Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.5 (thrombin)
33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Thrombin is a coagulation protease that activates platelets, leukocytes, endothelial and mesenchymal cells at sites of vascular injury, acting partly through an unusual proteolytically activated G-protein-coupled receptor. Knockout of the gene encoding this receptor provided definitive evidence for a second thrombin receptor in mouse platelets and for tissue-specific roles for different thrombin receptors. We now report the cloning and characterization of a new human thrombin receptor, designated protease-activated receptor 3 (PAR3). PAR3 can mediate thrombin-triggered phosphoinositide hydrolysis and is expressed in a variety of tissues, including human bone marrow and mouse megakaryocytes, making it a candidate for the sought-after second platelet thrombin receptor. PAR3 provides a new tool for understanding thrombin signalling and a possible target for therapeutics designed selectively to block thrombotic, inflammatory and proliferative responses to thrombin.
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PMID:Protease-activated receptor 3 is a second thrombin receptor in humans. 908 10

Recent studies of mice deficient in the thrombin receptor, protease-activated receptor 1 (PAR1), provided definitive evidence for the existence of a second thrombin receptor in mouse platelets. We recently identified a new thrombin receptor designated protease-activated receptor 3 (PAR3). The mRNA encoding a mouse homologue of PAR3 was highly expressed in mouse splenic megakaryocytes, making it a good candidate for the missing mouse platelet thrombin receptor. We now report that PAR3 protein is expressed on the surface of mouse platelets and that PAR3 antibodies partially inhibit activation of mouse platelets by thrombin but not U46619, a thromboxane receptor agonist. These observations suggest that PAR3 contributes to mouse platelet activation by thrombin.
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PMID:Antibodies to protease-activated receptor 3 inhibit activation of mouse platelets by thrombin. 959 61

A polypeptide corresponding to the extracellular domain of protease-activated receptor 3 (PAR-3) is hydrolyzed by thrombin slowly because of high K(M) (>100 microM). However, thrombin is found to bind two PAR-3, one without catalyzing hydrolysis or blocking the active site, while the other is hydrolyzed. In a solvent lacking Na(+), hydrolysis of a nitroanilide substrate is enhanced 1.6-fold by addition of PAR-3 polypeptide, with half-saturation at 2.5 microM. In contrast, the fibrinogen clotting activity of thrombin is inhibited completely by PAR-3, with a K(I) of 3 microM. None of the activities of thrombin are affected by addition of 50 microM PAR-4 polypeptide. Thus, PAR-3 in low concentrations binds thrombin in a configuration that blocks the anion-binding exosite but not the catalytic site, while hydrolysis of PAR-3, PAR-4, and other substrates that do not interact with exosite I persists. The allosteric effect of PAR-3 is characteristic of that of Na(+).
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PMID:PAR-3 is a low-affinity substrate, high affinity effector of thrombin. 1273 12

It has been proposed that the cleaved form of protease-activated receptor 3 (PAR3) acts as a cofactor for thrombin cleavage and activation of PAR4 on murine platelets, but the molecular basis of this physiologically important effect remains elusive. X-ray crystal structures of murine thrombin bound to extracellular fragments of the murine receptors PAR3 ((38)SFNGGPQNTFEEFPLSDIE(56)) and PAR4 ((51)KSSDKPNPR downward arrow GYPGKFCANDSDTLELPASSQA(81), downward arrow = site of cleavage) have been solved at 2.0 and 3.5 A resolution, respectively. The cleaved form of PAR3, traced in the electron density maps from Gln-44 to Glu-56, makes extensive hydrophobic and electrostatic contacts with exosite I of thrombin through the fragment (47)FEEFPLSDIE(56). Occupancy of exosite I by PAR3 allosterically changes the conformation of the 60-loop and shifts the position of Trp-60d approximately 10 A with a resulting widening of the access to the active site. The PAR4 fragment, traced entirely in the electron density maps except for five C-terminal residues, clamps Trp-60d, Tyr-60a, and the aryl-binding site of thrombin with Pro-56 and Pro-58 at the P2 and P4 positions and engages the primary specificity pocket with Arg-59. The fragment then leaves the active site with Gly-60 and folds into a short helical turn that directs the backbone away from exosite I and over the autolysis loop. The structures demonstrate that thrombin activation of PAR4 may occur with exosite I available to bind cofactor molecules, like the cleaved form of PAR3, whose function is to promote substrate diffusion into the active site by allosterically changing the conformation of the 60-loop.
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PMID:Crystal structures of murine thrombin in complex with the extracellular fragments of murine protease-activated receptors PAR3 and PAR4. 1760 3

