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 multifunctional serine proteinase that plays a key role in coagulation while exhibiting several other key cellular bioregulatory functions. The X-ray crystal structure of human alpha-thrombin was determined in its complex with the specific thrombin inhibitor D-Phe-Pro-Arg chloromethylketone (PPACK) using Patterson search methods and a search model derived from trypsinlike proteinases of known spatial structure (Bode, W., Mayr, I., Baumann, U., Huber, R., Stone, S.R., & Hofsteenge, J., 1989, EMBO J. 8, 3467-3475). The crystallographic refinement of the PPACK-thrombin model has now been completed at an R value of 0.156 (8 to 1.92 A); in particular, the amino- and the carboxy-termini of the thrombin A-chain are now defined and all side-chain atoms localized; only proline 37 was found to be in a cis-peptidyl conformation. The thrombin B-chain exhibits the characteristic polypeptide fold of trypsinlike serine proteinases; 195 residues occupy topologically equivalent positions with residues in bovine trypsin and 190 with those in bovine chymotrypsin with a root-mean-square (r.m.s.) deviation of 0.8 A for their alpha-carbon atoms. Most of the inserted residues constitute novel surface loops. A chymotrypsinogen numbering is suggested for thrombin based on the topological equivalences. The thrombin A-chain is arranged in a boomeranglike shape against the B-chain globule opposite to the active site; it resembles somewhat the propeptide of chymotrypsin(ogen) and is similarly not involved in substrate and inhibitor binding. Thrombin possesses an exceptionally large proportion of charged residues. The negatively and positively charged residues are not distributed uniformly over the whole molecule, but are clustered to form a sandwichlike electrostatic potential; in particular, two extended patches of mainly positively charged residues occur close to the carboxy-terminal B-chain helix (forming the presumed heparin-binding site) and on the surface of loop segment 70-80 (the fibrin[ogen] secondary binding exosite), respectively; the negatively charged residues are more clustered in the ringlike region between both poles, particularly around the active site. Several of the charged residues are involved in salt bridges; most are on the surface, but 10 charged protein groups form completely buried salt bridges and clusters. These electrostatic interactions play a particularly important role in the intrachain stabilization of the A-chain, in the coherence between the A- and the B-chain, and in the surface structure of the fibrin(ogen) secondary binding exosite (loop segment 67-80).(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The refined 1.9-A X-ray crystal structure of D-Phe-Pro-Arg chloromethylketone-inhibited human alpha-thrombin: structure analysis, overall structure, electrostatic properties, detailed active-site geometry, and structure-function relationships. 130 49

Human and bovine alpha-thrombin cleaved at the B-chain by chymotrypsin generates catalytically competent zeta-thrombins, which are comprised of two noncovalently linked fragments: a 36-(human) or 49-(bovine) residue A-chain linked by a disulfide to B-chain residues B1-148 (zeta 1-thrombin) and B-chain residues B149-259 (zeta 2-thrombin). Human and bovine D-Phe-Pro-Arg-CH2-zeta- and PhMeSO2-zeta-thrombins were prepared by reaction of the active-site histidine (H-B43) and serine (S-B205) with PPACK and PMSF, respectively. Unfolding and dissociation of the noncovalently linked polypeptide chains of either human or bovine D-Phe-Pro-Arg-CH2-zeta- and PhMeSO2-zeta-thrombins in 4.5 M guanidine-HCl and refolding upon 30-fold dilution in 50 mM sodium phosphate buffer pH 6.5, 750 mM NaCl, 0.1% PEG resulted in biphasic generation of catalytic activity. The slow phase was eliminated in the presence of the competitive inhibitor benzamidine-HCl. Unfolding and refolding mixtures of the appropriate inactive precursors generated the active chimeric thrombins bovine zeta 1-thrombin:human zeta 2-thrombin and human zeta 1-thrombin:bovine zeta 2-thrombin. Human zeta 1-thrombin and zeta 2-thrombin were isolated, and, upon recombining, the isolated fragments refolded to generate catalytically competent zeta-thrombin with an active-site content, specific activity toward Chromozym-TH, and a specificity constant (kcat/Km) for FPA release from fibrinogen that were all within 60% of those of native alpha-thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Catalytically competent human and bovine zeta-thrombin and chimeras generated from unfolded polypeptide chains. 130 87

