It is widely accepted that shear stress activates platelets. However, this may have two linked but separate causes: a direct effect of shear stress on individual platelets, and secondary inter-platelet activation dependent on the release of agonists caused by shear. Gel-filtered platelets were exposed to intermittent low shear at 20,000 and 200,000 platelets/microl and their activation was measured with a prothrombinase-based assay. At the lower count, activation was slow and essentially linear, but at the higher count, it rose exponentially with time, leading to 3-fold more prothrombinase activity. Inclusion of apyrase and/or prostaglandin I(2) slightly reduced activation at high platelet counts, but did not abolish the nonlinear kinetics, and antibodies against von Willebrand factor had no significant effect. The contributions of anionic phospholipid and factor Va to the prothrombinase activity were assessed by measurements in the presence of exogenous factor Va. The results strongly suggest that anionic phospholipid appearance is caused directly by shear exposure, but that factor Va release from the alpha-granules is a secondary event and largely the result of platelet-platelet signaling.