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)

Distinction between fibrinopeptide A (FPA) and larger polypeptides containing the FPA sequence is critical for the interpretation of clinical results with FPA immunoassay methods. Therefore, the immunochemical reactivity of 14 rabbit anti-FPA sera with six different FPA containing antigens was studied in detail. Antigens tested included: fibrinogen; fragment E of fibrinogen; the amino-terminal disulfide knot of fibrinogen; Aalpha 1(Ala)-51(Met); Aalpha 1(Ala)-23(Arg); and, FPA. Synthetic partial sequences of FPA were also tested. The 14 FPA-specific antisera were divided into 3 distinct categories with: I, FPA immunoreactivity of larger polypeptides containing FPA approximately 1/100 of FPA on a molar basis, II, FPA immunoreactivity of the larger polypeptides intermediate between I and III; and III, FPA immunoreactivity of the larger polypeptides approximately equal to that of FPA on a molar basis. The antigenic determinants of a category I antiserum (R 2) are included in Aalpha 7(Asp)-16(Arg) with Asp(7), Phe(8) and Arg(16) being essential. When attached to FPA, the sequence Gly(17)-Arg(23) decreases the immunoreactivity of FPA with category I antisera 100-fold. The practical consequence of these findings is that, when category III antisera are employed, both FPA and larger FPA-containing polypeptides are equally immunoreactive. Since thrombin treatment of the larger polypeptides does not alter their immunoreactivity, category III antisera cannot discriminate between FPA and the larger polypeptides. On the other hand, with category I antisera, although the immunoreactivity of FPA itself is unaltered by thrombin treatment, larger polypeptides [e.g., Aalpha 1(Ala)-23tArg)] show a 100-fold increase in immunoreactivity following thrombin treatment and thus can readily be identified and separately quantitated. It is concluded that antisera with the specificity of category I are essential for the specific and accurate measurement of FPA, and for its distinction from larger FPA-containing polypeptides, in clinical plasma samples.
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PMID:Specificity of antisera to human fibrinopeptide A used in clinical fibrinopeptide A assays. 6 Jul 91

Rates of hydrolysis of the newly developed peptide chromogenic substrates S-2160 (N-Bz-Phe-Val-Arg-pNA, HCl), S-2238 (H-D-Phe-Pip-Arg-pNA, 2HCl), S-2222 (N-Bz-Ile-Glu-Gly-Arg-pNA, HCl), and S-2251 (H-D-Val-Leu-Lys-pNA, 2HCl) from AB Kabi Peptide Research and Chromozym TH (Z-Gly-Pro-Arg-pNA, HCl) from Pentapharm Limited were tested against highly purified preparations of human plasmin, bovine trypsin, human alpha thrombin, and bovine factor Xa. S-2160, S-2238, and Chromozym TH are sensitive to thrombin, Chromozym TH and S-2238 exhibiting a substantially greater sensitivity than S-2160. All 3 substrates are insensitive to factor Xa but hydrolyzed to varying degrees by plasmin and trypsin. In contrast, S-2222 is sensitive to Xa and insensitive to thrombin. S-2251 is relatively plasmin-specific, being resistant to the clotting enzymes thrombin and Xa. S-2251 exhibits even greater sensitivity to the SK-plasmin complex than to plasmin. In addition, the substrate Chromozym PK (N-Bz-Pro-Phe-Arg-pNA, HCl) was evaluated and found to be relatively specific for plasma kallikrein. Assays for antithrombin III and heparin using S-2222 as the substrate and factor Xa as the enzyme, plasma plasminogen and plasmin inhibitors using S-2251 as the substrate, and plasma prekallikrein and kallikrein inhibitors using Chromozym PK as the substrate have been developed. Synthetic peptides mimicking amino acid sequences adjacent to proteolytic activation cleavage of plasma serine protease precursors appear to be sensitive and relatively specific tools applicable to kinetical and clinical studies of these enzymes and their inhibitors.
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PMID:Serine protease specificity for peptide chromogenic substrates. 14 72

