purpose of the invention of the present application can be achieved. For example, the speed of centrifugation is 90×g-100×g, and the time of centrifugation is 10min-20min.
[0041] Step 1 of the present application can be performed simultaneously using multiple sodium citrate anticoagulation tubes, and then multiple tubes, such as four to five tubes of platelet-rich plasma, are combined to obtain approximately 10 mL-12 mL of platelet-rich plasma.
[0042] The present application has no particular limitation on the size of the G10 polysaccharide gel chromatography column, as long as the purpose of the present application can be achieved. For example, the size of the G10 polysaccharide gel chromatography column is 1.7 cm×10 cm.
[0043] In some embodiments of the present application, step 2 further includes a step of confirming that the platelets are active, and the step of confirming that the platelets are active includes: using a platelet aggregometer to test the turbid platelet-rich tube containing ADP and the washing solution.
[0044] In some embodiments of the present application, the concentration of ADP is 150 μM-250 μM. The concentration of ADP described in the present application refers to the concentration of ADP in a mixture of ADP and a washing solution.
[0045] In some embodiments of the present application, the concentration of platelets in the washed human plasma platelets is (0.5-1.5)×10 8 Pieces / mL.
[0046] In some embodiments of the present application, in the Tyrodes buffer containing ATPase, the concentration of ATPase is 0.01 U / mL-0.03 U / mL.
[0047] The method for preparing washed human plasma platelets described in the present application can be performed at room temperature.
[0048] The second aspect of the present application provides a kit, comprising the prothrombinase complex described in the first aspect of the present application.
[0049] In some embodiments of the present application, the kit further comprises a chromogenic substrate; preferably, the chromogenic substrate comprises at least one of the chromogenic substrate S2238 and the chromogenic substrate S2765.
[0050] The chromogenic substrate S2238 and the chromogenic substrate S2765 described in this application can be purchased from Chromogenix, Stockholm, Sweden.
[0051] The third aspect of the present application provides the use of the prothrombinase complex described in the first aspect of the present application or the kit described in the second aspect of the present application in screening anticoagulant drugs or monitoring the effectiveness of platelet transfusion products.
[0052] Congenital FV deficiency is a bleeding disorder associated with mild to severe bleeding symptoms, with an incidence of 1 in 1 million in the general population. Patients with clinically significant FV deficiency, primarily involving mucous membranes, show very low or unmeasurable plasma FV levels. And for autoimmune FV deficiency (AiFVD), detection of anti-FV antibodies is necessary to avoid missing cases of non-neutralizing anti-FV autoantibodies. However, anti-FV antibodies are present in serum, and active measurements of the disease state are lacking.
[0053] The present application can confirm AiFVD cases and monitor the hemostasis status of patients after treatment by reconstituting the prothrombinase complex on washed platelets.
[0054] Considering the potential limitations of existing methods and the lack of formal analysis of FV content in platelet transfusion products, the kit provided in this application can be used to monitor the effectiveness of platelet transfusion products.
[0055] Reconstructing the prothrombinase complex on washed platelets is an effective method for monitoring FV and FXa activity. The prothrombinase complex plays a central role in the human coagulation cascade. In order to maintain the anticoagulation level required by patients, the research and pharmaceutical industries need to discover new anticoagulant drugs. This application can provide the best test target or kit for screening anticoagulant drug development candidates by reconstructing the prothrombinase complex on washed human plasma platelets.
[0056] The fourth aspect of the present application provides a method for detecting a prothrombinase complex, which comprises the steps of:
[0057] Add BSA (bovine serum albumin) to the detection buffer for pre-blocking, and then add the washed human plasma platelets, human coagulation factor FXa, human prothrombin, as well as a chromogenic substrate and an anticoagulant drug described in the first aspect of the present application to obtain a first reaction system; monitor the first reaction system at a wavelength of 405nm using a multi-well plate reader at room temperature for 20min-40min; or,
[0058] BSA is added to the detection buffer for pre-blocking, and then the PlatelinLS, human coagulation factor FXa, human coagulation factor FVa, human prothrombin, as well as chromogenic substrate and anticoagulant drugs described in the first aspect of the present application are added to obtain a second reaction system; the second reaction system is monitored at a wavelength of 405nm using a multiwell plate reader at room temperature for 20min-40min.
