PECAM-1 Y686 and S702 are inducibly phosphorylated, whereas Y707 is constitutively phosphorylated, in human platelets. (A) Left 2 lanes: Western blot analysis of PECAM-1 immunoprecipitates from HEK 293T transfectants expressing Y663,686F(11S→A)702S or Y663,686F(11S→A)707S forms of PECAM-1, which cannot be recognized by the pY686-specific antibody because of the Y686F substitution, demonstrated that recognition of PECAM-1 by pS702- and pS707-specific antibodies depended on the presence of a serine residue at position 702 or 707, respectively. The presence of PECAM-1 in all PECAM-1 immunoprecipitates was confirmed by binding of a phosphorylation-independent PECAM-1–specific antibody. Right 3 lanes: Representative Western blots illustrate stronger binding of pY686- and pS702-specific antibodies to PECAM-1 immunoprecipitated from thrombin receptor activating peptide (TRAP)–activated or TRAP-aggregated platelets relative to resting platelets. In contrast, pY707-specific antibodies bound equally well to PECAM-1 immunoprecipitated from resting or TRAP-activated or TRAP-aggregated platelets. Immunoprecipitation of equal amounts of PECAM-1 from resting or TRAP-activated or TRAP-aggregated platelets was confirmed by binding of a phosphorylation-independent PECAM-1–specific antibody. (B) Quantitative analysis of PECAM-1 pY686, pS702, and pS707 levels in resting, TRAP-activated, and TRAP-aggregated human platelets. Western blot band intensities were determined by densitometric analysis, and the ratios of phosphorylated PECAM-1 to total PECAM-1 were calculated. For each antibody, ratios were normalized to values observed in resting platelets. The means ± SEM calculated from 3 independent experiments are shown. *P < .05, **P < .01 relative to resting platelets.