The synthetic thrombin-inhibitor termed No. 205 (N-alpha-dansyl-L-arginine-4-ethyl-piperidine amide) found in our laboratories was studied kinetically using synthetic peptide substrates. The following results were obtained. 1. No. 205 inhibited thrombin competively with bz-Phe-Val-Arg-pNA and the Ki value obtained was extremely small, 3.7 x 10(-8) M. 2. No. 205 also inhibited trypsin competitively with bz-Phe-Val-Arg-pNA but the Ki value obtained was far larger than that for thrombin, 1.0 x 10(-5) M. 3. No. 205 inhibited F. Xa, plasmin and urokinase only to a small extent when estimated using 2 x 10(-4) M D-Val-Leu-Lys-pNA, bz-Ile-Glu-Gly-Arg-pNA and Glu-Gly-Arg-pNA, respectively. 4. No 205 differed from APPA in its specific inhibitory spectrum for thrombin as compared to trypsin, plasmin and F. Xa. The above results indicate that No. 205 is an extremely potent and highly selective reversible thrombin-inhibitor.
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PMID:Kinetic studies on the selectivity of a synthetic thrombin-inhibitor using synthetic peptide substrates. 15 13

The 1H and 13C spectra of four peptides, L-Phe-Val-Arg-pNA, D-Phe-Val-Arg-pNA, L-Phe-Pip-Arg-pNA and D-Phe-Pip-Arg-pNA (pNA = p-nitroaniline, Pip = pipecolic acid residue), have been examined, and deductions made about their conformation in solution. The D-Phe peptides, which are cleaved especially rapidly by thrombin in water, have structures (in deuterated DMSO) in which the aromatic ring of the D-Phe residue is folded back over the Val or Pip residue. This arrangement is not found in the L-Phe peptides. It is proposed that this feature (in which Phe could be situated near Val and near the Arg-Gly bond of the A alpha chain in the three-dimensional structure of fibrinogen) may be especially advantageous for binding to the enzyme.
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PMID:H and 13C nuclear magnetic resonance spectra of some peptides with fibrinogen-like reactivity. 42 3

An automated method is presented for the determination of thrombin generation in plasma during activation by thromboplastin. The thrombin generated is allowed to split the chromogenic substrate Tol-Gly-Pro-Arg-pNA yielding p-nitroaniline. The increase in absorbance at 410 nm is recorded. This assay may serve as a specific, precise and fast alternative for the conventional clotting tests: "Prothrombin time" and "Thrombotest".
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PMID:An automated amidolytic assay of thrombin generation: an alternative for the prothrombin-time test. 43 85

Using the chromogenic substrate, Tos-Gly-Pro-Arg-pNA-HCl (Chromozym TH, Boehringer Mannheim) plasma thrombin was estimated in six cases of envenomation by Australian elapid snakes. All patients manifested findings characteristic of defibrination due to envenomation by these snakes. Fibrin-fibrinogen degradation products were grossly elevated, as was plasma thrombin in all cases. Following treatment with antivenene, all abnormal coagulation parameters returned rapidly towards normal by 24 hours and plasma thrombin disappeared.
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PMID:Plasma thrombin assay using a chromogenic substrate in disseminated intravascular coagulation due to snake bite envenomation. 46 21

Thrombocytin, a platelet-activating enzyme from Bothrops atrox venom, has been purified to homogeneity by precipitation with sodium salicylate and chromatography on heparin--agarose. Thrombocytin is a single-chain glycoprotein with a molecular weight of 36 000 which contains 5.6% carbohydrate. It causes platelet aggregation, release of platelet serotonin, and activation of factor XIII. The most sensitive substrate for the amidolytic activity of thrombocytin was Tos-Gly-Pro-Arg-p-nitroanilide hydrochloride. The activity of thrombocytin on this substrate and on platelets was inhibited by diisopropyl fluorophosphate (DFP), soybean trypsin inhibitor, and several arginine chloromethyl ketones. Active site titration with nitrophenyl guanidinobenzoate demonstrated that approximately 86% of the preparation was in the active form. These experiments demonstrate the presence of serine and histidine in the active site of thrombocytin and suggest that thrombocytin is a classical serine protease with a platelet-activating activity similar to thrombin.
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PMID:Thrombocytin, a serine protease from Bothrops atrox venom. 1. Purification and characterization of the enzyme. 47 68

