PB1043 - Complementary roles of red blood cells and platelets in high-throughput whole-blood thrombin generation

Tuesday, July 12, 2022

6:30 PM – 7:30 PM

Location: Exhibition

Presentation Format: In-Person

Presenting Author(s)

Siyu Sun, MS

PhD student
Synapse rsearch institute
MAASTRICHT, Limburg, Netherlands

Co-Author(s)

JZ

Jinmi Zou

PhD
Synapse research insitute
MAASTRICHT, Limburg, Netherlands
JK

Joke Konings, PhD

Senior Scientist
Synapse Research Institute
Maastricht, Limburg, Netherlands
Dana Huskens, PhD

PhD, Research Project Leader
Synapse research insitute
MAASTRICHT, Limburg, Netherlands
JW

Jun Wan

PhD
Blood Research Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA
Chapel Hill, North Carolina, United States
CR

Chris Reutelingsperger

Professor, PhD
Maastricht university
MAASTRICHT, Limburg, Netherlands
Hugo ten Cate, MD, PhD, FAHA

Professor, MD, PhD
Thrombosis Expertise Center, Heart & Vascular Center, Maastricht University Medical Center, Maastricht, the Netherlands
Maastricht, Limburg, Netherlands
PG

Philip G. de Groot

Emeritus Professor
Department of Functional Coagulation, Synapse Research Institute, Maastricht, the Netherlands
MAASTRICHT, Limburg, Netherlands
Bd

Bas de Laat, MD, PhD

General Director
Department of Functional Coagulation, Synapse Research Institute, Maastricht, the Netherlands
Maastricht, Limburg, Netherlands
JW

Johan W. M. Heemskerk

Emeritus Professor
Synapse research insitute
MAASTRICHT, Limburg, Netherlands
Mark Roest, PhD

Clinical Director
Department of Platelet pathophysiology, Synapse Research Institute, Maastricht, the Netherlands
Maastricht, Limburg, Netherlands

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.