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)

Procoagulant, anticoagulant, and fibrinolytic activities are associated with endothelial cells and involve the production, secretion, and receptor mediated binding of proteins involved in these processes. The procoagulant aspect of endothelial cells function involves the production and release of von Willebrand Factor(vWF), the production of tissue factor, and the presence of Factor IX/IXa receptors on the cell surface. Secretion of vWf will promote the initial steps in thrombus formation by supporting platelet-platelet interaction and platelet-subendothelial matrix adhesion. Tissue factor which is undetectable in resting cells appears after exposure to various cytokines and initiates factor VIIa activation of factors IX and X. Receptors of Factor IX/IXa are also present and mediate the assembly of the prothrombinase complex on the endothelial cell surface. The anticoagulant pathway involves the cell surface protein thrombomodulin, protein C and its cofactor protein S. Thrombomodulin binds thrombin which activates protein C which in the presence of protein S cleaves and inactivates Factors V and VIII. Inactivation of these two coagulation cofactors halts the coagulation. Finally, endothelial cells also play a pivotal role in the fibrinolytic system. Production and regulated secretion of tissue plasminogen activator creates a profibrinolytic state in the endothelial cell environment. In addition, receptors for plasminogen and urokinase are also present, constituting a cell surface mediated fibrinolytic pathway. Plasminogen activator inhibitor type I, the primary inhibitor of tPA, is also produced by endothelial cells. Thus endothelial cells can promote and inhibit fibrinolysis, depending on the prevailing environmental conditions.
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PMID:[Endothelial cells and vascular hemostasis]. 131 12

Thrombomodulin (TM), the endothelial cell surface receptor for thrombin-mediated activation of protein C and of its anticoagulant system, is involved in maintaining vascular nonthrombogenicity, and depressed TM activity may induce intravascular fibrin formation. TM antigen was previously found by immunohistochemical methods in rabbit glomeruli. We therefore attempted to identify the corresponding TM activity in isolated detergent-solubilized rat and human glomeruli. Like purified lung TM, rat glomeruli extracts accelerated the hydrolysis by activated protein C of the chromogenic substrate S-2238 in the presence of 10 nM thrombin, as determined by spectrophotometry. One mg glomerular protein promoted the formation of 681 +/- 115 nmol activated protein C, the equivalent of the amount generated by 845 ng of purified rabbit TM. TM activity correlated with the protein content of the glomerular extracts (r = 0.94). These extracts prolonged rat plasma activated partial thromboplastin time. Incubation of glomeruli with tumor necrosis factor-alpha (TNF) or E. coli lipopolysaccharide depressed their TM-like activity in a dose and time dependent manner. Incubation with TNF suppressed their anticoagulant activity. In human glomeruli, TM activity was also found at a level which corresponded to their TM antigen content, and was determined by ELISA with mouse monoclonal antibody. These results indicate that measurement of glomerular TM activity might help to clarify the mechanisms of intraglomerular fibrin deposition in renal diseases.
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PMID:Quantification and modulation of thrombomodulin activity in isolated rat and human glomeruli. 131 19

Effects of zinc and calcium ions on the heparin-neutralizing abilities of histidine-rich glycoprotein (HRG) and platelet factor 4 (PF4) were examined. Both HRG and PF4 effectively neutralized the ability of heparin to accelerate the activated protein C (APC) and the thrombin inhibitions by protein C inhibitor (PCI). the heparin-neutralizing ability of HRG in the APC inhibition by PCI, however, was decreased in a Ca(2+)-dependent manner and apparently lost at 1 mM Ca2+, while it was enhanced by Zn2+ regardless of the presence or absence of Ca2+. The heparin-neutralizing ability of HRG in the thrombin inhibition by PCI was not affected by Ca2+. In contrast to HRG, there was no significant difference in the heparin-neutralizing ability of PF4 in the presence or absence of 1 mM Ca2+. These results strongly suggest additional physiological functions of HRG and PF4 as modulators of PCI.
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PMID:Modulation of protein C inhibitor activity by histidine-rich glycoprotein and platelet factor 4: role of zinc and calcium ions in the heparin-neutralizing ability of histidine-rich glycoprotein. 131 17