Thrombocytin, a serine protease from Bothrops atrox venom, caused platelet aggregation and release of platelet constituents at a concentration of 10(-7) M and clot retraction at a concentration of 2 x 10(-9) M. Thrombocytin was slightly more active when tested on platelets in plasma than on washed platelets suspended in Tyrode--albumin solution. Thrombin was 5 times more active than thrombocytin when tested on platelets in plasma and 50 times more active when tested on washed platelets. The patterns or release induced by thrombocytin and thrombin were similar. Prostaglandin E1 (10(-5) M) produced complete inhibition of platelet release induced by thrombocytin and thrombin. Indomethacin (10(-4) M) was without any effect. Antithrombin III, in the presence of heparin, inhibited the action of thrombocytin on platelets and on a synthetic peptide substrate (Tos-Gly-Pro-Arg-pNA.HCl). formation of an antithrombin III--thrombocytin complex was demonstrated on NaDodSO4--polyacrylamide gel electrophoresis. Hirudin and alpha 1-antitrypsin did not inactivate thrombocytin. Thrombocytin had a low fibrinogen-clotting activity (less than 0.06% that of thrombin). Thrombocytin also caused progressive degradation of the alpha chain of human fibrinogen, and it cleaved prothrombin, releasing products similar to intermediate 1 and fragment 1 produced by thrombin. Thrombocytin activated factor XIII by limited proteolysis and increased the procoagulant activity of factor VIII in a manner analogous to that of thrombin.
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PMID:Thrombocytin, a serine protease from Bothrops atrox venom. 2. Interaction with platelets and plasma-clotting factors. 47 69

The ability of heparin fractions of different molecular weight to potentiate the action of antithrombin III against the coagulation factors thrombin and Xa has been examined in purified reaction mixtures and in plasma. Residual thrombin and Xa have been determined by their peptidase activities against the synthetic peptide substrates H-D-Phe-Pip-Arg-pNA and Bz-Ile-Gly-Arg-pNA. High molecular weight heparin fractions were found to have higher anticoagulant activities than low molecular weight heparin when studied with both thrombin and Xa incubation mixtures in purified mixtures and in plasma. The inhibition of thrombin by heparin fractions and antithrombin III was unaffected by other plasma components. However, normal human plasma contained a component that inhibited the heparin and antithrombin III inhibition of Xa particularly when the high molecular weight heparin fraction was used. Experiments using a purified preparation of platelet factor 4 suggested that the platelet-derived heparin-neutralizing protein was not responsible for the inhibition.
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PMID:Evidence for a plasma inhibitor of the heparin accelerated inhibition of factor Xa by antithrombin III. 47 56

Fibrinogen Zurich I is characterized by an abnormal fibrin monomer polymerization. It consists of two fractions of molecules, one with a normal aggregation and one not aggregating at all and interfering with the aggregation of the normal population. Using a radioimmunoassay for fibrinopeptide A, only approximately half of the expected fibrinopeptide A could be recovered after thrombin or Defibrase proteolysis. The defective fibrinopeptide A release could be confirmed by measurement of the N-terminal Gly/Tyr ratio. It is likely that the abnormal fibrin monomer aggregation of the abnormal fraction of fibrinogen Zurich I is due to the defective fibrinopeptide A release of this fraction.
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PMID:Fibrinogen Zurich I: impaired release of fibrinopeptide A. 48 44


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