[0059] The anticoagulant drug described in the present application may be a candidate in the development of an anticoagulant drug or an existing anticoagulant drug; its concentration in the reaction system may be adjusted according to actual needs and is not particularly limited in the present application.
[0060] In some embodiments of the present application, the detection buffer comprises: 15mM-24mM HEPES, 140mM-160mM NaCl and 1.5mM-2.5mM Ca 2+ In some embodiments, the assay buffer comprises: 20 mM HEPES, 150 mM NaCl, and 2 mM Ca 2+ .
[0061] In some embodiments of the present application, the amount of BSA used is 0.2 vol%-0.8 vol% of the detection buffer.
[0062] In some embodiments of the present application, in the first reaction system: the concentration of platelets is (0.5-1.5)×10 7 / mL; the concentration of the chromogenic substrate is 0.2mM-0.4mM; the concentration of human coagulation factor FXa is 2nM-8nM; the concentration of human prothrombin is 0.5M-1.5M;
[0063] In the second reaction system: the concentration of the chromogenic substrate is 0.2mM-0.4mM; the concentration of the human coagulation factor FXa is 2nM-8nM; the concentration of the human coagulation factor FVa is 20pM-60pM; the concentration of the human prothrombin is 0.5M-1.5M; the amount of Platelin LS added to every 100μL of the second reaction system is 5μL-15μL.
[0064] The following examples are given to more specifically describe the embodiments of the present application. Various tests and evaluations were performed according to the following methods. In addition, unless otherwise specified, "parts" and "%" are based on mass.
[0065] The amino acid sequence of the anticoagulant protein molecule PA11 used in the following examples is: NLIQFGNMIQCANKGSRPSLDYADYGCYCGWGGSGTPVDELDRCCQVHDNCYEQAGKKGCFPKLTLYSWKCTGNVPTCNSKPGCKSFVCACDAAAAKCFAKAPYKKENYNIDTKKRCK (SEQ ID NO.1). The purification method of PA11 comprises the following steps: dissolving the lyophilized crude venom of Australian P. australis snake venom (about 1.5 g) in double distilled water, removing the insoluble matter by centrifugation, and removing the supernatant. Take a heparin 6B agarose column (5×18 cm), after balancing with two column volumes of 0.05M ammonium acetate buffer (pH 6.5), add the snake venom supernatant to the column, and elute the unbound protein using the same buffer. The bound components are eluted and separated by using a linear gradient of NaCl (0 to 0.5M). The eluted components were separated using YM3 membrane The protein was concentrated in a 500 mL concentration pool (Merck Millipore). A CM-6B agarose column (2×18 cm) was then prepared and balanced with 0.05 M ammonium acetate buffer (pH 6.5). The concentrated fraction showing the highest anticoagulant activity was added to the column and the protein was eluted with 0.5 M NaCl buffer at a flow rate of 1 mL / min. All purification steps were monitored by absorbance at 280 nm and all steps were performed at 4°C. Among the eluted fractions from the CM-6B agarose column, the main fraction with the highest anticoagulant activity was PA11, which was then eluted using a YM3 membrane. The PA11 was concentrated in a 500 mL concentration cell (Merck Millipore) to obtain about 5 mg / mL.
[0066] Example 1
[0067] Blood from healthy volunteers was collected in sodium citrate anticoagulation tubes (containing 3.8wt% sodium citrate solution, the volume ratio of sodium citrate solution to freshly collected human blood was 1:9), a total of four tubes, and then centrifuged at 100×g for 15 minutes to obtain platelet-rich plasma (PRP). The four tubes of PRP obtained were combined to obtain about 10 mL of PRP.
[0068] About 10 mL of PRP was loaded into a G10 sephadex chromatography column (1.7 cm × 10 cm) pre-equilibrated with Tyrodes buffer (pH 7.4, containing: 10 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 2.9 mM NaH2PO4, 5.6 mM glucose and 0.35 vol% BSA), and the G10 sephadex chromatography column was washed with the same buffer at room temperature, and all the washing solutions were collected in 1.5 mL plastic microtubes.
