Abstract
Factor VIII is a cofactor in the tenase enzyme complex which assembles on the membrane of activated platelets. A critical step in tenase assembly is membrane binding of factor VIII. Platelet membrane factor VIII-binding sites were characterized by flow cytometry using either fluorescein maleimide-labeled recombinant factor VIII or a fluorescein-labeled monoclonal antibody against factor VIII. Following activation by thrombin, most platelets bound factor VIII within 90 s. In addition, over the course of several minutes, membranous vesicles (microparticles) were shed from the platelet plasma membrane and each microparticle bound as much factor VIII as a stimulated platelet. Over 30 min, stimulated platelets (but not microparticles) lost the capacity to bind factor VIII. Factor VIII bound saturably to microparticles from platelets stimulated with thrombin, thrombin plus collagen, or the complement proteins C5b-9. The binding of factor VIII was compared to factor V, a structurally homologous coagulation cofactor. Analysis of microparticle binding kinetics yielded similar on and off rates for factor VIII and factor Va and KD values of 2-10 nM. In the presence of 20 nM factor Va, the binding of factor VIII to microparticles was increased, and there was a comparable increase in platelet tenase activity. At higher factor Va concentrations, factor VIII binding and tenase activity were inhibited. Conversely, factor VIII had a similar dose-dependent effect on factor Va binding and platelet prothrombinase activity. Synthetic phospholipid vesicles containing phosphatidylserine competed with microparticles for binding of factor VIII and factor Va. These studies indicate that activated platelets express a transient increase in high affinity receptors for factor VIII, whereas platelet-derived microparticles express a sustained increase in receptors. The binding characteristics of platelet membrane receptors for factor VIII are similar to those for factor Va.
Highlights
To examine the time courseof the loss of factor VIII-binding sites from platelet membranes, DiIC16(3)-labeled platelewtsere diluted to 4 X 107/mlin minimum essential medium, 0.1% bovine albumin, 0.02 M HEPES, pH 7.4, rather than solution 11
Lets and microparticleswere incubated with preformed antibody-factor VI11 complexes, as described under "Experimental Procedures." The data were fitted to a pseudo-first order binding model (Fig. 5A, inset) andyielded an association rate constant, k l, of2-4 x lo6 M" min" (TableI).Thisrate constant was similar to that obtained when the binding of factor Va was measured usingFITC-labeled V237 fluorescence (Table I).After ligandbinding hadreached equilibrium, the rates of dissociation of factor VI11 and factor Va from microparticles were estimated by measuring the decrease in particle fluorescence following a 100-folddilution of the samples (Fig. 5B).For factorVIII, the first ordedr issociation rate constant, kz,was 2 x lo-' min"
This study demonstrates that within 1 min after platelet stimulation by thrombin,factorVIII-bindingsitesareexpressed on the platelet plasma membrane and that with9i0n s plasma membrane vesicles are shed which have a high density of these sites
Summary
To examine the time courseof the loss of factor VIII-binding sites from platelet membranes, DiIC16(3)-labeled platelewtsere diluted to 4 X 107/mlin minimum essential medium, 0.1% bovine albumin, 0.02 M HEPES, pH 7.4, rather than solution 11. 80 min of incubation of activated platelets and microparticles with factorVI11 (15 nM)-FITC 1B3 was followed by a rapid 100-fold dilution and serial aliquot of platelets was removed fromthe incubation mixture, fluorescence measurements were obtained (lower panel).
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