Dynamic changes in the interaction between αIIbβ3 and α-actinin in platelets. Washed human platelets were stimulated with PAR1-AP (25μM) or PAR4-AP (150μM) under nonstirring conditions for the time indicated. (A) αIIbβ3 was immunoprecipitated from lysates prepared from human platelets stimulated with PAR1-AP. Immunoprecipitates were then subjected to SDS–polyacrylamide gel electrophoresis (PAGE) and immunoblotted with anti–α-actinin antibody. Immuno-blots were stripped and reprobed with anti-αIIbβ3 antibody. Preimmunoprecipitated lysates were also subjected to SDS-PAGE and immunoblotted with the same series of antibodies. (B) 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. PAC-1 binding/30 seconds was normalized for integrin expression levels. (C) Phycoerythrin-conjugated anti-CD62P was added to the activated platelets and incubated for only 30 seconds to evaluate granule secretion. (D) Intracellular calcium mobilization was assessed by monitoring Fluo3-AM fluorescence by flow cytometry. (E) αIIbβ3 was immunoprecipitated then immunoblotted with anti–α-actinin antibody. Immuno-blots shown are representative of 3 different experiments and analyzed by scanning densitometry and quantified with ImageJ (National Institutes of Health).