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

---

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

Monoclonal antibodies against thrombomodulin have become a useful means to study the structure and function of thrombomodulin. In this study, we used a monoclonal antibody against human thrombomodulin, named SZ-53, to investigate the function of thrombomodulin on the surface of cultured human umbilical vein endothelial cells. Preincubation of endothelial cells with SZ-53 before addition of thrombin not only inhibited thrombomodulin mediated activation of protein C, but also inhibited thrombin mediated release of t-PA and PGI2 from endothelial cells. The inhibitory effects depended on the concentration of SZ-53 IgG. According to our experimental results, we suggest that thrombomodulin on the surface of endothelial cells could participate in the regulation of thrombin mediated release of t-PA and PGI2 from these cells.
...
PMID:A monoclonal antibody (SZ-53) against thrombomodulin inhibits thrombin-mediated release of t-PA and PGI2 from endothelial cells. 166 94

We isolated a cDNA encoding a functional human thrombin receptor by direct expression cloning in Xenopus oocytes. mRNA encoding this receptor was detected in human platelets and vascular endothelial cells. The deduced amino acid sequence revealed a new member of the seven transmembrane domain receptor family with a large amino-terminal extracellular extension containing a remarkable feature. A putative thrombin cleavage site (LDPR/S) resembling the activation cleavage site in the zymogen protein C (LDPR/I) was noted 41 amino acids carboxyl to the receptor's start methionine. A peptide mimicking the new amino terminus created by cleavage at R41 was a potent agonist for both thrombin receptor activation and platelet activation. "Uncleavable" mutant thrombin receptors failed to respond to thrombin but were responsive to the new amino-terminal peptide. These data reveal a novel signaling mechanism in which thrombin cleaves its receptor's amino-terminal extension to create a new receptor amino terminus that functions as a tethered ligand and activates the receptor.
...
PMID:Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. 167 65

In serine proteases, residue 192, three residues prior to the active site Ser-195, plays an important role in determining substrate specificity. In trypsin (EC 3.4.21.4) and most trypsin-like enzymes with relatively broad specificity, this position is occupied by Gln. In thrombin (EC 3.4.21.5), an enzyme with restricted specificity, position 192 is occupied by Glu. The potential importance of Glu-192 in restricting the specificity of thrombin was investigated by isosterically replacing Glu-192 with Gln. Unlike trypsin, thrombin cleavage of peptides with acidic residues in positions P3 and P'3 [where P3 and P'3 refer to three residues removed from the Arg (P1) cleavage site on the amino and carboxyl side, respectively] is inefficient. Protein C, an anticoagulant zymogen, has Asp residues in positions P3 and P'3. Thrombomodulin, an endothelial cell protein, complexes with thrombin to activate protein C rapidly thus altering the specificity of thrombin. Compared to thrombin, the Glu-192----Gln mutant thrombin activates protein C 22 times more rapidly and cleaves the P7-P'5 peptide from the protein C activation site 19 times faster. Enhanced protein C activation results primarily from an increase in the catalytic rate constant rather than an improved Michaelis constant, a property that is shared by the thrombin-thrombomodulin complex. The Glu-192----Gln mutation does not influence fibrinopeptide A release and only increases the rate of fibrinopeptide B release 2.7-fold. These results demonstrate that Glu-192 plays a critical role in restricting the specificity of thrombin and suggest that thrombomodulin may function in part by altering the enzyme-substrate interaction near residue 192 in thrombin.
...
PMID:Glu-192----Gln substitution in thrombin mimics the catalytic switch induced by thrombomodulin. 167 22

About 30% of human plasma protein C is smaller than the predominant form as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It has been suggested that this species, referred to as beta protein C, is a degraded molecule. However, beta protein C is secreted in culture by the HepG2 cell line and is present in plasma collected directly into numerous proteinase inhibitors; the percentage of beta protein C does not change with time during culture or after blood collection. Neither thrombin, activated protein C, nor activated factor X converts the alpha form to beta in the presence or absence of calcium and phospholipids. The NH2-terminal sequences of the heavy chains of both forms are identical, and both release the same dodecapeptide and develop a functional active site when cleaved by thrombin. Both also react with antibodies to a synthetic COOH-terminal peptide. Timed digests with N-glycosidase are consistent with the interpretation that beta protein C has three N-linked oligosaccharide chains whereas alpha protein C has four. It is asparagine 329 that is not glycosylated in beta protein C since antibodies to a synthetic peptide based on the sequence around this amino acid react only with beta protein C. This site is unique in having cysteine instead of serine or threonine 2 residues distal. It is likely that the sulfhydryl group can substitute for the usual hydroxyl group as a hydrogen bond acceptor for the glycosylation reaction only until it forms a disulfide bond. The percentage of protein C that is glycosylated at this site may therefore depend at least in part on the rate of disulfide bond formation which may in turn be related to the rate of protein synthesis.
...
PMID:Beta protein C is not glycosylated at asparagine 329. The rate of translation may influence the frequency of usage at asparagine-X-cysteine sites. 169 79

