VWF activates PLC and αIIbβ3 through a Src kinase–dependent pathway. (Ai) Human platelet-rich plasma (3 × 10⁸ platelets/mL) and (Aii-iii) human washed platelets (3 × 10⁸ platelets/mL) were preincubated for 5 minutes with vehicle (dimethyl sulfoxide [DMSO] 0.1%), the αIIbβ3 antagonist, lotrafiban (10 μM), or the Src family kinase inhibitor PD173952 (25 μM) followed by stimulation with (i) ristocetin (Rist; 1.5 mg/mL), (ii) VWF-ristocetin (VWF-Rist; 10 μg/mL and 1 mg/mL) or (iii) thrombin (Thr; 0.05 IU/mL). Experiments were conducted in aggregometer cuvettes with stirring at 37°C. Arrows indicate addition of agonist. 100% represents maximal light transmission. (B) Human washed platelets loaded with [³²P]orthophosphoric acid (0.5 mCi/mL [18.5 MBq/mL]) were resuspended in plasma (1 × 10⁹ platelets/mL) and preincubated for 5 minutes with vehicle (DMSO 0.1%), the αIIbβ3 antagonist, lotrafiban (10 μM), or the Src kinase inhibitor, PP2 (10 μM), followed by stimulation with ristocetin (1.5 mg/mL) for 5 minutes. Reactions were stopped by addition of an equal volume of chloroform-methanol (1:1, vol/vol). Phospholipids were separated by thin layer chromatography and analyzed by autoradiography. Results are presented as fold increases over basal (mean ± SEM). Results are representative of 3 experiments.