Lymphocytes require sphingosine-1-phosphate (S1P) receptor-1 to exit lymphoid organs, but the source(s) of extracellular S1P and whether S1P directly promotes egress are unknown. By using mice in which the two kinases that generate S1P were conditionally ablated, we find that plasma S1P is mainly hematopoietic in origin, with erythrocytes a major contributor, whereas lymph S1P is from a distinct radiation-resistant source. Lymphocyte egress from thymus and secondary lymphoid organs was markedly reduced in kinase-deficient mice. Restoration of S1P to plasma rescued egress to blood but not lymph, and the rescue required lymphocyte expression of S1P-receptor-1. Thus, separate sources provide S1P to plasma and lymph to help lymphocytes exit the low-S1P environment of lymphoid organs. Disruption of compartmentalized S1P signaling is a plausible mechanism by which S1P-receptor-1 agonists function as immunosuppressives.

Platelet-dependent arterial thrombosis triggers most heart attacks and strokes. Because the coagulation protease thrombin is the most potent activator of platelets, identification of the platelet receptors for thrombin is critical for understanding thrombosis and haemostasis. Protease-activated receptor-1 (PAR1) is important for activation of human platelets by thrombin, but plays no apparent role in mouse platelet activation. PAR3 is a thrombin receptor that is expressed in mouse megakaryocytes. Here we report that thrombin responses in platelets from PAR3-deficient mice were markedly delayed and diminished but not absent. We have also identified PAR4, a new thrombin-activated receptor. PAR4 messenger RNA was detected in mouse megakaryocytes and a PAR4-activating peptide caused secretion and aggregation of PAR3-deficient mouse platelets. Thus PAR3 is necessary for normal thrombin responses in mouse platelets, but a second PAR4-mediated mechanism for thrombin signalling exists. Studies with PAR-activating peptides suggest that PAR4 also functions in human platelets, which implies that an analogous dual-receptor system also operates in humans. The identification of a two-receptor system for platelet activation by thrombin has important implications for the development of antithrombotic therapies.

Identification of the mechanisms by which the coagulation protease thrombin activates platelets is critical for understanding haemostasis and thrombosis. Thrombin activates cells at least in part by cleaving protease-activated G-protein-coupled receptors (PARs). PAR3 and PAR4 are thrombin receptors expressed in mouse platelets. Inhibition of thrombin binding to mPAR3 (ref. 4) and knockout of the mPAR3 gene inhibited mouse platelet activation at low but not high concentrations of thrombin. Thus PAR3 is important for thrombin signalling in mouse platelets. Expression of human PAR3 in heterologous expression systems reliably resulted in responsiveness to thrombin. Curiously, despite its importance for the activation of mouse platelets by thrombin, mouse PAR3 (mPAR3) did not lead to thrombin signalling even when overexpressed. We now report that mPAR3 and mPAR4 interact in a novel way: mPAR3 does not itself mediate transmembrane signalling but instead functions as a cofactor for the cleavage and activation of mPAR4 by thrombin. This establishes a paradigm for cofactor-assisted PAR activation and for a G-protein-coupled receptor's acting as an accessory molecule to present ligand to another receptor.

Platelets are critical in haemostasis and in arterial thrombosis, which causes heart attacks and other events triggered by abnormal clotting. The coagulation protease thrombin is a potent activator of platelets ex vivo. However, because thrombin also mediates fibrin deposition and because multiple agonists can trigger platelet activation, the relative importance of platelet activation by thrombin in haemostasis and thrombosis is unknown. Thrombin triggers cellular responses at least in part through protease-activated receptors (PARs). Mouse platelets express PAR3 and PAR4 (ref. 9). Here we show that platelets from PAR4-deficient mice failed to change shape, mobilize calcium, secrete ATP or aggregate in response to thrombin. This result demonstrates that PAR signalling is necessary for mouse platelet activation by thrombin and supports the model that mouse PAR3 (mPAR3) does not by itself mediate transmembrane signalling but instead acts as a cofactor for thrombin cleavage and activation of mPAR4 (ref. 10). Importantly, PAR4-deficient mice had markedly prolonged bleeding times and were protected in a model of arteriolar thrombosis. Thus platelet activation by thrombin is necessary for normal haemostasis and may be an important target in the treatment of thrombosis.

Protease-Activated Receptor-1 (PAR1) is the prototypical member of a family of G protein-coupled receptors that mediate cellular responses to thrombin and related proteases. Thrombin irreversibly activates PAR1 by cleaving the N-terminal exodomain of the receptor, which exposes a tethered peptide ligand that binds the receptor’s heptahelical bundle to effect G protein-activation. Here we report a 2.2Å resolution crystal structure of human PAR1 bound to vorapaxar, a PAR1 antagonist. The structure reveals an unusual mode of drug binding that explains how a small molecule binds virtually irreversibly to inhibit receptor activation by PAR1’s tethered ligand. In contrast to deep, solvent-exposed binding pockets observed in other peptide-activated GPCRs, the vorapaxar-binding pocket is superficial but has little surface exposed to the aqueous solvent. PARs are important targets for drug development. The structure reported here will aid development of improved PAR1 antagonists and discovery of antagonists to other members of this receptor family.

