Coengagement of PECAM-1 with IV.3 cross-linking leads to a reduction in tyrosine phosphorylation of platelet proteins, induction of tyrosine phosphorylation of PECAM-1, and recruitment of SHP-2 protein-tyrosine phosphatase. (A) Washed platelets were stimulated by FcγRIIa cross-linking with and without coengagement of PECAM-1 over 5 minutes at 37°C with stirring. Immunoblotting of 100 μg platelet whole proteins with horseradish peroxidase (HRP)-conjugated antiphosphotyrosine antibody, RC20, and enhanced chemiluminescence (ECL) development. Arrows indicate proteins with reduced antiphosphotyrosine content. (B, top panel) Washed platelets were stimulated by FcγRIIa cross-linking with and without coengagement of PECAM-1 over a time course of 0 to 5 minutes at 37°C with stirring. Immunoprecipitation of PECAM-1 with monoclonal anti-PECAM-1 antibody (PECAM-1.3) followed by immunoblotting with HRP-conjugated antiphosphotyrosine antibody (RC20) and ECL development. (B, middle panel) Blot was stripped and reprobed for SHP-2 antigen with polyclonal anti-SHP-2 antibody. (B, bottom panel) Blot was stripped and reprobed for PECAM-1 antigen content with polyclonal anti-PECAM-1 (SEW16) antibody.