P-selectin on activated platelets and stimulated endothelial cells mediat cell adhesion with monocytes and neutrophils. Since activated platelets induce tissue factor on mononuclear leukocytes, we examined the effect of P-selectin on the expression of tissue factor activity in monocytes.Purified P-selectin stimulated tissue factor expression on mononuclear leukocytes in a dose-dependent manner. Chinese hamster ovary (CHO) cells expressing P-selectin stimulated tissue factor procoagulant activity in purified monocytes, whereas untransfected CHO cells and CHO cells expressing E-selectin did not. Anti-P-selectin antibodies inhibited the effects of purified P-selectin and CHO cells expressing P-selectin on monocytes. Incubation of CHO cells expressing P-selectin with monocytes leads to the development of tissue factor mRNA in monocytes and to the expression of tissue factor antigen on the monocyte surface. These results indicate that P-selectin upregulates the expression of tissue factor on monocytes as well as mediates the binding of platelets and endothelial cells with monocytes and neutrophils. The binding of P-selectin to monocytes in the area of vascular injury may be a component of a mechanism that initiates thrombosis. stimulation of these cells by agonists such as thrombin, P-selectin is phosphorylated (26,27) and rapidly translocated to the plasma membrane (23). P-selectin is a lectin that binds to lineage-specific carbohydrates on the surface of monocytes and neutrophils (28)(29)(30). This protein binds to a mucinlike glycoprotein PSGL-1 that must be properly glycosylated to retain functional properties as the P-selectin ligand (31). P-selectin on platelets mediates the accumulation of leukocytes into the growing thrombus during experimental thrombosis in vivo (32). Inhibitory antibodies that block the interaction of P-selectin on platelets with the P-selectin ligand on leukocytes inhibit the uptake of leukocytes into the thrombus and inhibit the magnitude of thrombus formation. These experiments have demonstrated that P-selectin mediates monocyte and neutrophil interaction with activated platelets in vitro and in vivo. The potential exists for stimulation of leukocyte effector function by P-selectin binding. To evaluate this potential, we have examined the ability of P-selectin to upregulate tissue factor expression on monocytes. In the current study, we demonstrate that P-selectin induces the expression of tissue factor on monocytes exposed to P-selectin. Blood clotting is a host defense mechanism that, in parallel with the inflammatory and repair responses, preserves the integrity of the vascular system after tissue injury (1). Platelets, leukocytes, and endothelial cells are among the cellular components critical for this process. The plasma blood clotting proteins participate in a molecular cascade in which tissue injury activates blood coagulation, leading to the formation of a fibrin clot (2). The response to vascular injury culminates in the formation of a platelet plug, the deposition of ...

Eukaryotic cells have evolved a variety of mechanisms for dampening their responsiveness to hormonal stimulation in the face of sustained activation. The mechanisms for such processes, collectively referred to as desensitization, often involve alterations in the properties and number of cell-surface hormone receptors. It has been speculated that phosphorylation-dephosphorylation reactions, which are known to regulate the catalytic activities of enzymes, also regulate the function of receptors. Highly specific receptor kinases, such as rhodopsin kinase and beta-adrenergic receptor kinase, which show stimulus-dependent phosphorylation of receptors have been described. Direct evidence for a causal relationship between receptor phosphorylation and desensitization has been lacking however. Here we report that prevention of agonist-stimulated beta 2-adrenergic receptor (beta 2AR) phosphorylation by truncation of its serine and threonine-rich phosphate acceptor segment delays the onset of desensitization. We also show that selective replacement of these serine and threonine residues by alanine and glycine delays desensitization even further. These data provide the first direct evidence that one molecular mechanism of desensitization of G-protein-coupled receptors involves their agonist-induced phosphorylation.

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