title: Factor Xa-driven thrombin generation in plasma: dependency on the aminophospholipid density of membranes and inhibition by phospholipid-binding proteins.
abstract: 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 microM 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 four-fold 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.
authors.affiliation: Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, P.O. Box 616, 6200 MD Maastricht, The Netherlands.
authors.forename: Simone J H
authors.initials: SJ
authors.lastname: Wielders
authors.forename: Lisette
authors.initials: L
authors.lastname: Ungethüm
authors.forename: Chris P M
authors.initials: CP
authors.lastname: Reutelingsperger
authors.forename: Edouard M
authors.initials: EM
authors.lastname: Bevers
authors.forename: Theo
authors.initials: T
authors.lastname: Lindhout
mesh_terms: D000818:Animals; D017304:Annexin A5 / Q000494:pharmacology; D000954:Antigens, Surface / Q000494:pharmacology; D001792:Blood Platelets / Q000502:physiology; D002417:Cattle; D015943:Factor Va / Q000494:pharmacology; D015951:Factor Xa / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008894:Milk Proteins / Q000494:pharmacology; D010718:Phosphatidylserines / Q000032:analysis* / Q000502:physiology; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D053835:Unilamellar Liposomes / Q000737:chemistry*
chemical_list: D017304:Annexin A5; D000954:Antigens, Surface; C098942:MFGE8 protein, human; D008894:Milk Proteins; D010718:Phosphatidylserines; D053835:Unilamellar Liposomes; D015943:Factor Va; D013917:Thrombin; D015951:Factor Xa