Thrombomodulin is an endothelial glycoprotein that serves as a cofactor for protein C activation. To examine the ligand specificity of human thrombomodulin, we performed equilibrium binding assays with human thrombin, thrombin S205A (wherein the active site serine is replaced by alanine), meizothrombin S205A, and human factor Xa. In competition binding assays with CV-1(18A) cells expressing cell surface recombinant human thrombomodulin, recombinant wild type thrombin and thrombin S205A inhibited 125I-diisopropyl fluorophosphate-thrombin binding with similar affinity (Kd = 6.4 +/- 0.5 and 5.3 +/- 0.3 nM, respectively). However, no binding inhibition was detected for meizothrombin S205A or human factor Xa (Kd greater than 500 nM). In direct binding assays, 125I-labeled plasma thrombin and thrombin S205A bound to thrombomodulin with Kd values of 4.0 +/- 1.9 and 6.9 +/- 1.2 nM, respectively. 125I-Labeled meizothrombin S205A and human factor Xa did not bind to thrombomodulin (Kd greater than 500 nM). We also compared the ability of thrombin and factor Xa to activate human recombinant protein C. The activation of recombinant protein C by thrombin was greatly enhanced in the presence of thrombomodulin, whereas no significant activation by factor Xa was detected with or without thrombomodulin. Similar results were obtained with thrombin and factor Xa when human umbilical vein endothelial cells were used as the source of thrombomodulin. These results suggest that human meizothrombin and factor Xa are unlikely to be important thrombomodulin-dependent protein C activators and that thrombin is the physiological ligand for human endothelial cell thrombomodulin.
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PMID:Ligand specificity of human thrombomodulin. Equilibrium binding of human thrombin, meizothrombin, and factor Xa to recombinant thrombomodulin. 131 33

Loss of sensitivity to thrombin following an initial response is characteristic of a number of cell types, including platelets. It has recently been proposed that thrombin receptors resemble other G protein-coupled receptors, but that activation involves a novel mechanism in which thrombin cleaves the receptor, exposing a new N terminus that serves as the ligand for the receptor. Based upon this model, we have examined the mechanism of thrombin receptor desensitization by comparing the effects of thrombin with those of a peptide corresponding to the N-terminal sequence of the receptor following proteolysis by thrombin: SFLLRNPNDKYEPF or TRP42/55. Like thrombin, TRP42/55 stimulated pertussis toxin-sensitive inositol 1,4,5-trisphosphate formation, raised cytosolic Ca2+, and inhibited cAMP formation in the megakaryoblastic HEL cell line. Exposure to either thrombin or TRP42/55 desensitized the cells to both, but not to a third agonist, neuropeptide Y. The rate of recovery after desensitization depended upon the order of agonist addition. Resensitization of the cell to thrombin following a brief exposure to thrombin required up to 24 h and could be inhibited with cycloheximide. Resensitization to TRP42/55 after exposure to thrombin, or to thrombin after exposure to TRP42/55, on the other hand, was detectable within 30 min and could be inhibited by serine/threonine phosphatase inhibitors, but not by cycloheximide. Loss of responsiveness to thrombin and TRP42/55 was also observed following addition of the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). However, while the protein kinase inhibitor staurosporine completely prevented the desensitization caused by TPA, it had only a limited effect on the desensitization caused by TRP42/55. These results demonstrate that the G protein-mediated effects of thrombin can be reproduced by a receptor-derived peptide and suggest that desensitization occurs by at least two mechanisms. The first, which is seen with thrombin, but not TRP42/55, involves proteolysis and requires protein synthesis for recovery. The second, which occurs with TRP42/55 and TPA, as well as with thrombin, involves phosphorylation, possibly of the receptor itself. Although protien kinase C is activated by thrombin and is presumably responsible for the desensitization caused by TPA, it does not appear to play a major role in receptor desensitization caused by thrombin and TRP42/55. This suggests that other kinases, such as those which inactivate adrenergic receptors and rhodopsin, are involved in the down-regulation of thrombin receptor function.
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PMID:Homologous desensitization of HEL cell thrombin receptors. Distinguishable roles for proteolysis and phosphorylation. 131 26

