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)

Several laboratory methods are available to measure the anticoagulant activity of recombinant hirudin (r-hirudin), a potent thrombin inhibitor. These assays include clot-based, amidolytic, immunologic and physicochemical techniques. Although r-hirudin, like heparin, is an effective anticoagulant, the mechanism of action of the two agents is different. Thus it is not surprising that the global tests, such as the prothrombin time (PT), partial thromboplastin time (APTT) and the Heptest (Haemachem, Inc., St. Louis, Mo., USA), do not show adequate responses to r-hirudin. In the range of 0.5-10.0 microgram/ml, where full anticoagulation is achieved, as determined by animal models of thrombosis, these assays show little to no prolongation of the time to clot. In order to find a more suitable assay system, modifications of the above assays were evaluated. The diluted APTT and diluted Heptest showed linear concentration-dependent responses to lower levels of r-hirudin with an enhanced sensitivity than that of the classical assays. On the other hand, the diluted thrombin time was too sensitive. Whole-blood clotting assays, ACT and thrombelastograph, effectively measured r-hirudin levels up to 25 micrograms/ml. The amidolytic anti-factor IIa assay, specific for evaluating direct thrombin inhibition, was very effective particularly when modified to decrease the sample:thrombin ratio. This assay may be useful in quality control since it is biochemically defined, and reagents are easily standardized. The relevance of the results of the anti-IIa assay to clinical conditions, however, remains to be determined. Thrombin generation assays have limited value in monitoring the anticoagulant effect of r-hirudin since the effect of thrombin inhibition by r-hirudin on coagulation feedback mechanisms, and thus the effect on thrombin generation, appears to be minimal. Immunologic methods such as ELISA and RIA are under development, but they may only be useful for the direct quantitation of absolute levels of r-hirudin and not for monitoring the clinical anticoagulant action. Furthermore, these assays are only sensitive to sub-microgram/ml levels. Therefore, thrombin-based clotting and amidolytic assays may at present be the best choice for evaluating the functional, clinical antithrombotic effects of r-hirudin.
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PMID:Laboratory assays for the evaluation of recombinant hirudin. 130 Oct 38

A fraction separated from rat submandibular gland homogenates was found to contain a potent vasoconstrictor when tested on isolated rabbit aortic rings. The vasoconstrictor was purified by a series of chromatographic steps. The purified compound (2.77 x 10(-9) M) induced 40% of the maximum contractile response to 60 mM KCl. Constriction was slow in onset, long-lasting, rinse-resistant, and unchanged by de-endothelialization; in addition, it was dose-related and inhibited by both EGTA and verapamil, but it was not affected by DUP753, an angiotensin II receptor antagonist. The compound was found to be a protein having a pI of 7.36 and a molecular weight of approximately 29,000 and exhibiting partial immunologic identity to rat glandular kallikrein and rat tonin. After 2-mercaptoethanol treatment, it separated into heavy (approximately 19,900) and light (approximately 10,700) chains having amino-terminal sequences of AY(X)HNNDLMLL and VVGGYN(X)ETNSQ, respectively. We found that they correspond to the amino-terminal and internal sequence of a previously unidentified kallikrein-like serine protease whose mRNA, named S3, has been found in the rat submandibular gland and prostate. The vasoconstrictor is able to hydrolyze t-butoxycarbonyl-valine-proline-arginine-methylcoumarin amide (a thrombin substrate), although its Kcat/Km was only 0.02% that of rat thrombin. Both vasoconstrictor and enzymatic activity on t-butoxycarbonyl-valine-proline-arginine-methylcoumarin amide were completely suppressed by amidinophenylmethylsulfonyl fluoride and soybean trypsin inhibitor; however, they were unaffected by hirudin, a thrombin inhibitor. At pH 6.5, it released angiotensin II when incubated with sheep angiotensinogen, although it had approximately one-tenth the activity of tonin. The submandibular enzymatic vasoconstrictor is a kallikrein-like enzyme, having some properties of both tonin and thrombin. It directly contracts vascular smooth muscle, acting via a mechanism that requires intact enzymatic activity.
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PMID:A novel serine protease with vasoconstrictor activity coded by the kallikrein gene S3. 190 May 13

Hirudin is the most potent natural inhibitor of thrombin known to date. It is gaining popularity as an anticoagulant now that recombinant and synthesized forms are available. It is a monospecific and co-factor-independent thrombin inhibitor with otherwise inert pharmacological properties. Being a surprisingly weak immunogen, its administration has exhibited no side effects, particularly on platelets. Bleeding complications are not to be expected at therapeutic doses. Effective anticoagulatory doses can be easily predicted and laboratory control is no problem. Application of hirudin or derivatives thereof may be indicated for: prophylaxis and treatment of postoperative venous thrombosis and diffuse microthrombosis; prevention of arterial thrombosis, especially in cardiac surgery; enhancement of fibrinolytic therapy and/or angioplasty to prevent reocclusion; extracorporeal circulation; and plastic surgery. Hirudin may be a particularly useful alternative anticoagulatory agent in patients sensitized to heparin or in patients with hereditary or acquired antithrombin III deficiency. However, whether hirudin is really an effective therapeutic tool and whether it can replace heparin in certain clinical indications can be judged only after extended clinical experience has been accumulated.
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PMID:Hirudin, a new therapeutic tool? 191 33

