Factor Xa-driven thrombin generation in plasma: Dependency on the aminophospholipid density of membranes and inhibition by phospholipid-binding proteins

Simone J. H. Wielders; Lisette Ungethüm; Chris P. M. Reutelingsperger; Edouard M. Bevers; Theo Lindhout

doi:10.1160/TH07-05-0322

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2007; 98(05): 1056-1062
DOI: 10.1160/TH07-05-0322

Blood Coagulation, Fibrinolysis and Cellular Haemostasis

Schattauer GmbH

Factor Xa-driven thrombin generation in plasma: Dependency on the aminophospholipid density of membranes and inhibition by phospholipid-binding proteins

Simone J. H. Wielders

¹Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, The Netherlands

,

Lisette Ungethüm

¹Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, The Netherlands

,

Chris P. M. Reutelingsperger

¹Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, The Netherlands

,

Edouard M. Bevers

¹Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, The Netherlands

,

Theo Lindhout

¹Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, The Netherlands

› Author Affiliations

Abstract

Summary

Phosphatidylserine (PS) externalization of activated platelets plays a pivotal role in haemostasis and thrombosis. In the present study we have explored the relationship between the PS density of membranes and the rate of thrombin generation in plasma. Factor (F)Xa-initiated thrombin generation was measured in platelet-free plasma (PFP) containing either phospholipid vesicles of varying PS-content or non-stimulated platelets (reconstituted PRP). The duration of the initiation phase of FXa-driven thrombin generation decreased dramatically with increasing PS density. Concomitantly, the maximal rate of thrombin generation during the propagation phase (maxR) increased non-linearly, with the steepest incline between 5 and 10 mol% PS. Titration of FVa into plasma containing 2 mol% PS increased maxR proportionally and diminished the lag phase. In contrast, platelet-dependent thrombin generation was not influenced by addition of FVa. With increasing platelet concentration, the duration of the initiation phase drastically decreased, and maxR increased proportionally. At a physiologically relevant platelet concentration, maxR corresponded with the maxR found with 2 µM of 10 mol% PS. Annexin A5 (AnxA5) and lactadherin appeared to be powerful inhibitors of in-situ thrombin generation under all conditions examined, with AnxA5 being three- to fourfold more potent than lactadherin. In conclusion, maximal thrombin generation in plasma requires membranes with a density of 10–20 mol% PS. Our data further indicate that thrombin formed in situ induces externalization of PS to approx 10 mol% in a substantial platelet subpopulation.

Keywords

Platelet physiology - thrombin - phospholipids - coagulation factors - annexins

Full Text

References

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