Okadaic acid and calyculin A, specific and cell permeable inhibitors of protein phosphatase 1 and 2A, inhibited aggregation, secretion and delta [Ca++]i in thrombin stimulated platelets. The inhibitory effect of calyculin A (IC50: 3.6-4.8nM) was about two hundred times more potent than that of okadaic acid (IC50: 0.8-1.3 microM), which is consistent with the difference of the reported Ki values for protein phosphatase 1. These phosphatase inhibitors and PGI2 synergistically enhanced the phosphorylation of 50kDa protein (P50), which is solely related to the inhibition of platelet reaction. These results indicate that serine/threonine protein phosphatase 1 might play a role in platelet activation.
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PMID:The effects of okadaic acid and calyculin A on thrombin induced platelet reaction. 131 73

Protein C is a plasma, vitamin K-dependent zymogen of a serine protease that can inhibit blood coagulation. Protein C is regulated by a series of reactions known as the protein C pathway. The importance of this pathway is seen in the occurrence of thrombosis in individuals with deficiencies in elements of the pathway like protein C and protein S. Work on several steps in this pathway has revealed that mechanisms involved in activation of protein C and the expression of its anticoagulant activity have features that allow for the expression of the anticoagulant activity away from sites in which procoagulant reactions occur, but not systemically. Thrombin, the principal procoagulant enzyme at the site of an injury, is converted to an anticoagulant enzyme at distant sites through its interaction with the endothelial cell protein thrombomodulin. Structural and functional studies have revealed the importance of several domain structures in the modulation of thrombin activity. Structural features of both activated protein C and its substrates (coagulation factors V and VIII) are such that they require the localization of enzyme and substrate on the surface of phosphatidyl serine containing membranes for optimum activity.
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PMID:Regulation of blood coagulation by the protein C system. 131 8

The binding of a variety of agonists to their receptors leads to the breakdown of membrane phospholipids and the formation of intracellular second messengers. Hydrolysis of inositol phospholipids by phospholipase C results in the formation of two second messengers, inositol-1,4,5-trisphosphate which mobilizes intracellular calcium and the neutral lipid diacylglycerol (DAG) which binds to and activates protein kinase C (PKC). PKC is actually a family of homologous serine/threonine protein kinases which play a central role in regulation of growth, differentiation and secretion reactions in a variety of cell types. In addition to these feedforward roles of PKC, it is thought to play an important feedback role, regulating early events in signal transduction. To explore these feedback functions we have examined the effect of PKC inhibitors on second messenger formation in thrombin-stimulated human platelets (a rapidly responding system) and the effect of PKC overexpression on second messenger formation and mitogenesis in rat fibroblasts (a system where sustained signaling occurs). Treatment of platelets with inhibitors of PKC potentiates DAG mass formation in response to thrombin while prior activation of PKC with phorbol esters blocks DAG mass formation, consistent with PKC playing a negative feedback role, inhibiting inositol phospholipid breakdown. DAG can also be formed by the sequential hydrolysis of phosphatidylcholine by phospholipase D and phosphatidic acid phosphohydrolase. This is a minor reaction in the rapidly responding platelet system, but may play a role in sustained signaling events. We have found that fibroblasts which overexpress the beta 1 isozyme of PKC display greatly enhanced DAG formation and phospholipase D activation in response to phorbol ester treatment. Upon stimulation of fibroblasts with thrombin, phospholipase D activation is also enhanced by PKC overexpression while formation of inositol phosphates is suppressed. These data suggest that PKC may act as a switch, terminating inositol phospholipid hydrolysis and activating the hydrolysis of phosphatidylcholine. Furthermore, we have observed a strong correlation between activation of phospholipase D and mitogenesis, suggesting an important role for this enzyme in long-term cellular responses to activation.
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PMID:Regulation of phospholipid hydrolysis and second messenger formation by protein kinase C. 132 4

We have examined the biological activities of thrombin and the thrombin-receptor-related polypeptides, S42FLLRNPNDKYEPF55(TRP42-55), S42FLLRNPND50(TRP42-50), and A42FLLRNPND50(A42-TRP42-50) as well as an arginine-containing basic peptide beginning with the SF motif (SFRGHITR), in rat aortic (RA) rings and in a gastric guinea pig longitudinal (LM) smooth muscle preparation. In the RA preparation, thrombin, as well as the three receptor-related peptides caused a relaxation in tissue that was precontracted with noradrenaline; the basic peptide, SFRGHITR, was inactive either as an agonist or as an antagonist to TRP42-55. In the LM bioassay, which unlike the RA preparation did not persistently desensitize in response to thrombin, all three receptor-related peptides, like thrombin, caused a prompt phasic reproducible contraction. The basic peptide, SFRGHITR, was inactive. In the LM assay, TRP42-55, TRP42-50 and A42-TRP42-55 all caused comparable contractile responses. We conclude that the gastric LM smooth muscle possesses a thrombin receptor and provides a convenient and reliable assay for the activities of thrombin receptor-related peptides. Our data also demonstrated that neither the C-terminal hirudin-related pentapeptide nor the N-terminal serine hydroxyl group are required for the biological activity of the thrombin receptor-derived peptide previously described (TRP42-55). Based on our findings we suggest that only a small portion of the N-terminal sequence of TRP42-55 may be required for thrombin-like biological activity.
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PMID:Actions of thrombin and thrombin receptor peptide analogues in gastric and aortic smooth muscle: development of bioassays for structure-activity studies. 132 91

