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The accumulation of thrombin at sites of vascular injury provides one of the chief means for recruiting platelets into a growing hemostatic plug. Studies completed over the past 10 years show that platelet responses to thrombin are mediated by a subset of G protein-coupled receptors known as protease-activated receptors. These receptors are activated on cleavage by thrombin, initiating the intracellular signaling events needed to transform mobile, nonadhesive platelets into cells that can participate in the growth of an immobile hemostatic plug. How this is accomplished is the subject of this review.

Platelets are critical in haemostasis and in arterial thrombosis, which causes heart attacks and other events triggered by abnormal clotting 1 , 2 , 3 , 4 , 5 . The coagulation protease thrombin is a potent activator of platelets ex vivo 6 . However, because thrombin also mediates fibrin deposition and because multiple agonists can trigger platelet activation 7 , 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) 8 . 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.

Platelet-dependent arterial thrombosis triggers most heart attacks and strokes. Because the coagulation protease thrombin is the most potent activator of platelets 1 , 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 2 , 3 , 4 , 5 , 6 , but plays no apparent role in mouse platelet activation 7 , 8 , 9 . PAR3 is a thrombin receptor that is expressed in mouse megakaryocytes 10 . 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.

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.

How does a protease act like a hormone to regulate cellular functions? The coagulation protease thrombin (EC 3.4.21.5) activates platelets and regulates the behavior of other cells by means of G protein-coupled protease-activated receptors (PARs). PAR1 is activated when thrombin binds to and cleaves its amino-terminal exodomain to unmask a new receptor amino terminus. This new amino terminus then serves as a tethered peptide ligand, binding intramolecularly to the body of the receptor to effect transmembrane signaling. The irreversibility of PAR1's proteolytic activation mechanism stands in contrast to the reversible ligand binding that activates classical G protein-coupled receptors and compels special mechanisms for desensitization and resensitization. In endothelial cells and fibroblasts, activated PAR1 rapidly internalizes and then sorts to lysosomes rather than recycling to the plasma membrane as do classical G protein-coupled receptors. This trafficking behavior is critical for termination of thrombin signaling. An intracellular pool of thrombin receptors refreshes the cell surface with naive receptors, thereby maintaining thrombin responsiveness. Thus cells have evolved a trafficking solution to the signaling problem presented by PARs. Four PARs have now been identified. PAR1, PAR3, and PAR4 can all be activated by thrombin. PAR2 is activated by trypsin and by trypsin-like proteases but not by thrombin. Recent studies with knockout mice, receptor-activating peptides, and blocking antibodies are beginning to define the role of these receptors in vivo.

We studied the activation of human platelets by thrombin and proteinase activated receptor (PAR)‐activating peptides (PAR‐APs) [SFLLRNPNDKYEPF‐amide (TRAP), TFLLR‐amide (PAR1AP) and AYPGKF‐amide (PAR4AP)]. PAR agonist‐induced platelet aggregation, glycoprotein (GP) Ib and GPIIb/IIIa surface expression and ADP release were measured by light aggregometry, flow cytometry and chemiluminescence. Aggregation inhibitors, including prostacyclin (PGI2), nitric oxide‐releasing agent (S‐nitroso‐glutathione, GSNO), aspirin, apyrase, and phenanthroline were used to study the susceptibility of PAR agonist‐induced aggregation to pharmacological inhibition. Thrombin was the most potent platelet agonist, followed by PAR1AP, TRAP and PAR4AP. The aggregatory potencies of PAR‐APs were not modified by the aminopeptidase inhibitor, amastatin. Subthreshold concentrations of PAR1AP potentiated the effects of PAR4AP to stimulate maximal aggregation. Both PGI2 and GSNO reduced PAR agonist‐induced aggregation and diminished GPIIb/IIIa up‐regulation. PAR agonist‐induced aggregation was aspirin‐insensitive indicating a minor role for TXA2. In contrast, phenanthroline and apyrase significantly enhanced the anti‐aggregatory effects of aspirin against thrombin‐, PAR1AP‐ and TRAP‐induced aggregation suggesting the involvement of ADP‐ and MMP‐2‐dependent pathways. PAR4AP‐induced aggregation (but not PAR1AP‐induced aggregation) was entirely ADP‐dependent (abolished by apyrase) and resistant to phenanthroline (MMP‐2‐independent). Thus, the mechanisms of PAR1 and 4‐induced platelet aggregation are distinct and depend differentially on their ability to interact with pathways of aggregation, along with the subsequent activation of GPIIb/IIIa receptors. British Journal of Pharmacology (2002) 135, 1123–1132; doi:10.1038/sj.bjp.0704559

