Studies from our laboratory have demonstrated that von Willebrand factor (VWF) stimulates thrombin generation in platelet-rich plasma. The precise role of VWF and fibrin in this reaction, however, remained to be clarified. In the present study we utilized thrombin-free planar fibrin layers and washed platelets to examine the relationship between platelet-fibrin interaction and exposure of coagulation-stimulating phosphatidylserine (PS) under conditions of low and high shear stress. Our study confirms that platelet adhesion to fibrin at a shear rate of 1000 s(-1) requires fibrin-bound VWF. The cytosolic calcium concentration ([Ca(2+)]i) of stationary platelets was not elevated and PS exposing platelets were virtually absent (2 +/- 2%). However, thrombin activation resulted in a marked increase in the number of PS exposing platelets (up to 85 +/- 14%) along with a transient elevation in [Ca(2+)]i from 0.05 micro mol L(-1) up to 1.1 +/- 0.2 micro mol L(-1). Platelet adhesion to fibrin at a shear rate of 50 s(-1) is mediated by thrombin but not by fibrin-bound VWF. The [Ca(2+)]i of these thrombin-activated platelets was elevated (0.2 +/- 0.1 micro mol L(-1)), but only a minority of the platelets (11 +/- 8%) exposed PS. The essential role of VWF in this thrombin-induced procoagulant response became apparent from low shear rate perfusion studies over fibrin that was incubated with VWF and botrocetin. After treatment with thrombin, the majority of the adherent platelets (57 +/- 23%) exposed PS and had peak values of [Ca(2+)]i of about 0.6 micro mol L(-1). Taken together, these results demonstrate that thrombin-induced exposure of PS and high calcium response on fibrin-adherent platelets depends on shear- or botrocetin-induced VWF-platelet interaction.