Buried water molecules in the structurally homologous family of eukaryotic serine proteases were examined to determine whether buried waters and their protein environments are conserved in these proteins. We found 16 equivalent water sites conserved in trypsin/ogen, chymotrypsin/ogen, elastase, kallikrein, thrombin, rat tonin and rat mast cell protease, and 5 additional water sites in enzymes which share the primary specificity of trypsin. Based on an alignment of 30 serine protease sequences, it appears that the protein environments of these 21 conserved buried waters are highly conserved. The protein environments of buried waters are comprised primarily of atoms from highly conserved residues or main chain atoms from nonconserved residues. In one instance, the protein environment of a water is conserved even in the presence of an unlikely Pro/Ala substitution. We also note 3 instances in which a histidine side chain substitutes for water, suggesting that the structural role of water at these sites is satisfied by the presence of an alternative hydrogen bonding partner. Buried waters appear to be integral structural components of these proteins and should be incorporated into protein structures predicted on the basis of sequence homology to this family, including the catalytic domains of coagulation proteases.
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PMID:Buried water in homologous serine proteases. 133 31

Platelet-dependent activation of polymorphonuclear neutrophils (PMNL) was investigated with a lumi-aggregometer in heparinized whole blood and platelet-PMNL suspensions. The lumi-aggregometer allowed us to simultaneously monitor increases in impedance or light transmission as consequences of platelet aggregation and luminol-enhanced chemiluminescence (CL) as a measure of the oxidative burst in PMNL. Aggregation and platelet-PMNL contacts were also checked by light and electron microscopy. In whole blood, adenosine diphosphate (ADP) and the thromboxane A2 mimetic U 46619 induced the aggregation (increase in impedance) and the CL, which were both suppressed by EDTA, arginyl-glycyl-aspartyl-serine (RGDS) peptide, and the absence of stirring. In contrast, FMLP caused only CL that was unaffected by EDTA, RGDS peptide, and nonstirring. Similar observations were obtained with mixed suspensions containing washed platelets and PMNL at their physiologic concentrations. ADP, U 46619, and thrombin induced both aggregation (increase in light transmission) and CL, whereas FMLP caused CL but only very weak aggregation. Exogenous fibrinogen strongly enhanced the effects of ADP and U 46619. Iloprost, EDTA, RGDS peptide, red blood cell (RBC) ghosts, and nonstirring inhibited the effects induced by the platelet agonists, but were ineffective on the CL induced by FMLP. Treatment of platelets with aspirin did not affect the CL of PMNL induced by platelets. Microscopic examination, the requirements of stirring, Ca2+, and fibrinogen, and the inhibitory effects of RGDS peptide and RBC ghosts show that stimulated platelets activate PMNL in a contact-dependent manner that depends on fibrinogen binding. This was confirmed by the immunochemical demonstration of fibrinogen (but not of fibronectin) in the contact spaces between activated platelets and PMNL. Because supernatants and lysates of resting or thrombin-stimulated platelets did not induce the CL of PMNL, soluble agonists did not appear to be involved. Nonstimulated washed platelets also caused CL of PMNL that required stirring and Ca2+ and was inhibited by RBC ghosts. No CL occurred in unstimulated stirred whole blood, suggesting that a preactivation of platelets during the preparation may be responsible for the effects of unstimulated washed platelets. The results show that platelets provide a strong stimulus for PMNL that requires intercellular contact. Fibrinogen exposure on the platelet surface seems to be necessary for the activation of PMNL by stimulated platelets.
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PMID:Contact-induced neutrophil activation by platelets in human cell suspensions and whole blood. 135 74


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