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Complementary roles of red blood cells and platelets in high-throughput whole-blood thrombin generation

1Maastricht University, MAASTRICHT, Limburg, Netherlands, 2Department of biochemistry, Maastricht university, MAASTRICHT, Limburg, Netherlands, 3Synapse Research Institute, Maastricht, the Netherlands, MAASTRICHT, Limburg, Netherlands, 4 Synapse Research Institute, Maastricht, Limburg, Netherlands, 5Blood Research Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA, Chapel Hill, North Carolina, United States, 6Departments of Biochemistry and Internal Medicine, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, the Netherlands;, Maastricht, Limburg, Netherlands, 7Department of Functional Coagulation, Synapse Research Institute, Maastricht, the Netherlands;, Maastricht, Limburg, Netherlands, 8Department of Functional Coagulation, Synapse Research Institute, Maastricht, the Netherlands, Maastricht, Limburg, Netherlands, 9Department of Biochemistry, CARIM, Maastricht University, Maastricht, the Netherlands; Synapse Research Institute Maastricht, The Netherlands, Maastricht, Limburg, Netherlands, 10Synapse Research Institute, Maastricht, the Netherlands, Maastricht, Limburg, Netherlands

Abstract Number: PB1043

Meeting: ISTH 2022 Congress

Theme: Coagulation and Natural Anticoagulants » Regulation of Coagulation

Background: A whole blood (WB) thrombin generation (TG) method was developed, allowing continuous and high-throughput TG measurements. This method approaches physiology and promises new insights in the interactions of various blood cells and the coagulation process.

Aims: To investigate the contribution of both platelets and red blood cells (RBC) during different phases of clot formation applying WB-TG.

Methods: Application of the automated 96-well plate-based tissue factor (TF) induced WB-TG under conditions blocking or promoting platelet activation and/or RBC eryptosis. Platelet collagen receptors (GPVI) or thrombin receptors (PARs) were antagonized, and procoagulant phosphatidylserine (PS) was blocked with annexin A5. Parallel TG measurements were performed with platelet-rich plasma (PRP).

Results: In TF-triggered WB and PRP, TG parameters were optimal at 6 mM CaCl2 plus 3 mM MgCl2 for WB-TG, and 11 mM CaCl2 plus 5.5 mM MgCl2 for PRP-TG. At ≤1 pM TF, GPVI-induced platelet activation caused a smaller increase in TG peak in WB than in PRP. Higher concentrations of dabigatran or rivaroxaban were needed to inhibit TG in WB than in PRP. Similarly, combined inhibition of PAR1/4 slightly reduced WB-TG, and more potently reduced PRP-TG. In a reconstituted system, prior treatment of RBCs with wildtype annexin A5 (but not mutated annexin A5) downregulated the WB-TG in the initiation phase. Markedly, in WB, dose-dependent addition of annexin A5, first enhanced the GPVI-induced TG and then annulled all TG. Furthermore, annexin A5 delayed the onset of platelet ATP release in WB.

Conclusion(s): In WB, the role of agonist-induced platelet activation on TG is complemented by thrombin-generating RBC, in a manner downregulated by annexin A5. These findings are particularly relevant in RBC-rich clotting in venous thromboembolism.

To cite this abstract in AMA style:

Sun S, zou J, Konings J, Huskens D, Wan J, Reutelingsperger C, ten Cate H, de Groot P, de Laat B, Heemskerk J, Roest M. Complementary roles of red blood cells and platelets in high-throughput whole-blood thrombin generation [abstract]. https://abstracts.isth.org/abstract/complementary-roles-of-red-blood-cells-and-platelets-in-high-throughput-whole-blood-thrombin-generation/. Accessed November 30, 2023.

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