The initial outgrowth of neuritogenesis in mouse NB2a/d1 neuroblastoma cells may be regulated by thrombin or a thrombin-like protease, present either in serum or adsorbed to the plasma membrane, since neuritogenesis is induced by serum deprivation and treatment with the specific thrombin inhibitor, hirudin (Shea et al., 1991, J. Neurochem., 56:842). Cultured astroglial cells secrete factors that promote neuritogenesis, including protease inhibitors active against thrombin, leading to suggestions that the inhibition of specific neuronal surface proteases by the surrounding glial environment may represent an initial step in axonal outgrowth in situ. To examine the relative importance of glial-derived protease inhibitory activities on neurine outgrowth, we tested the neurite promoting effect of glial-conditioned medium (GCM) on NB2a/d1 cells. Like serum deprivation and hirudin treatment, GCM induced neurite outgrowth within 4 hr. Exogenous thrombin inhibited the effect of GCM, and cell-free enzyme assays confirmed the presence of thrombin-inhibitory activity in GCM, suggesting that GCM induces neuritogenesis by inhibition of a thrombin-like protease. Unlike neurites induced by serum removal or hirudin addition, which are rapidly resorbed following serum replenishment or hirudin depletion, however, GCM-induced neurites continued to elongate after GCM removal. Furthermore, cultures treated simultaneously with GCM and thrombin exhibited delayed outgrowth of neurites following GCM removal which were insensitive to further thrombin treatment. These findings indicate that the initial elaboration of neurites can be mediated by glial-derived protease inhibitor(s) active against a thrombin-like protease, but indicate the requirement of additional glial-derived factors for the maintenance and continued elaboration of these neurites.
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PMID:Transient neuritogenesis in NB2a/d1 neuroblastoma cells induced by glial-derived protease inhibitors. 191 50

Increase of TAT is reflected by the generation of thrombin in hypercoagulable state. TAT might increase in DIC characterized by the formation of disseminated micro-thrombosis. DIC was classified into three groups according to the results of screening tests (FDP, platelet count, fibrinogen, prothrombin time). TAT values significantly increased in the stage of pre-DIC compared with the control group consisting of DIC prone underlying disease. Pre-DIC was easily detected by an increase of TAT during the clinical course. Management of high TAT began with the use of an anticoagulant such as heparin under the condition of sufficient ATIII level. The lowering effect of TAT was easily obtained by the anticoagulant. In ATIII-deficient DIC, the high TAT reduced with the substitution of ATIII concentrate, though a transient increase of TAT was found during the administration of ATIII. To reduce the high TAT under the deficient state of ATIII, MD805, a synthetic thrombin inhibitor, was introduced to avoid further consumption of ATIII. The TAT was decreased by the use of MD805 without administration of ATIII. MD805 could be used as an effective anticoagulant in high TAT due to DIC under an ATIII-deficient state. Although the TAT improved with an adequate anticoagulation in DIC, spontaneous bleeding sometimes appeared as a complication associated with the high level of alpha 2 plasmin inhibitor plasmin complex. In this case, the combined use of tranexamic acid relieved the bleeding.
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PMID:[Thrombin.antithrombin III complex]. 192 Aug 62

Since thrombin causes an endothelium-dependent relaxation of precontracted pig coronary arteries, the ability of thrombocytin, a serine proteinase from the venom of the common lancehead, Bothrops atrox, to induce endothelium-dependent changes in the vascular tone was investigated. Relaxation of pig coronary rings did not appear in vessels denuded of the endothelium. Thrombocytin (0.1-2.0 micrograms/ml) caused an endothelium-dependent, reversible, transient relaxation of PGF2 alpha-precontracted arteries which could be blocked by heparin and relatively high concentrations of alpha-NAPAP, a synthetic competitive thrombin inhibitor. Indomethacin and hirudin did not influence the relaxant effect. Both the thrombocytin- and bradykinin-induced relaxation were diminished by the guanylate cyclase inhibitor methylene blue and by NG-monomethyl-L-arginine. The thrombocytin-induced relaxation was absent in de-endothelialized vessels. Thrombocytin was able to induce aggregation of human blood platelets in Tyrode's solution at the same concentration range as used for the relaxation. Batroxobin neither relaxed precontracted arteries nor aggregated human blood platelets in vitro. The present studies show that the serine proteinase thrombocytin is not only able to aggregate platelets but may also release endothelium-derived relaxing factor from the vascular endothelium.
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PMID:Endothelium-dependent relaxant effect of thrombocytin, a serine proteinase from Bothrops atrox snake venom, on isolated pig coronary arteries. 192 73