Nephrotic syndrome is characterized by massive proteinuria and injury of specialized glomerular epithelial cells called podocytes. Studies have shown that, whereas low-concentration thrombin may be cytoprotective, higher thrombin concentrations may contribute to podocyte injury. We and others have demonstrated that ex vivo plasma thrombin generation is enhanced during nephrosis, suggesting that thrombin may contribute to nephrotic progression. Moreover, nonspecific thrombin inhibition has been shown to decrease proteinuria in nephrotic animal models. We thus hypothesized that thrombin contributes to podocyte injury in a protease-activated receptor-specific manner during nephrosis. Here, we show that specific inhibition of thrombin with hirudin reduced proteinuria in two rat nephrosis models, and thrombin colocalized with a podocyte-specific marker in rat glomeruli. Furthermore, flow cytometry immunophenotyping revealed that rat podocytes express the protease-activated receptor family of coagulation receptors in vivo High-concentration thrombin directly injured conditionally immortalized human and rat podocytes. Using receptor-blocking antibodies and activation peptides, we determined that thrombin-mediated injury depended upon interactions between protease-activated receptor 3 and protease-activated receptor 4 in human podocytes, and between protease-activated receptor 1 and protease-activated receptor 4 in rat podocytes. Proximity ligation and coimmunoprecipitation assays confirmed thrombin-dependent interactions between human protease-activated receptor 3 and protease-activated receptor 4, and between rat protease-activated receptor 1 and protease-activated receptor 4 in cultured podocytes. Collectively, these data implicate thrombinuria as a contributor to podocyte injury during nephrosis, and suggest that thrombin and/or podocyte-expressed thrombin receptors may be novel therapeutic targets for nephrotic syndrome.
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PMID:Thrombin-Induced Podocyte Injury Is Protease-Activated Receptor Dependent. 2842 76

Thrombin participates in procoagulation, anticoagulation, and platelet activation. This enzyme contains anion binding exosites, ABE I and ABE II, which attract regulatory biomolecules. As prothrombin is activated to thrombin, pro-ABE I is converted into mature ABE I. Unexpectedly, certain ligands can bind to pro-ABE I specifically. Moreover, knowledge of changes in conformation and affinity that occur at the individual residue level as pro-ABE I is converted to ABE I is lacking. Such changes are transient and were not captured by crystallography. Therefore, we employed nuclear magnetic resonance (NMR) titrations to monitor development of ABE I using peptides based on protease-activated receptor 3 (PAR3). Proton line broadening NMR revealed that PAR3 (44-56) and more weakly binding PAR3G (44-56) could already interact with pro-ABE I on prothrombin. ¹H-¹⁵N heteronuclear single-quantum coherence NMR titrations were then used to probe binding of individual ¹⁵N-labeled PAR3G residues (F47, E48, L52, and D54). PAR3G E48 and D54 could interact electrostatically with prothrombin and tightened upon thrombin maturation. The higher affinity for PAR3G D54 suggests the region surrounding thrombin R77a is better oriented to bind D54 than the interaction between PAR3G E48 and thrombin R75. Aromatic PAR3G F47 and aliphatic L52 both reported on significant changes in the chemical environment upon conversion of prothrombin to thrombin. The ABE I region surrounding the 30s loop was more affected than the hydrophobic pocket (F34, L65, and I82). Our NMR titrations demonstrate that PAR3 residues document structural rearrangements occurring during exosite maturation that are missed by reported X-ray crystal structures.
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PMID:Deciphering Conformational Changes Associated with the Maturation of Thrombin Anion Binding Exosite I. 2911 72

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