Effects of the blockade of P2Y₁₂ on α-actinin and αIIbβ3. Washed human platelets were incubated with thrombin (0.2 U/mL). The P2Y₁₂ antagonist, AR-C69931MX (1μM), was added after 2 minutes of thrombin stimulation. (A) FITC–PAC-1 was added to the activated platelets after stimulation and incubated for 30 seconds to obtain the PAC-1 binding velocity at the time indicated. (B) αIIbβ3 was immunoprecipitated then immunoblotted with anti–α-actinin antibody. (C) Tyrosine-phosphorylated proteins were immunoprecipitated then immunoblotted with anti–α-actinin antibody. Preimmunoprecipitated lysates were also subjected to SDS–polyacrylamide gel electrophoresis and immunoblotted with anti–phospho-VASP (Ser239) antibody or anti-VASP antibody. Error bars represent SEMs of 3 experiments.