bjective To reveal the mechanism of mechanical regulation of platelet surface CD40L expression mediated by vWF-A1 in flow filed. Methods The prokaryotic systems expressing proteins, flow cytometry, and parallel plate flow chamber technique with cellular immunofluorescent antibody staining were selected, so as to investigate the activation and subsequent expression of CD40L induced by vWF-A1 in platelets under different shear environments. Results The vWF-A1 did not activate platelets under resting conditions. Fluid shear stress (FSS) was the initiating switch for vWF-A1-mediated platelet CD40L expression. With the increase of shear stress accumulation (SSA), the expression level of platelet CD40L showed a trend of increasing and then decreasing, and when SSA reached 2 Pa·min, the expression of platelet CD40L reached its peak. Conclusions The expression of CD40L on platelet surface is regulated by vWF-A1, FSS and SSA. SSA promote the ability of platelets to express CD40L.