Changes in α-actinin phosphorylation and its interaction with αIIbβ3 in platelets from patients with Glanzmann thrombasthenia or P2Y₁₂ deficiency. (A) Platelet lysates from patients with Glanzmann thrombasthenia or patients with P2Y₁₂ deficiency were subjected to SDS–polyacrylamide gel electrophoresis and immunoblotted with anti-αIIbβ3 antibody or anti–α-actinin antibody. (B) Washed platelets were stimulated with PAR1-AP (25μM) or PAR4-AP (150μM) for the time indicated. Tyrosine-phosphorylated proteins were immunoprecipitated then immunoblotted with anti–α-actinin antibody. (C) Washed normal platelets were stimulated with PAR4-AP (150μM) for the time indicated after incubation with AR-C69931MX (1μM) for 2 minutes. αIIbβ3 was immunoprecipitated then immunoblotted with anti–α-actinin antibody. (D) Washed normal platelets were stimulated with PAR4-AP (150μM) for the time indicated after incubation with AR-C69931MX (1μM) for 2 minutes. 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. Error bars represent SEMs of 3 experiments.