Wheat germ agglutinin-induced platelet activation via platelet endothelialcell adhesion molecule-1: Involvement of rapid phospholipase C gamma 2 activation by Src family kinases

Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) is a 130K tran smembrane glycoprotein that belongs to the immunoglobulin gene superfamily and is expressed on the surface of hematological or vascular cells, includi ng platelets and endothelial cells. Although the importance of this adhesio n molecule in various cell-cell interactions is established, its function i n platelets remains ill-defined. In the process of clarifying them mechanis m by which the lectin wheat germ agglutinin (WGA) activates platelets, we u nexpectedly discovered that PECAM-1 is involved in signal transduction path ways elicited by this N-acetyl-D-glucosamine (NAGlu)-reactive lectin. WGA, which is a very potent platelet stimulator, elicited a rapid surge in Syk a nd phospholipase C (PLC)-gamma2 tyrosine phosphorylation and the resultant intracellular Ca2+ mobilization; collagen, as reported, induced these respo nses, but in a much slower and weaker manner. WGA strongly induced tyrosine phosphorylation of a 130-140K protein, which was confirmed to, be PECAM-1 by immunoprecipitation and immunodepletion studies. WGA-induced PECAM-1 tyr osine phosphorylation occurred rapidly, strongly and in a manner independen t of platelet aggregation or cell-cell contact; these characteristics of PE CAM-1 phosphorylation were not mimicked at all by receptor-mediated platele t agonists. In addition, WGA was found to associated with PECAM-1 itself, a nd anti-PECAM-1 antibody, as well as NAGlu, specifically inhibited WGA-indu ced platelet aggregation. In PECAM-1 immunoprecipitates, Src family tyrosin e kinases existed, and a kinase activity was detected, which increased upon WGA stimulation. Furthermore, the Src family kinase inhibitor PP2 inhibite d WGA-induced platelet aggregation, Ca2+ mobilization, and PLC-gamma2 tyros ine phosphorylation. Finally,. WGA induced PECAM-1 tyrosine phosphorylation and cytoskeletal reorganization in vascular endothelial cells. Our results , suggest that (i) PECAM-1 is involved in WGA-induced platelet activation, (ii) PECAM-1 clustering by WGA activates unique and strong platelet signali ng pathways, leading to a rapid PLC activation via Src family kinases, and (iii) WGA is a useful tool for elucidating PECAM-1-mediated signaling with wide implications not confined to platelets.