Disseminated intravascular coagulation (DIC) is a common and life-threatening complication in sepsis. Sepsis-associated DIC is recognized as the systemic activation in coagulation with suppressed fibrinolysis that leads to organ dysfunction in combination with systemic intravascular inflammation. In this process, thrombin contributes a key role in connecting both coagulation and inflammation. Endothelial injury, a result of sepsis, causes DIC due to the effect of multiple activated factors that include neutrophils, platelets, and damage-associated molecular patterns. Recent advances in the understanding of pathophysiology have made it possible to diagnose sepsis-associated DIC at earlier timing with better accuracy. However, progress in the treatment is still limited, and new therapeutics for sepsis-associated DIC are needed.

Direct thrombin inhibitors (DTIs) are a new class of anticoagulants that bind directly to thrombin and block its interaction with its substrates. Four parenteral DTIs have been approved by the FDA — hirudin and argatroban for heparin-induced thrombocytopenia, bivalirudin as an alternative to heparin in percutaneous coronary intervention, and desirudin as prophylaxis against venous thromboembolism in hip replacement. This article discusses the clinical data on this important new class of medications. Direct thrombin inhibitors are a new class of anticoagulants that bind directly to thrombin and block its interaction with its substrates. Four parenteral DTIs have been approved by the FDA. This article discusses the clinical data on this important new class of medications. Direct thrombin inhibitors (DTIs) are a new class of anticoagulants that bind directly to thrombin and block its interaction with its substrates. Some DTIs — such as recombinant hirudins, bivalirudin, and ximelagatran, either alone or in combination with melagatran — have undergone extensive evaluation in phase 3 trials for the prevention and treatment of arterial and venous thrombosis. The evidence concerning the clinical applicability of other DTIs, such as argatroban and dabigatran, is limited to phase 2 studies. Four parenteral DTIs have been approved by the Food and Drug Administration (FDA) in North America: hirudin and argatroban for the treatment . . .

An inflammatory process in the central nervous system (CNS) is believed to play an important role in the pathway leading to neuronal cell death in a number of neurodegenerative diseases including Parkinsons disease, Alzheimers disease, prion diseases, multiple sclerosis and HIV-dementia. The inflammatory response is mediated by the activated microglia, the resident immune cells of the CNS, which normally respond to neuronal damage and remove the damaged cells by phagocytosis. Activation of microglia is a hallmark of brain pathology. However, it remains controversial whether microglial cells have beneficial or detrimental functions in various neuropathological conditions. The chronic activation of microglia may in turn cause neuronal damage through the release of potentially cytotoxic molecules such as proinflammatory cytokines, reactive oxygen intermediates, proteinases and complement proteins. Therefore, suppression of microglia-mediated inflammation has been considered as an important strategy in neurodegenerative disease therapy. Several anti-inflammatory drugs of various chemical ingredients have been shown to repress the microglial activation and to exert neuroprotective effects in the CNS following different types of injuries. However, the molecular mechanisms by which these effects occur remain unclear. In recent years, several research groups including ours have attempted to explain the potential mechanisms and signaling pathways for the repressive effect of various drugs, on activation of microglial cells in CNS injury. We provide here a comprehensive review of recent findings of mechanisms and signaling pathways by which microglial cells are activated in CNS inflammatory diseases. This review article further summarizes the role of microglial cells in neurodegenerative diseases and various forms of potential therapeutic options to inhibit the microglial activation which amplifies the inflammation-related neuronal injury in neurodegenerative diseases.