Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a cell surface glycoprotein receptor expressed on a range of blood cells, including platelets, and on vascular endothelial cells. PECAM-1 possesses adhesive and signaling properties, the latter being mediated by immunoreceptor tyrosine-based inhibitory motifs present on the cytoplasmic tail of the protein. Recent studies in vitro have demonstrated that PECAM-1 signaling inhibits the aggregation of platelets. In the present study we have used PECAM-1-deficient mice and radiation chimeras to investigate the function of this receptor in the regulation of thrombus formation. Using intravital microscopy and laserinduced injury to cremaster muscle arterioles, we show that thrombi formed in PECAM-1-deficient mice were larger, formed more rapidly than in control mice, and were more stable. Larger thrombi were also formed in control mice that received transplants of PECAM-1-deficient bone marrow, in comparison to mice that received control transplants. A ferric chloride model of thrombosis was used to investigate thrombus formation in carotid arteries. In PECAM-1-deficient mice the time to 75% vessel occlusion was significantly shorter than in control mice. These data provide evidence for the involvement of platelet PECAM-1 in the negative regulation of thrombus formation. IntroductionPlatelet endothelial cell adhesion molecule-1 (PECAM-1, CD31) is a 130-kDa membrane glycoprotein that is expressed on a range of blood cells including platelets, monocytes, neutrophils, B lymphocytes, some T lymphocyte subsets, and also on vascular endothelial cells. [1][2][3][4] This member of the immunoglobulin superfamily has been reported to be associated with a wide range of functions, depending on the cell of interest. These include transendothelial migration of leukocytes, 5-7 integrin regulation, [8][9][10][11][12][13][14][15][16] modulation of T-and Blymphocyte antigen receptor signaling, 17,18 B-lymphocyte development, 19 vasculogenesis, 20 apoptosis, 21,22 and protection against endotoxic shock. 23 Several lines of investigation have recently determined that PECAM-1 is involved in the negative regulation of platelet function in vitro. The activation of PECAM-1 prior to the stimulation of platelets results in the inhibition of platelet aggregation and the inhibition of activatory signaling mechanisms. 24,25 Of particular note, therefore, are the observations that mouse platelets deficient in PECAM-1 are hyperresponsive to stimulation with collagen and demonstrate enhanced aggregation, secretion, and adhesion to this agonist. 26 Platelets from PECAM-1-deficient mice have also been shown to form larger thrombi in vitro under physiologic flow conditions. 25 PECAM-1 participates in homophilic ligand-binding interactions [27][28][29] ; indeed, such interactions between PECAM-1 molecules on the same cell and between cells are believed to underlie most of its identified functions. Additional potential ligand-binding interactions have been reported, such as with integrin ␣ v ␤ 3 ...

Platelet responses to collagen are mediated by the combined actions of the integrin ␣ 2 ␤ 1 , which serves as a major collagenbinding receptor, and the GPVI/FcR␥-chain complex, which transmits collagen-specific activation signals into the cell interior through the action of an immunoreceptor tyrosine-based activation motif within the cytoplasmic domain of the FcR␥-chain. Despite much progress in identifying components of the signaling pathway responsible for collagen-induced platelet activation, virtually nothing is known about the regulatory elements that modulate this important hemostatic event. PECAM-1, a recently recognized member of the inhibitory receptor family, contains a functional immunoreceptor tyrosine-based inhibitory motif within its cytoplasmic domain that, when tyrosine phosphorylated, recruits and activates the protein-tyrosine phosphatase, SHP-2. To test the hypothesis that PECAM-1 functions to regulate GPVI/FcR␥-chain-mediated platelet activation, the responses of wild-type versus PECAM-1-deficient murine platelets to GPVI-specific agonists were compared. Four distinct GPVI/FcR␥-chain-dependent responses were found to be significantly exaggerated in platelets derived from PE-CAM-1-deficient mice, including Mg ؉؉ -independent adhesion to immobilized fibrillar collagen, collagen-induced platelet aggregation, platelet aggregation induced by the GPVI-specific agonist collagen-related peptide, and GPVI/FcR␥-chain-induced dense granule secretion. Together, these data provide compelling evidence that PECAM-1 modulates platelet responses to collagen, and they implicate this novel member of the inhibitory receptor family in the regulation of primary hemostasis. IntroductionPlatelet endothelial cell adhesion molecule-1 (PECAM-1, CD31) is a 130-kd member of the immunoglobulin (Ig) superfamily that is expressed on the surface of circulating platelets, endothelial cells, neutrophils, monocytes, and certain T-lymphocyte subsets. The extracellular domain of PECAM-1 is composed of 6 extracellular Ig-like homology units, 1 the amino-terminal 2 of which mediates PECAM-1-PECAM-1 homophilic interactions. 2,3 Antibodies to the extracellular domain have been shown to have profound physiologic and cell biologic effects, including delaying leukocyte transendothelial migration 4-6 and inhibiting angiogenesis. 7 The extracellular domain of PECAM-1 also serves as a portal for entry into endothelial cells of certain strains of Plasmodium falciparuminfected erythrocytes. 8,9 The PECAM-1 cytoplasmic domain also plays a key biologic role because a large number of extracellular stimuli (reviewed in Newman 10 ) have been shown to result in the phosphorylation of 2 key tyrosine residues, located at positions 663 and 686 of the cytoplasmic domain. 11 The sequence surrounding each of these 2 tyrosine residues conforms to an immunoreceptor tyrosine-based inhibitory motif (ITIM) that, when phosphorylated, provides a major docking site for the src homology 2 (SH2) domaincontaining protein tyrosine phosphatase (PTP), SHP-2. 11-14 ...