Previous studies have strongly suggested that the initial attachment (adhesion) of platelets to subendothelium or collagen at high shear rates is mediated by the binding of von Willebrand factor (vWf) to platelet glycoprotein Ib (GPIb). The finding in the present study that incubating human umbilical artery subendothelium with a monoclonal antibody to the GPIb binding site of vWf markedly inhibited platelet adhesion is fully consistent with this hypothesis. Platelet adhesion (and thrombus formation) also requires GPIIb-IIIa, and previous studies have suggested that the binding of vWf to GPIIb-IIIa may be involved in this process. To explore this further, we utilized two monoclonal antibodies (152B-20 and 152B-6) that recognize the arg1744 gly1745 asp1746 (RGD) sequence of vWf. These antibodies selectively inhibit vWf binding to GPIIb-IIIa and have no cross-reactivity with other RGD-containing proteins that can bind to GPIIb-IIIa. When added to citrated human blood, these antibodies (152B-20 in particular) inhibited platelet adhesion and thrombus formation on rabbit subendothelium at a shear rate of 2600 sec-1 but not at 400 sec-1. The results of the study thus provide direct evidence that the binding of vWf to GPIIb-IIIa is important for platelet adhesion and thrombus formation on subendothelium at high shear rates and that the arg1744 gly1745 asp1746 sequence in the mature vWf is involved in this process.