High FVIII concentrations interfere with GPVI-mediated platelet activation in vitro.

BACKGROUND: The recruitment of activated FVIII at the surface of activated platelets is a key step towards the burst of thrombin and fibrin generation during thrombus formation at the site of vascular injury. It involves binding to phosphatidyl-serine (PS) and, possibly, to fibrin bound to αIIbβ3. Seminal work had shown the binding of FVIII to resting platelets, yet without a clear understanding of a putative physiological relevance.

OBJECTIVES: To characterize the FVIII-platelet interaction and its potential modulation on platelet function.

METHODS: FVIII was incubated with washed platelets. The effects on platelet activation (spontaneously or triggered by collagen and thrombin) were studied by flow cytometry and light transmission aggregometry. We explored the involvement of downstream pathways by studying phosphorylation profiles (western blot). The FVIII-GPVI interaction was investigated by ELISA, confocal microscopy and proximity ligation assay.

RESULTS: FVIII bound to the surface of resting and activated platelets in a dose-dependent manner. FVIII at supra-physiological concentrations did not induce platelet activation but rather specifically inhibit collagen-induced platelet aggregation and altered GPVI-dependent phosphorylation. FVIII rid of its chaperon protein, von Willebrand factor (VWF), interacted in close proximity with GPVI at the platelet surface.

CONCLUSIONS: We showed that VWF-free FVIII binding to, or close to, GPVI modulates platelet activation in vitro. This may represent a yet uncharacterized negative feedback loop to control overt platelet activation. Whether local activated FVIII concentrations achieved during platelet accumulation and thrombus formation at the site vascular injury in vivo are compatible with such a function remains to be determined.