cPLA₂α is constitutively associated with αIIbβ3 in human and mouse platelets. (A,C) Coimmunoprecipitation of αIIbβ3 and cPLA₂α was assayed in washed human platelets treated with 300 μg/mL of fibrinogen + 1 mM of MnCl₂ for 0 to 15 minutes at 37°C as indicated, or with 1 U/mL of thrombin for 5 minutes. Each experiment was performed 3 times with identical results. NSS indicates normal sheep serum. (B) cPLA₂α^−/− or wild-type littermate platelets were incubated in the presence or absence of 300 μg/mL of fibrinogen + 1 mM of MnCl₂ for 5 minutes at 37°C. Then αIIbβ3 immunoprecipitates were examined by Western blotting for the presence of cPLA₂α. NRS indicates normal rabbit serum. This experiment was performed twice. (D,E) cPLA₂α and vimentin coimmunoprecipitate with αIIbβ3. Washed human platelets were incubated in the presence or absence of 300 μg/mL of fibrinogen + 1 mM of MnCl₂ for 10 minutes at room temperature. Platelets were lysed in NP-40 lysis buffer, and lysates were immunoprecipitated with an antibody to cPLA₂α (D) or β3 (E). Immunoblots were probed with an antibody to vimentin, cPLA₂α, or β3. This experiment was performed twice.