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)

A clotting enzyme associated with the hemolymph coagulation system of Japanese horseshoe crab (Tachypleus tridentatus) was highly purified from the amebocyte lysate. The method for purification consisted of gel-filtration of the lysate on a pyrogen-free Sepharose CL-6B column and affinity chromatography of the endotoxin-treated clotting enzyme on a benzamidine-Sepharose 4B column. Through these procedures, about 3 mg of the purified enzyme was obtained from 70 ml of the lysate and about 390-fold purification was achieved. The purified preparation was found to give a single major band, respectively, on polyacrylamide-gel disc electrophoresis at pH 3.2 in the presence of 6 M urea and on sodium dodecyl sulfate (SDS)-gel electrophoresis in the presence and absence of 2-mercaptoethanol. It also gave a single symmetrical peak on QAE-Sephadex A-25 column chromatography. The molecular weight of the clotting enzyme was estimated to be approximately 42,000 for the unreduced sample by SDS-gel electrophoresis. For the reduced sample, it was 30,000, suggesting that the protein consists of plural polypeptide chains bridged by disulfide(s). The Tachypleus clotting enzyme was a glycoprotein, as shown by the positive periodic acid-Schiff reaction for the protein band on SDS-gel and the amino acid analysis. The purified clotting enzyme transformed Tachypleus coagulogen to coagulin gel and hydrolyzed a chromogenic peptide substrate, Tos-Ile-Glu-Gly-Arg-p-nitroanilide for Factor Xa, liberating p-nitroaniline. The enzyme was sensitive to DFP and benzamidine. It was also inhibited partially by PCMB. Antithrombin III and alpha 2-plasmin inhibitor (alpha 2-antiplasmin) were very effective inhibitors of this enzyme among ten kinds of naturally occurring proteinase inhibitors tested. The clotting enzyme had a restricted specificity towards protein substrates and activated only prothrombin among plasma zymogens including Factor IX, Factor X, fibrinogen, plasminogen and prekallikrein. The cleavage sites of bovine prothrombin for this enzyme were the same Arg-Thr and Arg-Ile linkages as those for Factor Xa, resulting in the formation of alpha-thrombin. These results indicate that the horseshoe crab clotting enzyme is a Factor Xa-like serine proteinase rather than alpha-thrombin. It seems likely that the Tachypleus clotting enzyme is a prototype of mammalian serine proteinases participating in blood coagulation.
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PMID:A clotting enzyme associated with the hemolymph coagulation system of horseshoe crab (Tachypleus tridentatus): its purification and characterization. 714 17

Five purified concentrates--Nanotiv (Kabi Pharmacia), Immunine (Immuno), Factor IX VHP (Biotransfusion), Alphanine (Alpha Therapeutic Corporation), and Mononine (Armour Pharmaceutical Company)--were characterized biochemically and their in vivo pharmacokinetic and thrombogenic properties evaluated. The results were compared with those for two prothrombin complex concentrates (PCCs): Preconativ (Kabi Pharmacia) and Prothromplex TIM4 (Immuno). The measured values for factor IX coagulant activity (FIX:C) generally agreed with the manufacturers' labeled values. The purified concentrates were virtually devoid of other vitamin K-dependent coagulation factors, the inhibitor proteins C and S, and either fibrinogen, fibronectin, or immunoglobulins. Indicators of thrombin generation (i.e., prothrombin fragments F1 + 2 and thrombin-antithrombin complex) were present in varying amounts in all preparations. The level of specific activity in the purified concentrates exceeded that in the PCCs by a factor of 50- to 100-fold. Pharmacokinetic variables were studied in severe hemophilia B patients: Nanotiv was compared with Preconativ; Immunine was compared with Prothromplex TIM4 in crossover studies; and Mononine was tested in a single-drug study. No differences were apparent between Nanotiv, Preconativ, and Mononine, but recovery rates were lower, clearance rates higher, and FIX:C half-life shorter for Immunine and Prothromplex TIM4, although the disparate results might have been attributable to methodologic differences. Purified factor IX concentrates were used successfully as cover for surgery and in immune tolerance induction without observable adverse effects.
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PMID:Properties of factor IX concentrates. 757 97

A prospective cross-over study was carried out on 19 patients with haemophilia B. comparing the pharmacokinetics of a purified factor IX concentrate prepared by metal chelate affinity chromatography (9MC) with a conventional three-factor prothrombin complex concentrate (9A). The highly purified factor IX concentrate was shown to have a half-life comparable to the PCC; the in vivo recovery of the purified concentrate was significantly greater than that of the complex (P < 0.01). The 20% change in the value of the International Standard for Factor IX Concentrate, introduced in 1988, might have been expected to lower the recovery values. However, the in vivo recovery for both concentrates was somewhat higher than reported previously, particularly in the older literature. In nine patients, serial assays for fibrinopeptide A, prothrombin fragment F1+2 and thrombin-antithrombin complexes (TAT) were performed to assess the potential thrombogenicity of the two concentrates. Evidence was obtained that there was significantly less activation of coagulation following administration of purified factor IX (9MC), as compared to the activation that occurred after the PCC.
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PMID:A cross-over pharmacokinetic and thrombogenicity study of a prothrombin complex concentrate and a purified factor IX concentrate. 798 19