The haemostatic parameters were studied within 14 days of acute myocardial infarction (AMI) in 103 patients randomly allocated into a group receiving low-dose heparin or into a group treated without anticoagulants. Patients with isotopic evidence of deep vein thrombosis were excluded from the analysis. An important formation of thrombin-antithrombin III complex (TAT) in the plasma was detected in the early stage of the disease. It was accompanied by an activation of plasma intrinsic fibrinolysis (IF), an elevation of fibrinogen and its degradation products (FDP) and a reduction of extrinsic plasma fibrinolytic activity (EF) together with normal levels of factor X, antithrombin III (AT III), protein C and alpha-2-antiplasmin. Sequentially studies periods of the disease revealed a diminution of TAT complex concentration in the plasma on the seventh day of AMI together with a rise of the both plasma fibrinolytic activities (IF, EF) as well as an elevation of fibrinogen and its degradation products, returning to the initial values on the 14 day of AMI. In the patients treated with heparin the augmentation of TAT complex in the plasma was prolonged until the fifth day of AMI. Moreover, heparin administration was connected with significantly higher levels of AT III and protein C along with a lower concentration of factor X and FDP on the seventh day of the disease. The fluctuation of fibrinolytic activities (IF, EF) in the plasma was heparin-independent. The present results indicate that low-dose heparin treatment modulates the plasmatic fluctuation of TAT complex as well as factor X, AT III and protein C levels in patients with acute myocardial infarction.
...
PMID:Fluctuation of thrombin-antithrombin III complex in patients with acute myocardial infarction: influence of low-dose heparin administration. 169 24

Fetomodulin is a surface marker protein of differentiated F9 embryonal carcinoma cells. Gene cloning has recently identified it as thrombomodulin which binds thrombin and proteolytically activates protein C. Activity assays and RNA blotting were adopted to analyze F9 cell differentiation with specific reference to another well-characterized marker, tissue plasminogen activator. Retinoic acid induced primitive endoderm differentiation of F9 cells and simultaneously activated tissue plasminogen activator synthesis. This differentiation, however, did not result in fetomodulin expression. When primitive endoderm cells were exposed to 1 mM dibutyryl cyclic AMP, the tissue plasminogen activator level rose further within 6 hr. In contrast, the cofactor activity of fetomodulin stayed below a detectable level for as long as 15 hr and then increased with time. Expression of the two marker proteins appeared to be regulated differently.
...
PMID:Differential expression of fetomodulin and tissue plasminogen activator to characterize parietal endoderm differentiation of F9 embryonal carcinoma cells. 169 71

Binding of thrombin to cultured endothelial cells has been studied in the presence of fibrinogen and alpha 2-macroglobulin. Both fibrinogen and alpha 2-macroglobulin inhibit the interaction of thrombin with endothelial cells. Whereas fibrinogen decreases the rate of activation by the thrombin-thrombomodulin complex of protein C, thrombomodulin inhibits the rate of inactivation by alpha 2-macroglobulin thrombin. alpha 2-macroglobulin also binds to endothelial cells; (Kd = 3 x 10(-7) M with 3 x 10(5) binding sites/cell), and the rate of binding of the alpha 2-macroglobulin to endothelial cells is faster than its complex formation with the thrombin. The data suggest that essentially the cell-bound form of fibrinogen and alpha 2-macroglobulin influences thrombin binding and functions.
...
PMID:Interaction of thrombin with endothelial cells in the presence of fibrinogen and alpha 2-macroglobulin. 170 95