[0069] The turbid platelet-rich tube containing ADP (200 μM) was tested using a platelet aggregometer to confirm that the platelets were active. The active platelets were pooled and then centrifuged at 90×g for 15 minutes.
[0070] The platelets obtained by centrifugation were resuspended in Tyrodes buffer containing 0.02 U / mL apyrase (purchased from Sigma-Alrich) and the platelet count was 1×10 8 / mL, and washed human plasma platelets were obtained.
[0071] Add BSA to the assay buffer (containing: 20 mM HEPES, 150 mM NaCl and 2 mM Ca 2+ ), the amount of BSA used was 0.5 vol% of the detection buffer, and pre-blocked at 4°C overnight. Then 10 μL of washed human platelets (final platelet count 1×10 7 / mL), human coagulation factor FXa (purchased from Calbiochem, the final concentration in the reaction system is 5nM), chromogenic substrate S2238 (purchased from Chromogenix, Stockholm, Sweden, the final concentration in the reaction system is 0.3mM), different concentrations of PA11 (the concentrations are 0nM, 2.5nM, 6.2nM, 12.4nM, 31nM, 62nM, 124nM, 248nM and 496nM) and human prothrombin (purchased from Haematologic Technologies Inc.USA, the final concentration in the reaction system is 1M), and a reaction system with a final volume of 100μL is obtained. The reaction system was monitored at a wavelength of 405nm for 30 minutes at room temperature using a multiwell plate reader to monitor the generation of thrombin. The results are shown in FIG. Figure 2 shown. Figure 2 The graph shows the pNA (peptide nucleic acid) generation progress curve of the prothrombinase complex in the presence of different concentrations of PA11, where the "cross" line represents the actual pNA concentration measured experimentally, and the solid line represents the fitted pNA concentration; the concentrations of the anticoagulant PA11 from top to bottom are 0nM, 2.5nM, 6.2nM, 12.4nM, 31nM, 62nM, 124nM, 248nM and 496nM. Figure 2 It can be seen that in the presence of different concentrations of PA11, the pNA (peptide nucleic acid) generation rate is not the same, indicating the successful combination of the prothrombinase complex in human proplatelets, and showing that PA11 has high activity in inhibiting the prothrombinase complex.
[0072] Example 2
[0073] Add BSA to the assay buffer (containing: 20 mM HEPES, 150 mM NaCl and 2 mM Ca 2+ ), the amount of BSA used was 0.5 vol% of the detection buffer, and pre-blocking was performed at 4°C overnight. Then, 10 μL of Platelin LS (purchased from Organon Teknika Corporation LLC, Munich, Germany), human coagulation factor FVa (purchased from Haematologic Technologies Inc. USA, the final concentration in the reaction system is 40 pM), human coagulation factor FXa (purchased from Calbiochem, the final concentration in the reaction system is 5 nM), chromogenic substrate S2238 (purchased from Chromogenix, Stockholm, Sweden, the final concentration in the reaction system is 0.3 mM), different concentrations of PA11 (the concentrations are 0 nM, 2.5 nM, 6.2 nM, 12.4 nM, 31 nM, 62 nM, 124 nM, 248 nM and 496 nM respectively) and human prothrombin (purchased from Haematologic Technologies Inc. USA, the final concentration in the reaction system is 1 M) were added to obtain a reaction system with a final volume of 100 μL. The generation of thrombin was then monitored at 405 nm using a multiwell plate reader at room temperature for 30 minutes. Figure 3 shown. Figure 3 The figure shows the progress curve of pNA (peptide nucleic acid) generation on 10 μL Platelin LS surface and at different concentrations of PA11, where the "cross" line represents the actual pNA concentration measured experimentally, and the solid line represents the fitted pNA concentration. Figure 3It can be seen that in the presence of the same concentration of PA11, the pNA generation rate is roughly the same, but it shows that PA11 has no obvious inhibitory ability on the prothrombinase complex formed on the surface of PlatelinLS.
[0074] Figure 2 and Figure 3 By comparison, the prothrombinase complex formed on human platelets has advantages in detecting FV ability and screening anticoagulant drugs.
