Abstract Objective To reveal the mechanism of mechanistic regulation of platelet surface CD40L expression mediated by vWF-A1 in flow fluid environment. Methods Selected prokaryotic systems expressing proteins, flow cytometry, and parallel plate flow chamber technique with cellular immunofluorescent antibody staining to investigate platelets' induced activation by vWF-A1 and subsequent expression of CD40L in different shear environments. Results vWF-A1 does not activate platelets under resting conditions; fluid shear is 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 it reached 20 dyn.min/cm2, the expression of platelet CD40L reached its peak. Conclusions The expression of CD40L on platelet surface is regulated by vWF-A1, fluid shear stress, and SSA. SSA promote the ability of platelets to express CD40L.