Sclerotherapy of bleeding esophageal varices in liver cirrhotics is a common procedure, but little is known about the possible entry of sclerosants into the systemic circulation. We injected a mixture of thrombin, sodium tetradecyl, and cefazolin and studied the effect of this sclerosant on selected hemostasis parameters. Twenty-four patients with liver cirrhosis (Child's Classification C) were studied 29 times. Blood samples were drawn before and immediately after the injection of the sclerosant. In seven patients we collected a sample 30 minutes and 24 hours after treatment. Before injection, almost all patients had elevated D-dimer, t-PA and PAI-1 levels. Fibrinogen, antithrombin, alpha-2 antiplasmin, and protein C were decreased. Only thrombin/antithrombin III complex (TAT) levels were within normal ranges. Immediately after the injection, TAT, D-dimer, and t-PA levels rose significantly (P less than 0.001, P less than 0.01, P less than 0.001), PAI-1 and PC levels decreased (P less than 0.01), while antithrombin, alpha-2 antiplasmin, and fibrinogen concentrations were unchanged. TAT and D-dimer levels were still elevated after 24 hours (P less than 0.05). These data indicate that thrombin entered the systemic circulation (elevated TAT) and that the hemostasis system was briefly systemically activated (elevated D-dimer). In spite of these changes in the hemostasis system, clinically there were no detectable thrombotic or hemorrhagic complications.
...
PMID:Hemostasis activation during esophageal variceal sclerotherapy with thrombin in cirrhotics. 171

Antibody inhibitors against human thrombin are rare and have remained poorly characterized. We report the case of a 40-yr-old patient who developed a potent thrombin inhibitor revealed by mild bleeding symptoms and marked prolongation of most laboratory clotting times. After two years of evolution, he died from cerebral hemorrhage. The inhibitor, a polyclonal IgG, was associated with hematological and immunological criteria of autoimmune disorder. Antithrombin IgG was isolated from the patient's plasma by protein A- and thrombin-affinity chromatography. Fab fragments inhibited amidolytic activity of alpha thrombin, and thrombin-thrombomodulin catalyzed protein C activation with a Ki of approximately 10(-8) M in a noncompetitive manner. Alpha to gamma conversion of thrombin resulted in a moderate loss of affinity for the inhibitor. Upon complex formation of thrombin with staphylocoagulase or alpha 2-macroglobulin (alpha 2M), inhibition was decreased by two orders of magnitude and acquired an apparent competitive character. In Western blot experiments, the antibody reacted with active alpha-thrombin, did not react with chloromethylketone-inhibited thrombin and reacted with a lower affinity with iPr2P-thrombin. The inhibitor did not block thrombin binding to benzamidine-, heparin-, or fibrin-Sepharose, but displaced proflavin from its complex with thrombin. Taken together, these results indicate that the patient's autoantibody recognized a conformational structure which includes, at least in part, the apolar binding site adjacent to the catalytic site of thrombin.
...
PMID:An acquired antithrombin autoantibody directed toward the catalytic center of the enzyme. 171 42

By monitoring the activation of protein C and the regulation of factor Xa-catalyzed thrombin formation by the activated protein C (APC) on the surface of human umbilical vein endothelial cells (HUVEC), we found that functional protein C was synthesized in cultured HUVEC and expressed thereon in the presence of vitamin K. Furthermore, without exogenously added protein S, time-dependent and saturable accumulation of APC (20 fmol APC/10(5) cells) on the surface of HUVEC was observed. During prothrombin activation by the complex of membrane-bound factor Xa and endogenous factor Va formed on the surface of HUVEC, APC was generated, and the rate of thrombin formation decreased. Treatment of HUVEC with an antibody that inhibits the APC-catalyzed inactivation of endogenous factor Va clearly quenched the activity of surface-associated APC. Immunostaining of HUVEC with a horseradish peroxidase (HRP)-conjugated antibody that solely recognizes human protein C confirmed the presence of protein C on the surface of HUVEC. Northern blot analysis revealed that an about 1.8 kb mRNA species derived from HUVEC was hybridized with 32P-labeled protein C cDNA, as in the case of those from HepG2, which are known to synthesize normal protein C. The increase in the amount of protein C mRNA in HUVEC in parallel with cell growth provided supporting evidence for the synthesis of protein C during the culture of HUVEC. These results indicate that blood coagulation is regulated by endogenously generated and activated protein C, together with or without protein S, through inactivation of factor Va on the surface of endothelial cells.
...
PMID:Synthesis of protein C in human umbilical vein endothelial cells. 171 50


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---