We have studied the mechanism of interaction between soluble von Willebrand factor (vWF), labeled with fluorescein isothiocyanate (FITC), and platelets exposed to shear in a cone-and-plate viscometer. A flow cytometer calibrated with fluorescent bead standards was used to calculate the number of molecules associated with each platelet in suspension. To validate the methods and reagents used, binding of the same labeled vWF was assessed in the presence of ristocetin or ␣-thrombin and found to be saturable, with a narrow and symmetric distribution on >90% of the platelets. As expected, essentially all bound ligand interacted exclusively with platelet membrane glycoprotein (GP) Ib␣ in the presence of ristocetin and with GP IIb-IIIa after stimulation with ␣-thrombin. In contrast, only a minor proportion (<20%) of the platelets exposed to shear were found to bind vWF, with no evidence for saturation and markedly decreased interaction when the platelet count was below 100,000 l. Moreover, shear-induced vWF binding was blocked equally effectively by selected monoclonal antibodies against either GP Ib␣ or GP IIb-IIIa or against the respective binding sites in vWF. Thus, both receptors are involved in the process, possibly through initial transient interactions mediated by GP Ib␣ that lead to platelet activation and subsequent irreversible binding supported by GP IIb-IIIa. While the levels of shear stress theoretically applied to platelets in these experiments are above those thought to occur in the normal circulation, our findings demonstrate a unique vWF binding mechanism that is not mimicked by other known modulators and correlates with platelet aggregation. Similar processes may occur in response to lower shear stress when platelets are exposed to thrombogenic surfaces and agonists generated at sites of vascular injury during thrombus formation.
The role of von Willebrand factor (vWF)1 in platelet thrombus formation, particularly under conditions of high shear, is well established and supported by experimental evidence as well as clinical observations (1). It is known that platelets have two distinct binding sites for vWF, glycoprotein (GP) Ib␣ in the GP Ib-IX-V complex and the integrin ␣ IIb  3 (GP IIb-IIIa complex) (2). Binding of soluble normal vWF to GP Ib␣ in the absence of flow requires the presence of exogenous modulators, like ristocetin (3) or botrocetin (4), while interaction with GP IIb-IIIa can only occur after platelet activation (5). In addition to vWF, the GP IIb-IIIa receptor can also bind fibrinogen, fibronectin (6), and vitronectin (7), but the respective role of these proteins in thrombogenesis is still a topic for investigation.Exposure of platelets to levels of shear higher than thought to occur in the normal circulation results in aggregation if soluble vWF is present (8); no other adhesive ligand can support this process (9). Shear-induced platelet aggregation may represent an important pathophysiological function of vWF, but the underlying mechanism is still poorly understood. It has been pro...