A parallel-plate flow chamber and epifluorescence video microscopy with digital image processing techniques were used to investigate the mechanisms of mural thrombus formation and to evaluate new potentially useful antithrombotic agents under controlled physiological flow. We examined the role of the interaction of a plasma multimeric protein, von Willebrand factor (vWF), with the two platelet receptors, glycoprotein (GP) Ib and the GP IIb-IIIa complex, in mediating platelet adhesion and subsequent platelet aggregation on a surface of bovine fibrillar type I collagen exposed to citrated, or heparinized, whole blood with flourescently-labeled platelets at wall shear rates of 100 sec\sp−1 to 3000 s\sp−1. Using blood anticoagulated with citrate, it was found that platelet adhesion under arterial flow conditions was extensively reduced: (a) in the absence of vWF; (b) by blocking platelet GP Ib with a monoclonal antibody or with a recombinant vWF fragment (rvWF\sp445−733) that contains the GP Ib binding domain; or (c) by blocking the GP Ib binding site in vWF with a $>$2.5 kD polymeric form of aurin tricarboxylic acid (ATA). Monoclonal antibodies against either the RGD sequence in vWF (GP IIb-IIIa binding site) or GP IIb-IIIa itself predominantly inhibited platelet aggregation. The inhibitory effect of the recombinant vWF fragment was completely reversed in the presence of therapeutic levels of unfractionated porcine heparin, but not by low molecular weight heparin. Polymeric ATA inhibited platelet adhesion to collagen in the presence of either citrate, unfractionated porcine heparin, or low molecular weight heparin. We conclude that: (a) the vWF/GP Ib interaction is essential for platelet adhesion to collagen; (b) binding of vWF and other ligands to GP IIb-IIIa primarily contribute to subsequent platelet aggregation; and (c) the recombinant vWF fragment and polymeric ATA are potentially useful agents in suppressing platelet adhesion in areas of the arterial circulation where blood vessels are injured and subendothelium is exposed. Our study suggests that the antithrombotic effect of the vWF fragment, but not polymeric ATA, may be compromised by the concurrent administration of unfractionated porcine heparin. We also tested the inhibitory effect of two novel antiplatelet agents, G-110 and G-112, on thrombus formation from heparinized whole blood on collagen-coated surfaces under controlled flow. Our study, together with others on related carbamoylpiperidine compounds, corroborates the nature of pivotal features in their molecular structure which enhance desirable antithrombotic effects.