Journal
JOURNAL OF THROMBOSIS AND HAEMOSTASIS
Volume 2, Issue 10, Pages 1806-1815Publisher
WILEY
DOI: 10.1111/j.1538-7836.2004.00919.x
Keywords
factor VIII; fibrin; GPIIbIIIa; platelets
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Funding
- NHLBI NIH HHS [R01 HL310481, R01 HL57867, P01 HL42443] Funding Source: Medline
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Factor (F)VIII functions as an enzymatic cofactor on the membranes of stimulated platelets. However, thrombin stimulates platelets to express only a small number of binding sites for FVIII. We wished to determine whether molecules that are likely to be present in a developing thrombus stimulate platelets to up-regulate FVIII binding site expression. Flow cytometry was utilized to measure binding of fluoresce-inlabeled FVIIIa to activated platelets and a FXase assay was utilized to measure platelet-dependent function. Various agonists as well as normal and mutant fibrinogens and fibrin were evaluated as co-stimuli. Thrombin-stimulated platelets expressed 214 +/- 67 binding sites for thrombin-activated FVIII (FVIIIa) and none of the established soluble agonists enhanced binding site exposure. However, the presence of 5 mug mL(-1) fibrin increased the number of FVIIIa binding sites/platelet three- to eight-fold (1470 +/- 130, range 600-1800) with a parallel increase in platelet-based FXase assay. Binding site upregulation was not stimulated by fibrinogen and was blocked by inhibitors of GPIIbIIIa. Mutant fibrin lacking the gamma-chain C-terminal four residues was ineffective while fibrin with altered RGD sequences did stimulate expression of FVIIIa binding sites indicating that co-stimulation is mediated by the fibrin 7-chain termini. Fibrin-enhanced expression of FVIIIa binding sites was not supported by D364H fibrin, which does not aggregate normally, and was blocked by the GPRP peptide, which inhibits fibrin polymerization. Polymerized fibrin can function as a platelet co-stimulus, up-regulating expression of binding sites for FVIIIa.
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