Thrombin receptor occupancy modulates aggregation efficiency and platelet surface expression of vWF and thrombospondin at low thrombin concentrations

Previous studies evaluating requirements for occupancy of thrombin receptor s in normal platelet secretion and aggregation, using the thrombin antagoni sts hirudin and PPACK (D-Phe-Pro-Arg-chloromethyketone), have suggested tha t at low thrombin activating concentrations (0.025-0.13 U/ml), occupancy wa s required only in the first 45-60 s following activation. In our study, we differentiate between thrombin receptor occupancy requirements for surface expression of secreted adhesive proteins, for activation of GPIIb-IIIa rec eptors, and for aggregation of washed platelets (WP) in laminar shear flow. Platelets activated with 0.05 U/ml thrombin for 10 min to allow maximal se cretion (hereafter referred to as "pre-activated platelets"), then sheared, showed a 50-70% decrease in platelet counts after 60 s of shear. Treatment of pre-activated platelets with hirudin or PPACK produced a 65% reduction of capture efficiencies, or, (reflecting experimental/theoretical initial r ates of aggregation), as well as a 30-40% decrease in the surface expressio n of von Willebrand factor (VWF) and thrombospondin (TSP). However, a-granu le membrane P-selectin expression and numbers of activated GPIIb-IIIa recep tors were comparable for treated and non-treated platelets. No significant difference in any of the parameters tested was observed when platelets were similarly pre-activated with 0.2 U/ml thrombin, due to treatment with thro mbin antagonists. Binding of soluble FITC-vWF (GRGDSP-sensitive) to pre-act ivated, thrombin antagonist treated platelets, was greatly reduced (greater than or equal to 80%). Soluble Fg was shown to bind to antagonist-treated pre-activated platelets, but could not significantly enhance platelet aggre gation Although occupancy of thrombin receptors by catalytically active thr ombin is required transiently for secretion and activation of platelets, th ere is a further requirement for thrombin occupancy at low thrombin concent rations, far optimizing initial rates of platelet a,aggregation, surface ex pression of vWF and TSP, and activated GPIIb-IIIa ligand recognition.