The importance of von Willebrand factor (vWf) in the formation of platelet-fibrin thrombi on expanded polytetrafluoroethylene (ePTFE) surfaces was studied in an in vitro system, perfusing non-anticoagulated human blood over ePTFE grafts for 3 min at varying shear rates (100, 500 and 1500/s shear). Platelet (111In) and fibrin (125I) deposition was assessed on ePTFE surfaces in the presence and relative absence of vWf, achieved by use of polyclonal anti-vWf antibody (anti-vWf Ab). A total of 29 perfusions were performed. Increasing shear rate was associated with greater platelet deposition in the presence of vWf (p < 0.001). This shear-dependent rise in platelet deposition was not observed when vWf was blocked by anti-vWf Ab (P < 0.1), confirming the role of vWf in platelet deposition at high shear rates. Fibrin deposition increased with increasing shear rate in the presence of vWf (P < 0.01). Inhibiting vWf abolished the shear-dependent increase in fibrin deposition. These data suggest that vWf plays a critical role in platelet and fibrin thrombus formation on ePTFE surfaces. These effects are particularly important under conditions of high shear rate. These mechanisms may lead to the observed pathologic thrombus formation and platelet-dependent neointimal processes occurring at areas of high shear rate within the anastomotic regions of ePTFE grafts.