Roles of Src, PI 3-kinase, and PKC in calcium fluxes induced by platelet adhesion to dA1VWF. Platelets were studied in the presence of inhibitors of signaling through αIIbβ3 and ADP and thromboxane A₂ receptors. (A-B) Platelets under static conditions were allowed to adhere to dA1VWF in the presence or absence of (A) Src family kinase inhibitors PP2 (5 μM) or SU-6656 (1 μM), or 5 μM PP3 as a negative control, or (B) 100 nM wortmannin to block PI 3-kinase, wortmannin vehicle control, 12 μM bisindolylmaleimide I (Bis I) to block PKC, or 12 μM bisindolylmaleimide V (Bis V), an inactive congener. Results are expressed arbitrarily as the percent of cells showing intracellular calcium levels more than 2-fold to 10-fold over baseline. (C-D) Shear flow. Platelets in homologous blood were perfused over immobilized multimeric VWF, and (Ca²⁺)i of surface-interacting platelets was monitored as described in “Materials and methods.” Shown are the inhibitory effects of PP2 on the percent of activated platelets (C) and on α and β Ca²⁺ peaks in activated platelets (D). PP3 was used as negative control. Values shown are the mean ± SD of 3 to 4 experiments.