The purified factor IX concentrates Nanotiv (Kabi Pharmacia), Immunine (Immuno), Factor IX VHP (Bio-transfusion), Alphanine (Alpha) and Mononine (Armour) have been studied in vitro and compared with the prothrombin complex concentrates (PCCs) Preconativ (Kabi Pharmacia) and Prothromplex TIM4 (Immuno). The measured values for factor IX coagulant activity (IX:C) were in good agreement with the manufacturer's label values. In contrast to the PCCs, most of the purified concentrates were virtually devoid of other vitamin K-dependent coagulation factors, the inhibitors protein C and S as well as fibrinogen, fibronectin and immunoglobulins. Indicators of thrombin generation, namely prothrombin fragments 1 and 2 (F 1 + 2) and thrombin-antithrombin complex (TAT), were present in varying amounts in all preparations. The specific activity in the purified concentrates exceeded that in the PCCs by a factor of 50-100. Some differences in purity were found between the purified concentrates. In vivo, Nanotiv was compared with Preconativ and Immunine with Prothromplex TIM4 in crossover studies in patients with severe hemophilia B, and Mononine was tested in a single drug study. Most of the preparations yielded postinfusion increases in TAT, but not in F 1 + 2. Pharmacokinetic variables were analyzed with non-linear curve-fitting combined with model-independent methods. In retrospective comparisons, there were no apparent differences between Nanotiv, Preconativ and Mononine, whereas in vivo recovery seemed lower and the apparent clearance higher for Immunine and Prothromplex. Purified factor IX concentrates were successfully used as cover for surgery or in immune tolerance induction.
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PMID:Biochemical and in vivo properties of high purity factor IX concentrates. 812 33

The human coagulation system continuously generates very small quantities of Factor Xa and thrombin. Current evidence suggests that basal level activation of the hemostatic mechanism occurs via Factor VIIa-dependent activation of Factor X, but direct proof has not been available for the participation of tissue factor in this pathway. To examine this issue, we infused relatively high concentrations of recombinant Factor VIIa (approximately 50 micrograms/kg body wt) into normal chimpanzees and observed significant increases in the plasma levels of Factor IX activation peptide, Factor X activation peptide, and prothrombin activation fragment F1+2. Metabolic turnover studies with radiolabeled Factor IX activation peptide, Factor X activation peptide, and F1+2 indicate that elevated levels of the activation peptides are due to accelerated conversion of the three coagulation system zymogens into serine proteases. The administration of a potent monoclonal antibody to tissue factor, which immediately neutralizes function of the Factor VIIa-tissue factor complex in vitro, abolishes the activation of Factor X and prothrombin mediated by the infused recombinant protein, and also suppresses basal level activation of Factor IX and Factor X. The above results suggest that recombinant Factor VIIa functions as a prohemostatic agent by interacting with endogenous tissue factor sites, but definitive proof will require studies in hemophilic animals using relevant hemostatic endpoints.
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PMID:The activation of factor X and prothrombin by recombinant factor VIIa in vivo is mediated by tissue factor. 837 80

Coagulation reactions normally occur on cell membranes in vivo. Using a cell-based in vitro model system, we have shown that where a factor is located, not simply how much is activated, is critically important in determining its role in hemostasis. Factor Xa activated on a tissue factor (TF)-bearing cell is not equivalent to factor Xa activated on a platelet surface. Factor IX and factor VIII are required for hemostasis because they combine to generate factor Xa on the platelet surface. Factor X activation by factor VIIa/TF does not compensate for a lack of factor IX or VIII because the factor Xa activated by VIIa/TF is located on the wrong surface for efficient thrombin generation.
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PMID:Cellular interactions in hemostasis. 890 66