Human blood monocytes (Mo) and monocyte-derived macrophages (M psi) possess cytotoxic effects against tumor cell lines when appropriately stimulated by various biological response modifiers, e.g., gamma interferon (gamma IFN) and muramyltripeptide (MTP). Activated Mo/M psi represent a new tool for the treatment of human malignancies, termed "adoptive cellular immunotherapy". Activated Mo/M psi express tissue factor procoagulant activity (PCA), which is a physiological trigger of blood coagulation. PCA was evaluated in vitro using a modification of the one-stage recalcification clotting time, and hemostatic changes were studied in vivo in cancer patients. Nine patients with peritoneal carcinomatosis were injected intraperitoneally with activated Mo and 11 patients with non-small cell lung carcinomas were infused intravenously with activated M psi. Hemostatic changes were followed using activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), fibrinogen level, antithrombin III (ATIII) and protein C (PC) activities. Fibrinolytic activity was estimated by euglobulin lysis time and assays for plasminogen and fibrin/fibrinogen degradation products (FDP). These assays were performed before and after each autologous infusion and on days 2 and 3. Activated Mo and M psi expressed potent PCA (85.5 +/- 7.5 U/ml for MTP activated Mo and 50 +/- 5.3 U/ml for gamma IFN activated M psi suspensions). In both groups of patients, APTT, PT, and TT underwent no significant variations. There was no significant consumption of ATIII or PC, and fibrinolysis was not activated during the study period. In the group injected intraperitoneally with MTP-activated Mo, fibrinogen showed a significant and progressive increase in relation to the development of an inflammatory reaction, reaching a maximum average value of 6.1 g/l at the end of the therapy with a concomitant increase in FDP levels. This increase was not observed after intravenous therapy with gamma IFN-activated M psi. No patient suffered from hemorrhagic or thrombotic events. In our experience, repeated injections of activated Mo or M psi expressing potent tissue factor PCA did not induce significant in vivo activation of the coagulation system in cancer patients.
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PMID:Hemostatic changes in human adoptive immunotherapy with activated blood monocytes or derived macrophages. 132 42

Thrombomodulin (TM) is a surface glycoprotein that forms a 1:1 complex with thrombin, thereby interacting to form the basis of a major physiologically relevant natural anticoagulant mechanism. Although initially described as a vascular endothelial cell receptor, TM has been reported to be present in several other cells, including megakaryocytes, platelets, monocytes, and several cultured cells. Other investigators have reported that neutrophils (PMN) may play a role in the hemostatic mechanism by supporting transformation of prothrombin to thrombin. To determine whether PMN might contribute further to the regulation of the coagulation system, we have evaluated these cells for the expression of TM. Large numbers of human leukocytes were isolated by standard techniques, and the PMN fraction was extracted and shown to be free of platelets and monocytes. Membrane preparations were affinity purified on an anti-TM-Affigel-10 matrix and the eluted material was examined by Western blotting, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and silver staining. The purified material was identical in apparent molecular weight to TM from human placenta and human umbilical vein endothelial cells (HUVEC). Using a sensitive and specific immunoassay, we estimated that there are a minimum of 5,220 +/- 1,658 molecules of TM per PMN, as compared with more than 50,000 in HUVEC. Northern analysis of RNA from PMN indicates that specific messenger RNA for TM, as identified by a single 3.8-kb band, is identical to that from HUVEC, and thereby confirms that PMN can also synthesize the receptor. Localization of TM in PMN was attempted by immunofluorescence, and the receptor was visualized only in permeabilized PMN, but was not seen on the surface of nonpermeabilized cells. Flow cytometry was also used, and could detect TM in 10% to 15% of nonpermeabilized PMN, whereas the antigen was present in greater than 80% of permeabilized cells. Biologic function of the PMN-derived TM, as tested by thrombin-dependent activation of protein C, was absent. Our results suggest that TM is synthesized by PMN, but under nonstimulated conditions, the protein is largely excluded from the membrane surface, and lacks the ability to promote activation of protein C by thrombin. TM from PMN may provide a further link between inflammation and thrombosis and may also be a significant source of plasma TM.
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PMID:Human neutrophils synthesize thrombomodulin that does not promote thrombin-dependent protein C activation. 132 11

