Exposed subendothelial collagen acts as a substrate for platelet adhesion and thrombus formation after vascular injury. Synthetic collagen-derived triple-helical peptides, designated collagen-related peptide (CRP), GFOGER, and VWF-III, can specifically engage the platelet collagen receptors, glycoprotein VI and integrin alpha(2)beta(1), and plasma von Willebrand factor (VWF), respectively. Hitherto, the role of these 3 collagen-binding axes has been studied indirectly. Use of these uniform peptide substrates, rather than collagen fibers, provides independent control of each axis. Here, we use confocal imaging and novel image analysis techniques to investigate the effects of receptor-ligand engagement on platelet binding and activation during thrombus formation under flow conditions. At low shear (100s(-1) and 300s(-1)), both GFOGER and CRP are required for thrombus formation. At 1000s(-1), a combination of either CRP or GFOGER with VWF-III induces comparable thrombus formation, and VWF-III increases thrombus deposition at all shear rates, being indispensable at 3000s(-1). A combination of CRP and VWF-III is sufficient to support extensive platelet deposition at 3000s(-1), with slight additional effect of GFOGER. Measurement of thrombus height after specific receptor blockade or use of altered proportions of peptides indicates a signaling rather than adhesive role for glycoprotein VI, and primarily adhesive roles for both alpha(2)beta(1) and the VWF axis.