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IntroductionPlatelet endothelial cell adhesion molecule-1 (PECAM-1; also known as CD31) is a 130-kDa member of the immunoglobulin gene (Ig) superfamily expressed on the surface of circulating platelets and leukocytes and at the intercellular junctions of all continuous endothelium. 1-3 Extracellular Ig homology domain 1 possesses homophilic binding properties 4,5 and functions to mediate leukocyte transendothelial migration 6,7 and angiogenesis, 8 while the cytoplasmic domain harbors a functional 9-11 immunoreceptor tyrosine-based inhibitory motif (ITIM) that, when tyrosine phosphorylated, has been shown to recruit and activate the protein-tyrosine phosphatase, SHP-2, in a number of cellular systems, including human platelets, 12 bovine aortic vascular endothelial cells, 13 and rat basophilic leukemia cells. 14 Owing, in part, to its cytoplasmic ITIM, PECAM-1 has recently been assigned to the Ig-ITIM family of inhibitory receptors. 15 In addition to its role in vascular cell adhesion and signaling, there is growing evidence that PECAM-1 may be able to transduce signals that suppress programmed cell death. The first evidence of a role for PECAM-1 in apoptosis was provided by the studies of Noble et al, 16 who found that monocytes promoted the survival of serum-starved endothelial cells. Interestingly, when the anti-PECAM-1 monoclonal antibody (mAb), PECAM-1.3, was included in the monocyte/endothelial cell coculture, the cytoprotective role of added monocytes was lost. Because PECAM-1.3 inhibits PECAM-1 homophilic interactions, 4,17 the authors speculated that monocyte PECAM-1-endothelial cell PECAM-1 homophilic interactions might contribute to endothelial cell survival. Further evidence that engagement of PECAM-1 can result in the transduction of a survival signal was provided by studies showing that the rate and extent of serum deprivation-induced apoptosis of endothelial cells is lessened if endothelial cells are first attached to immobilized PECAM-1/IgG 18 or treated with an anti-PECAM-1 monoclonal antibody. 19 While the latter investigation, like the study of Noble et al, found a correlation between PECAM-1-induced cell survival and increased transcript levels of the antiapoptotic gene, A1, the molecular mechanisms by which PECAM-1 might exert its cytoprotective effects have not to date been examined.Two major cell death pathways-termed the extrinsic and intrinsic pathways of apoptosis-exist in mammalian cells (reviewed by Hengartner 20 ). The extrinsic pathway is initiated by engagement and aggregation of tumor necrosis factor (TNF) family death receptors (such as The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 U.S.C. section 1734. CD95/Fas) which, through a series of death domain-containing adaptor molecules, recruit and directly activate cytosolic caspase 8, which in turn converts procaspase 3 to caspase 3-the central executioner of the apoptotic process. Th...

The integrin family is composed of a series of 24 αβ heterodimer transmembrane adhesion receptors that mediate cell-cell and cell-extracellular matrix interactions. Adaptor molecules bearing immunoreceptor tyrosine-based activation motifs (ITAMs) have recently been shown to cooperate with specific integrins to increase the efficiency of transmitting ligand-binding-induced signals into cells. In human platelets, Fc receptor γ-chain IIa (FcγRIIa) has been identified as an ITAM-bearing transmembrane receptor responsible for mediating "outside-in" signaling through αIIbβ3, the major adhesion receptor on the platelet surface. To explore the importance of FcγRIIa in thrombosis and hemostasis, we subjected FcγRIIa-negative and FcγRIIa-positive murine platelets to a number of well-accepted models of platelet function. Compared with their FcγRIIa-negative counterparts, FcγRIIa-positive platelets exhibited increased tyrosine phosphorylation of Syk and phospholipase Cγ2 and increased spreading upon interaction with immobilized fibrinogen, retracted a fibrin clot faster, and showed markedly enhanced thrombus formation when perfused over a collagen-coated flow chamber under conditions of arterial and venous shear. They also displayed increased thrombus formation and fibrin deposition in in vivo models of vascular injury. Taken together, these data establish FcγRIIa as a physiologically important functional conduit for αIIbβ3-mediated outside-in signaling, and suggest that modulating the activity of this novel integrin/ITAM pair might be effective in controlling thrombosis.

Unfractionated heparin (UFH) is a widely used anticoagulant that has long been known to potentiate platelet responses to subthreshold doses of platelet agonists. UFH has been reported to bind and induce modest conformational changes in the major platelet integrin, ␣IIb␤3, and induce minor changes in platelet morphology. The mechanism by which UFH elicits these platelet-activating effects, however, is not well understood. We found that both human and murine platelets exposed to UFH, either in solution or immobilized onto artificial surfaces, underwent biochemical and morphologic changes indicative of a potentiated state, including phosphorylation of key cytosolic signaling molecules and cytoskeletal changes leading to cell spreading. Low molecular weight heparin and the synthetic pentasaccharide, fondaparinux, had similar plateletpotentiating effects. Human or mouse platelets lacking functional integrin ␣IIb␤3 complexes and human platelets pretreated with the fibrinogen receptor antagonists eptifibatide or abciximab failed to become potentiated by heparin, demonstrating that heparin promotes platelet responsiveness via its ability to initiate ␣IIb␤3-mediated outside-in signaling. Taken together, these data provide novel insights into the mechanism by which platelets become activated after exposure to heparin and heparin-coated surfaces, and suggest that currently used glycoprotein IIb-IIIa inhibitors may be effective inhibitors of nonimmune forms of heparin-induced platelet activation.