X-ray diffraction studies of human thrombin revealed that compared with trypsin, two insertions (B and C) potentially limit access to the active site groove. When amino acids Glu146, Thr147, and Trp148, adjacent to the C-insertion (autolysis loop), are deleted the resulting thrombin (des-ETW) has dramatically altered interaction with serine protease inhibitors. Whereas des-ETW resists antithrombin III inactivation with a rate constant (Kon) approximately 350-fold slower than for thrombin, des-ETW is remarkably sensitive to the Kunitz inhibitors, with inhibition constants (Ki) decreased from 2.6 microM to 34 nM for the soybean trypsin inhibitor and from 52 microM to 1.8 microM for the bovine pancreatic trypsin inhibitor. The affinity for hirudin (Ki = 5.6 pM) is weakened at least 30-fold compared with recombinant thrombin. The mutation affects the charge stabilizing system and the primary binding pocket of thrombin as depicted by a decrease in Kon for diisopropylfluorophosphate (9.5-fold) and for N alpha-p-tosyl-L-lysine-chloromethyl ketone (51-fold) and a 39-fold increase in the Ki for benzamidine. With peptidyl p-nitroanilide substrates, the des-ETW deletion results in changes in the Michaelis (Km) and/or catalytic (kcat) constants, worsened as much as 85-fold (Km) or 100-fold (kcat). The specific clotting activity of des-ETW is less than 5% that of thrombin and the kcat/Km for protein C activation in the absence of cofactor less than 2%. Thrombomodulin binds to des-ETW with a dissociation constant of approximately 2.5 nM and partially restores its ability to activate protein C since, in the presence of the cofactor, kcat/Km rises to 6.5% that of thrombin. This study suggests that the ETW motif of thrombin prevents (directly or indirectly) its interaction with the two Kunitz inhibitors and is not essential for the thrombomodulin-mediated enhancement of protein C activation.
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PMID:Interaction of thrombin des-ETW with antithrombin III, the Kunitz inhibitors, thrombomodulin and protein C. Structural link between the autolysis loop and the Tyr-Pro-Pro-Trp insertion of thrombin. 132 50

Mediterranean spotted fever, a tick-borne rickettsiosis caused by Rickettsia conorii, may lead to small-vessel or deep-vein thrombosis. In order to evaluate the role of endothelial cell alteration in this lesion, we infected human endothelial cells derived from umbilical veins with R. conorii. We report the induction of two previously unreported prothrombotic mechanisms in rickettsial disease: (i) a progressive decline in thrombomodulin antigen and (ii) early expression of tissue factor, and, as described for R. rickettsii infection, later release of von Willebrand factor from Weibel-Palade bodies. Thrombomodulin expression in infected endothelial cells, measured by the thrombin-dependent activation of protein C or flow cytometric analysis, decreased steadily between 4 and 24 h after inoculation with rickettsiae. R. conorii infection induced tissue factor expression, measured by clotting assay and flow cytometric analysis, which was detectable 2 h postinoculation, reached its maximum 4 h postinoculation, and progressively decreased thereafter. Infection resulted in a relatively late release of von Willebrand factor antigen into the culture medium. A double-label immunofluorescence assay for the simultaneous evaluation of von Willebrand factor and R. conorii showed that the depletion of cytoplasmic von Willebrand factor stored in Weibel-Palade bodies was due to a direct effect of the intracellular R. conorii. These disturbances of endothelial function observed with R. conorii-infected cells may provide a paradigm for the elucidation of thrombotic pathobiology with Mediterranean spotted fever.
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PMID:von Willebrand factor release and thrombomodulin and tissue factor expression in Rickettsia conorii-infected endothelial cells. 132 57