[0075] The prothrombinase complex provided in the present application can monitor the activity of FV and FXa, which can be used to confirm AiFVD cases and monitor the hemostasis status of patients after treatment; or to monitor the effectiveness of platelet transfusion products; or to screen anticoagulant drugs from candidates.
[0076] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present application shall be included in the scope of protection of the present application.
Claims
1. A prothrombinase complex, wherein: The prothrombinase complex comprises: washed human plasma platelets, human coagulation factor FXa and human prothrombin; or The prothrombinase complex comprises: Platelin LS, human coagulation factor FXa, human coagulation factor FVa and human prothrombin.
2. The prothrombinase complex according to claim 1, wherein The concentration of platelets in the washed human plasma platelets is (0.5-1.5)×10 8 Pieces / mL; The solvent in the washed human plasma platelets includes Tyrodes buffer containing ATPase; the concentration of ATPase is 0.01U / mL-0.03U / mL.
3. The prothrombinase complex according to claim 1 or 2, wherein The method for preparing washed human plasma platelets comprises the steps of: Step 1: Place freshly collected human blood in a sodium citrate anticoagulation tube and centrifuge to obtain platelet-rich plasma; Step 2: adding the platelet-rich plasma to a G10 sephadex chromatography column pre-equilibrated with a Tyrodes buffer, washing the G10 sephadex chromatography column with the Tyrodes buffer, and collecting the washing solution; Step 3: The washing solution is centrifuged and then resuspended in a Tyrodes buffer containing ATPase to obtain the washed human plasma platelets.
4. The prothrombinase complex according to claim 3, wherein The step 2 further includes a step of confirming that the platelets are active, wherein the step of confirming that the platelets are active includes: using a platelet aggregometer to test the turbid platelet-rich tube containing ADP and the washing solution; Preferably, the concentration of ADP is 150 μM-250 μM.
5. A kit comprising the prothrombinase complex according to any one of claims 1 to 4.
6. The kit according to claim 5, wherein The kit further comprises a chromogenic substrate; preferably, the chromogenic substrate comprises at least one of the chromogenic substrate S2238 and the chromogenic substrate S2765.
7. Use of the prothrombinase complex according to any one of claims 1 to 4 or the kit according to claim 5 or 6 in screening anticoagulant drugs or monitoring the effectiveness of platelet transfusion products.
8. A method for detecting a prothrombinase complex, wherein: Includes steps: BSA is added to the detection buffer for pre-blocking, and then washed human plasma platelets, human coagulation factor FXa, human prothrombin, and a chromogenic substrate and an anticoagulant drug according to any one of claims 1 to 4 are added to obtain a first reaction system; the first reaction system is monitored at a wavelength of 405 nm using a multi-well plate reader at room temperature for 20 min to 40 min; or, BSA is added to the detection buffer for pre-blocking, and then the Platelin LS, human coagulation factor FXa, human coagulation factor FVa, human prothrombin, as well as a chromogenic substrate and an anticoagulant drug according to claim 1 are added to obtain a second reaction system; the second reaction system is monitored at a wavelength of 405 nm using a multiwell plate reader at room temperature for 20 minutes to 40 minutes.
9. The detection method according to claim 8, wherein: The detection buffer includes: 15mM-24mM HEPES, 140mM-160mM NaCl and 1.5mM-2.5mM Ca 2+ ; Preferably, the amount of BSA used is 0.2 vol%-0.8 vol% of the detection buffer.
10. The detection method according to claim 8 or 9, wherein: In the first reaction system: the concentration of platelets is (0.5-1.5)×10 7 / mL; the concentration of the chromogenic substrate is 0.2mM-0.4mM; the concentration of the human coagulation factor FXa is 2nM-8nM; the concentration of the human prothrombin is 0.5M-1.5M; In the second reaction system: the concentration of the chromogenic substrate is 0.2mM-0.4mM; the concentration of the human coagulation factor FXa is 2nM-8nM; the concentration of the human coagulation factor FVa is 20pM-60pM; the concentration of the human prothrombin is 0.5M-1.5M; the amount of Platelin LS added to every 100μL of the second reaction system is 5μL-15μL.