VWF binding to GPIb simultaneously activates PECAM-1 and the FcRγ-chain. Human or murine platelets were stirred in the presence or absence of VWF/botrocetin for the indicated time period, lysed, and subjected to immunoprecipitation/SDS/immunoblot analysis. (A) PECAM-1 immunoprecipitates analyzed for antigen level and tyrosine phosphorylation state demonstrate time-dependent VWF-induced activation of PECAM-1. (B) Quantitation of PECAM-1 tyrosine phosphorylation expressed as a ratio of phosphorylated protein to the amount of PECAM-1 antigen immunoprecipitated. Note that both murine and human PECAM-1 increase in tyrosine phosphorylation as a function of time following addition of VWF/botrocetin. (C) GST-Syk-SH2-SH2 pull-down of tyrosine phosphorylated FcRγ from resting and VWF/botrocetin-stimulated murine platelet lysates. FcRγ-chain tyrosine phosphorylation occurred more quickly in PECAM-1-negative compared with PECAM-1-positive platelets. Equal loading was insured by including loading control (right lane). (D) Scanning densitometric analyses of the gel shown in panel C represent the mean ± SD of data drawn from 3 independent experiments and show that tyrosine phosphorylation of FcRγ is consistently greater, especially at early time points, in VWF/botrocetin-activated platelets derived from PECAM-1 knock-out versus wild-type mice (P = .04 at 2 minutes after stimulation). These data suggest that PECAM-1 regulates GPIb/V/IX signaling by affecting the phosphorylation state of the FcRγ-chain ITAM in murine platelets. ^*P < .05.