To elucidate the role of the COOH-terminal region of antithrombin III, we studied the effects of synthetic peptides corresponding to its sequence on the amidolytic and proteolytic activities of thrombin and Factor Xa in the presence or absence of the inhibitor, antithrombin III. The peptides ANRPFLVFI and IIFMGRVANP corresponding to residues Ala404 to Ile412 and Ile420 to Pro429, respectively, blocked the inhibition by antithrombin III. The effect of IIFMGRVANP was reduced in the presence of heparin. Both peptides at a concentration of 1 mM blocked complex formation between antithrombin III and thrombin or Factor Xa. The two peptides, particularly IIFMGRVANP, directly enhanced the amidolytic activity of thrombin and Factor Xa on the synthetic substrate Boc-Ala-Gly-Arg-MCA (where Boc is t-butoxycarbonyl and MCA is 4-methylcoumarin), which corresponds to residues P3-P1 of the reactive site of antithrombin III, and also on other substrates due to increased Vmax. IIFMGRVANP also shortened the thrombin-induced fibrinogen clotting time, whereas ANRPFLVFI inhibited the thrombin-catalyzed activation of protein C both in the presence and absence of thrombomodulin. The direct effect of ANRPFLVFI and IIFMGRVANP on thrombin was confirmed by enhancement of the incorporation of dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide into thrombin. These findings suggest that the COOH-terminal region of antithrombin III interacts with thrombin and Factor Xa to increase the reactivity of the enzyme, which may enhance acyl-bond formation between the inhibitor and the enzyme.
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PMID:The role of the COOH-terminal region of antithrombin III. Evidence that the COOH-terminal region of the inhibitor enhances the reactivity of thrombin and factor Xa with the inhibitor. 133 Oct 47

We have previously demonstrated that human aortic endothelium exhibits morphologic heterogeneity in situ, and this heterogeneity can be reproduced in culture. In this study, we have compared prothrombotic properties of cultured endothelial cells (EC) from areas of human aorta at high risk for atherosclerosis (HP-EC) with EC from areas at low risk (LP-EC). Using paired cultures from the same donors, we have found that the expression of cell surface thrombomodulin (TM)--as measured by the ability to generate activated protein C (APC) from protein C in the presence of thrombin--is relatively reduced on HP-EC compared to LP-EC (respectively, 4.98 +/- 4.43 vs. 5.83 +/- 4.37 pM APC/min/cm2; p = .03, n = 12). Furthermore, HP-EC more efficiently assemble the prothrombinase complex on their cellular surface, resulting in an increased rate of thrombin generation from prothrombin (9.81 +/- 3.10 (HP-EC) vs. 7.96 +/- 3.20 nM thrombin/min/cm2 (LP-EC); p less than .03, n = 7). The combination of reduced TM expression and increased prothrombinase complex assembly on HP-EC suggests a prothrombotic phenotype in these cells. These findings may be important in the pathogenesis of thrombosis associated with atherosclerotic plaques.
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PMID:Prothrombotic phenotype diversity of human aortic endothelial cells in culture. 133 14

NMR experiments were carried out to study the interaction of thrombin with a synthetic peptide, ESKATNATLDPR, derived from the newly-identified platelet receptor for thrombin [Vu, T.-K. H., Hung, D. T., Wheaton, V. I., & Coughlin, S. R. (1991) Cell 64, 1057-1068]. On the basis of the observation of the thrombin-induced line broadening and transferred NOEs, binding of the peptide was found to be located exclusively within residues LDPR of the proteolytic cleavage site LDPR/S essential for receptor activation by thrombin. Measurement of transferred NOEs and molecular modeling indicate that the side chain of the Asp(P3) residue may form a hydrogen bond with thrombin and, by doing so, it is brought near a positively-charged thrombin residue Arg(221A), thereby partially neutralizing the negative charge of an Asp residue at this site of protein substrates. The hydrophobic side chains of residues Leu(P4) and Pro(P2) reside on the same side of the peptide backbone as indicated by transferred NOEs and were found by modeling to fit into a hydrophobic cage around the thrombin active site. These results suggest that the interaction of thrombin with protein substrates such as prothrombin, protein C, protein S, the platelet receptor, and the A alpha- and B beta-chains of fibrinogen all follow the same canonical binding mode in that the substrate forms an antiparallel beta-strand with thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Solution structure of a platelet receptor peptide bound to bovine alpha-thrombin. 133 64


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