Interstitial fibrin deposition is a common histologic feature of tubulointerstitial diseases, which suggests that the coagulation system is activated. Thrombin, generated during the activation of the coagulation cascade, is a powerful activating factor for different cell types. Although proximal tubular cells are potential targets for this coagulation factor, no information is available on the effect of thrombin on these cells. Thus, the expression of protease-activated receptor-1 (PAR-1), the main thrombin receptor, was investigated in human proximal tubular cells (hPTC) in vivo and in vitro. A diffuse expression of PAR-1 was observed by immunohistochemistry along the basolateral membrane of PTC in normal human kidney. This observation was confirmed in vitro in cultured hPTC. Because tubular damage and monocyte infiltration are two hallmarks of tubulointerstitial injury, the effect of thrombin on DNA synthesis and monocyte chemotactic peptide-1 (MCP-1) gene and protein expression was evaluated in cultured hPTC. Thrombin induced a significant and dose-dependent increase in thymidine uptake and a striking upregulation of MCP-1 mRNA expression and protein release into the supernatant. Although PAR-1 is a G protein-coupled receptor, its activation in hPTC, as in other cell systems, resulted in a transient increase in cellular levels of tyrosine-phosphorylated proteins. An increased level of tyrosine-phosphorylated c-src suggested the activation of this cytoplasmic tyrosine kinase in response to thrombin and its potential role in thrombin-induced protein-tyrosine phosphorylation. Interestingly, thrombin-induced DNA synthesis and MCP-1 gene expression were completely blocked by genistein, a specific tyrosine kinase inhibitor, but not by its inactive analogue daidzein, demonstrating a central role for tyrosine kinase activation in the thrombin effects on hPTC. Moreover, the specific src inhibitor PP1 abolished the thrombin effect on DNA synthesis. In conclusion, thrombin might represent a powerful regenerative and proinflammatory stimulus for hPTC in acute and chronic tubulointerstitial diseases.
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PMID:Regenerative and proinflammatory effects of thrombin on human proximal tubular cells. 1082 Jan 65

In acute coronary events, plaque rupture and the subsequent formation of the catalytic tissue factor-factor VIIa complex is considered to initiate coagulation. It is unknown whether clotting factors XI and IX are activated in acute coronary events. Therefore, we prospectively investigated the activation of clotting factors XI and IX as well as activation of the contact system and the common pathway in 50 patients with acute myocardial infarction (AMI), in 50 patients with unstable angina pectoris (UAP), and in 50 patients with stable angina pectoris (SAP). Factor XIa-C1 inhibitor complexes, which reflect acute activation of factor XI, were detected in 24% of the patients with AMI, 8% of the patients with UAP, and 4% of the patients with SAP (P<0.05), whereas factor XIa-alpha(1)-antitrypsin complexes, which reflect chronic activation, were observed equally in all 3 study groups. Factor IX peptide levels were significantly higher in the patients with AMI and UAP compared with the patients with SAP (P<0.01). No differences regarding markers of the common pathway were demonstrated. Fibrinopeptide A levels were elevated in patients with AMI compared with patients with UAP and those with SAP (P<0.01). Factor XIIa- or kallikrein-C1 inhibitor complexes were not increased. In conclusion, this is the first demonstration of the activation of clotting factors XI and IX in patients with acute coronary syndromes. Because these clotting factors are considered to be important for continuous thrombin generation and clot stability, their activation might have clinical and therapeutic consequences.
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PMID:Activation of clotting factors XI and IX in patients with acute myocardial infarction. 1107 57

Venous thromboembolic disease, including deep vein thrombosis and pulmonary embolism, is a cause of significant mortality and morbidity. In the US, approximately 260000 cases are diagnosed annually. Current drugs for the prevention and treatment of venous thromboembolism (VTE) include heparin, low-molecular-weight heparins and warfarin. Since they possess several disadvantages, researchers are investigating improved anticoagulants. To understand how any promising anticoagulant would work, a review of the pathophysiology and regulation of the coagulation cascade is provided. The more prominent drugs reviewed include tissue factor pathway inhibitor protein, nematode anticoagulant protein, Factor IX inhibitors, anti-Factor Xa inhibitors (DX-9065a (Daiichi Seiyaku Co Ltd), YM-60828 (Yamanouchi Pharmaceutical Co Ltd), fondaparinux, idraparinux (Sanofi-Synthelabo/NV Organon)), selective thrombin inhibitors (oral heparin, ximelagatran (AstraZeneca plc)) and enhancers of natural anticoagulants (activated protein C, ART-123 (Asahi Kasei Pharma Corp)).
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PMID:New anticoagulants for venous thromboembolic disease. 1473 Apr 67

Factor IX (FIX) deficiency results in haemophilia B and high dose recombinant activated factor VII (rFVIIa) can decrease bleeding. Previously, we showed that FIX deficiency results in a reduced rate and peak of thrombin generation. We have now used plasma and an in vitro coagulation model to examine the effect of these changes in thrombin generation on fibrin clot structure and stability. Low FIX delayed the clot formation onset and reduced the fibrin polymerisation rate. Clots formed without FIX were composed of thicker fibrin fibres than normal. rFVIIa shortened the clot formation onset time and improved the fibre structure of haemophilic clots. We also examined clot formation in the presence of a fibrinolytic challenge by including tissue plasminogen activator or plasmin in the reaction milieu. In these assays, normal FIX levels supported clot formation; however, clots did not form in the absence of FIX. rFVIIa partially restored haemophilic clot formation. These results were independent of the effects of the thrombin-activatable fibrinolysis inhibitor. Our data suggest that rFVIIa enhances haemostasis in haemophiliacs by increasing the thrombin generation rate to both promote formation of a structurally normal clot and improve clot formation and stability at sites with high endogenous fibrinolytic activities.
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PMID:High dose factor VIIa improves clot structure and stability in a model of haemophilia B. 1635 42


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