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.

Of the four known protease-activated receptors (PARs), PAR1 and PAR4 are expressed by human platelets and mediate thrombin signaling. Whether these receptors are redundant, interact, or play at least partially distinct roles is unknown. It is possible that PAR1 and/or PAR4 might confer responsiveness to proteases other than thrombin. The neutrophil granule protease, cathepsin G, is known to cause platelet secretion and aggregation. We now report that this action of cathepsin G is mediated by PAR4. Cathepsin G triggered calcium mobilization in PAR4-transfected fibroblasts, PAR4-expressing Xenopus oocytes, and washed human platelets. An antibody raised against the PAR4 thrombin cleavage site blocked platelet activation by cathepsin G but not other agonists. Desensitization with a PAR4 activating peptide had a similar effect. By contrast, inhibition of PAR1 function had no effect on platelet responses to cathepsin G. When neutrophils were present, the neutrophil agonist fMet-Leu-Phe triggered calcium signaling in Fura-2-loaded platelets. Strikingly, this neutrophildependent platelet activation was blocked by the PAR4 antibody. These data show that PAR4 mediates platelet responses to cathepsin G and support the hypothesis that cathepsin G might mediate neutrophil-platelet interactions at sites of vascular injury or inflammation.Proteases regulate cellular behaviors in part via a family of G protein-coupled protease-activated receptors (PARs) 1 for which the thrombin receptor, PAR1, is the prototype. PAR1 is activated by an unusual mechanism in which thrombin cleaves the receptor amino-terminal exodomain to expose a new amino terminus that then serves as a tethered ligand (1, 2). The synthetic peptide SFLLRN, which mimics the first six amino acids of this newly unmasked amino terminus, can activate PAR1 independent of receptor cleavage and thrombin. The cognate P1Ј-P6Ј peptides of other PARs have been useful as agonists for probing the role of these receptors in various cellular responses (3-5).Four distinct PARs are now known. PAR1, PAR3, and the recently characterized PAR4 can be activated by thrombin (1, 5-7). PAR2 is activated by trypsin and trypsin-like enzymes (4). The importance of platelet activation in myocardial infarction and other thrombotic diseases has prompted considerable focus on identifying the PARs that mediate platelet activation by thrombin. Available data suggest that thrombin signaling in human platelets is mediated largely by hPAR1 and hPAR4 (8). PAR1 is necessary for platelet activation at low concentrations of thrombin, and PAR4 can mediate signaling at higher concentrations. The presence of two receptors raises the question of whether PAR1 and PAR4 are redundant, interact, or serve at least partially distinct functions in human platelets. In particular, it is possible that PAR4 might confer responsiveness to proteases other than thrombin.Platelet PARs may be exposed to a variety of proteases at sites of inflammation or coagulation, and leukocytes are one potential source. For example, neutr...

Endothelial surface expression of P-selectin and subsequent leukocyte rolling in venules can be induced by mast cell-derived histamine and binding of thrombin to protease-activated receptor-1 (PAR1). We hypothesized that activation of endothelial PAR2 by mast cell tryptase or other proteases also contributes to inflammatory responses. Leukocyte rolling flux and rolling velocity were assessed by intravital microscopy of the cremaster muscles of wild-type mice following perivenular micropipette injections of a control (LSIGRL) or PAR2-activating (SLIGRL) oligopeptide. Injection of SLIGRL increased mean rolling leukocyte flux fraction from 34 ± 11 to 71 ± 24% (p < 0.05) and decreased mean rolling velocity from 63 ± 29 to 32 ± 2 μm/s (p < 0.05). No significant changes occurred with control peptide injection. To further evaluate the role of PAR2 in inflammatory responses, PAR2-deficient mice were generated by gene targeting and homologous recombination. Perivenular injections of SLIGRL resulted in only a small increase in rolling leukocyte flux fraction (from 21 ± 8 to 30 ± 2%) and no change in rolling velocity. Leukocyte rolling after surgical trauma was assessed in 9 PAR2-deficient and 12 wild-type mice. Early (0–15 min) after surgical trauma, the mean leukocyte rolling flux fraction was lower (10 ± 3 vs 30 ± 6%, p < 0.05) and mean rolling velocity was higher (67 ± 46 vs 52 ± 36 μm/s, p < 0.01) in PAR2-deficient compared with control mice. The defect in leukocyte rolling in PAR2-deficient mice did not persist past 30 min following surgical trauma. These results indicate that activation of PAR2 produces microvascular inflammation by rapid induction of P-selectin-mediated leukocyte rolling. In the absence of PAR2, the onset of inflammation is delayed.

The 94-to 97-kDa mouse macrophage membrane protein that recognizes oxidized low density lipoprotein and phosphatidylserine-rich liposomes is identical to macrosialin, the mouse homologue of human CD68

We recently reported that oxidized low density lipoprotein (OxLDL), but not acetyl LDL (AcLDL), inhibited the binding and phagocytosis of nonopsonized, oxidatively damaged red blood cells (OxRBCs) by mouse peritoneal macrophages, implying the involvement of a "scavenger receptor" other than the AcLDL receptor. Numerous studies establish that loss of plasma membrane phospholipid asymmetry, which increases phosphatidylserine expression on the outer leaflet of the membrane, can play a key role in macrophage recognition of damaged and apoptotic cells. We report here that this recognition is in part attributable to the same mouse macrophage receptor that recognizes OxLDL.