Fibronectin Is Not the Only Important Molecule Required for Fibrinogen/VWF-Independent Platelet Aggregation: Study of Thrombosis in a New Strain of Triple Deficient Mice.

Abstract

Fibrinogen (Fg) has been considered essential for platelet aggregation. We demonstrated, however, that thrombi do form in Fg-deficient mice and in mice doubly deficient for both fibrinogen and von Willebrand factor (Fg/VWF−/−). We further reported that β3 integrin and thrombin are critical for this Fg/VWF-independent platelet aggregation. In Fg−/− or Fg/VWF−/− mice, platelet fibronectin (Fn) content is increased 3–5 fold. Furthermore, thrombus growth and stability are impaired in plasma Fn conditional deficient (M×-Cre, Fnflox/flox) mice. These data are consistent with the most recent studies of Fn assembly and suggest that Fn may support platelet thrombus formation. To examine whether Fn is the alternative key molecule which mediates platelet aggregation and thrombus formation in Fg/VWF−/− mice, we developed a novel strain of triple knockout (TKO) mice by breeding Fg/VWF−/− mice with M×-Cre+/− Fnflox/flox conditional knockout mice. Cre- littermates delivered from the same parents were used as a control. Fn depletion was induced by i.p. injections of polyinonic-polycytidylic acid. We found that TKO mice are viable with dramatically decreased levels of Fn in both the plasma (<2% of control) and platelets (<20% of control) as determined by immunoblot. No significant difference was found in peripheral blood cell counts or platelet surface adhesion proteins (GPIbα, P-selectin, β3 and β1 integrins) as compared with control mice. Unexpectedly, TKO platelet aggregation induced by thrombin and thrombin receptor activation peptide (TRAP) was enhanced in both platelet rich plasma and PIPES buffer (P<0.05). This phenomenon was also observed in a parallel-plate flow chamber. Significantly more TKO aggregates formed when fluorescently labelled whole blood was perfused over a collagen surface at 500s−1 (P<0.001). We also studied thrombus formation in TKO and Cre- control mice using intravital microscopy. Our preliminary data showed no obvious decrease in thrombus formation in TKO mice and injured arterioles occluded in one of two experimental mice. Our data suggest that Fn is not the only important molecule required for platelet aggregation and thrombus formation in Fg/VWF−/− mice. Further investigation of how Fn may inhibit Fg/VWF-independent platelet aggregation and the identification of what ligand(s) support this novel aggregation pathway may unveil an alternative haemostatic pathway in the absence of Fg and VWF.