Summary We report an unexpected role for protease signaling in neural tube closure and formation of the central nervous system. Mouse embryos lacking protease-activated receptor 1 and 2 showed defective hindbrain and posterior neuropore closure and developed exencephaly and spina bifida, important human congenital anomalies. Par1 and Par2 were expressed in surface ectoderm, Par2 selectively along the line of closure. Ablation of Gi/z and Rac1 function in these Par2-expressing cells disrupted neural tube closure, further implicating G protein-coupled receptors and identifying a likely effector pathway. Cluster analysis of protease and Par2 expression patterns revealed a group of membrane-tethered proteases often co-expressed with Par2. Among these, matriptase activated Par2 with picomolar potency, and hepsin and prostasin activated matriptase. Together, our results suggest a role for protease-activated receptor signaling in neural tube closure and identify a local protease network that may trigger Par2 signaling and monitor and regulate epithelial integrity in this context.

The coagulation protease thrombin triggers fibrin formation, platelet activation, and other cellular responses at sites of tissue injury. We report a role for PAR1, a protease-activated G protein-coupled receptor for thrombin, in embryonic development. Approximately half of Par1-/- mouse embryos died at midgestation with bleeding from multiple sites. PAR1 is expressed in endothelial cells, and a PAR1 transgene driven by an endothelial-specific promoter prevented death of Par1-/- embryos. Our results suggest that the coagulation cascade and PAR1 modulate endothelial cell function in developing blood vessels and that thrombin's actions on endothelial cells-rather than on platelets, mesenchymal cells, or fibrinogen-contribute to vascular development and hemostasis in the mouse embryo.

Defining the relative importance of protease-activated receptors (PARs) for thrombin signaling in mouse endothelial cells is critical for a basic understanding of thrombin signaling in these cells and for the rational use of knockout mice to probe the roles of thrombin's actions on endothelial cells in vivo. We examined thrombin-and PAR agonist-induced increases in cytoplasmic calcium, phosphoinositide hydrolysis, extracellular signalregulated kinase (ERK) phosphorylation, and gene expression in endothelial cells from wild-type and PAR-deficient mice. IntroductionThrombin triggers a host of responses in endothelial cells that may contribute to hemostasis, inflammation, and development of embryonic blood vessels. [1][2][3] For example, thrombin causes the release of von Willebrand factor, the mobilization of P-selectin to the endothelial surface, 4 and the production of platelet-activating factor 5,6 and chemokines 7,8 -events likely to be involved in recruiting platelets and leukocytes to sites of vascular injury. [9][10][11][12] Thrombin triggers changes in the junctional complexes between endothelial cells and cell rounding, and it increases the permeability of endothelial monolayers. [13][14][15][16] Thrombin also stimulates endothelial cell migration and the production of growth factors and their receptors, cytokines, and matrix proteins-events that may be involved in the proper formation and maintenance of blood vessels during embryonic development. 3,[17][18][19][20] Identifying the thrombin receptors that mediate endothelial cell activation is a necessary step toward defining the relative importance of endothelial cell responses to thrombin in vivo.Thrombin triggers cellular responses at least in part through G-protein-coupled protease-activated receptors (PARs). 1 In the mouse, PAR1 and PAR4 can each mediate thrombin responses; PAR2 is not activated by thrombin, and PAR3 does not itself mediate transmembrane signaling but instead functions as a cofactor that promotes PAR4 activation by thrombin in mouse platelets. 21,22 PARs are activated by the proteolytic unmasking of a tethered peptide ligand that resides in the receptor's N-terminal exodomain, and synthetic peptides that mimic this sequence function as agonists that activate PARs independent of receptor cleavage. 23,24 Activation of PAR1 with such a peptide is sufficient to trigger most, if not all, of the known endothelial responses to thrombin. However, it is unknown whether PAR1 accounts for all thrombin signaling in endothelial cells. Indeed, recent studies with PAR1 and factor V knockout mice suggest that other targets of thrombin-perhaps other endothelial PARs-are important for embryonic development and possibly vascular diseases. 3 We now report studies that address the relative importance of different PARs for thrombin signaling in vascular endothelial cells and whether PARs account for thrombin signaling in these cells. Materials and methods MaterialsThe peptides TFLLRN, YAPGKF, AYPGKF, and SLIGRL were synthesized as carboxyl terminal amide...