Shear stress induces large VWF multimers to bind platelets. Reactions were performed as indicated with (+) or without (−) VWF (30 μg/mL), formalin-fixed platelets (106/μL), and fluid shear stress (16 dyne/cm2, 10 minutes). Samples were diluted with an equal volume of reaction buffer and centrifuged for 5 minutes at 6400g. The platelet pellets (or any other pelleted material for reactions without platelets) were washed 3 times by resuspension in reaction buffer and centrifugation. The washed platelets or other contents were dispersed in Laemmli sample buffer, heated at 80°C for 5 minutes, and centrifuged, and supernatant corresponding to 20 μL of the initial reaction volume was analyzed by Western blotting after 1.5% SDS-agarose gel electrophoresis. The 20 ng of input VWF analyzed for reference corresponds to approximately 3% of the VWF initially present in the samples of reactions analyzed in lanes 1 to 4.