The efficacy and specificity of a novel synthetic thrombin inhibitor, DuP 714, on thrombin-induced elevation of cytoplasmic calcium, fibrinogen binding and aggregation in human platelets were examined. Thrombin (0.5 U/ml) stimulated an increase in [125I]fibrinogen binding in gel-filtered platelets which was blocked by DuP 714 with an IC50 value of 2 nM. Thrombin (1 U/ml)-induced elevation of intracellular [Ca2+]i was also blocked by DuP 714 with an IC50 value of 67 nM. A much higher concentration of thrombin (25 U/ml) was used to stimulate aggregation with heparinized platelet-rich plasma. Under these conditions, micromolar concentrations of DuP 714 were needed to inhibit thrombin. In all of these preparations, DuP 714 at concentrations as high as 10(-5) M had no intrinsic effects and did not affect the responses induced by arachidonate, ADP, collagen, epinephrine, vasopressin and serotonin. These data indicate that DuP 714 is a potent and specific thrombin inhibitor capable of arresting the actions of thrombin on human fibrin formation and platelet aggregation and secretion. It may serve as a potential antithrombotic agent for various forms of thrombotic disorders.
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PMID:Inhibition of the thrombin-platelet reactions by DuP 714. 193 Jan 90

Structural and functional properties of alpha-protease nexin I (alpha-PNI) expressed in Chinese hamster ovary cells were studied. All three cysteines were in the reduced form, showing that the potential disulfide bridge between residues Cys117 and Cys131 was not formed. Heparin association rate enhancements were from ka = 8.3 x 10(5) to 0.7-1.6 x 10(9) M-1 s-1 for the interaction of PNI with thrombin, from ka = 5.1 x 10(3) to 3.5 x 10(5) M-1 s-1 for interaction with Factor Xa, and from ka = 2.2 x 10(6) to 1.0 x 10(7) M-1 s-1 for interaction with trypsin; there was no rate enhancement of the plasmin interaction (ka = 1.0 x 10(5) M-1 s-1). The minimal heparin pentasaccharide had no effect on these interactions. Cleavage of the reactive center loop of PNI by three different proteases gave the typical stressed to relaxed change in thermal stability, but unlike with antithrombin III, there was no loss of heparin affinity. A similar difference from antithrombin was that PNI-thrombin complexes retained normal heparin affinity. These results are compatible with a role for protease nexin I as a cell-associated thrombin inhibitor that remains bound to the cell surface even after complexing with the protease, as compared with the role of antithrombin III as a circulating inhibitor of thrombin that becomes activated on binding to the microvasculature and is released on complex formation.
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PMID:Protease specificity and heparin binding and activation of recombinant protease nexin I. 193 53

Esterolytically inactive diisopropyl fluorophosphate-conjugated thrombin (DIP-alpha-thrombin) stimulated 3H-thymidine incorporation and proliferation of growth-arrested vascular smooth muscle cells (SMCs), similar to native alpha-thrombin. Half-maximal mitogenic response of SMCs was obtained at 1 nM thrombin and was specifically blocked by the leech-derived, high-affinity thrombin inhibitor, hirudin. Native thrombin and a variety of thrombin species that were chemically modified to alter thrombin procoagulant or esterolytic functions were found to induce 3H-thymidine incorporation to a similar extent. Exposure of SMCs to DIP-alpha-thrombin caused a rapid and transient expression of the c-fos protooncogene, determined by Northern blot analysis. These results indicate that thrombin is a potent mitogen for SMCs through a distinct non-enzymatic domain. Binding of 125I-alpha-thrombin to SMC cultures revealed an apparent dissociation constant of 6 nM and an estimated 5.4 x 10(5) binding sites per cell. This binding was inhibited to the same extent by native thrombin and by its nonenzymatic form, DIP-alpha-thrombin. Moreover, the chemotactic fragment of thrombin (CB67-129), which failed to elicit a mitogenic response, competed for 125I-alpha-thrombin binding to SMCs. Cross-linking analysis of 125I-alpha-thrombin to SMCs revealed a specific cell-surface binding site 55 kDa in size. Finally, thrombin immobilized to a naturally produced extracellular matrix retained potent mitogenic activity toward SMCs. These observations lend support to the possibility that in vivo, subendothelial basement membranes sequester thrombin (as well as other bioactive molecules), which may stimulate localized and persistent growth of arterial SMCs. Thrombin may thus be involved directly in progression of atherosclerotic plaque formation.
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PMID:Thrombin immobilized to extracellular matrix is a potent mitogen for vascular smooth muscle cells: nonenzymatic mode of action. 196 93

Heparin cofactor II (HC) is a plasma serine proteinase inhibitor (serpin) that inhibits the coagulant proteinase alpha-thrombin. We have recently demonstrated that proteolysis of HC by catalytic amounts of polymorphonuclear leukocyte proteinases (elastase or cathepsin G) generates leukocyte chemotaxins (Hoffman, M., Pratt, C. W., Brown, R. L., and Church, F. C. (1989) Blood 73, 1682-1685). One of four peptides produced when HC is degraded by neutrophil elastase has chemotactic activity for both monocytes and neutrophils with maximal migration comparable to formyl-Met-Leu-Phe, the "gold standard" bacterially derived chemotaxin. The amino-terminal sequence of this HC peptide is Asp-Phe-His-Lys-Glu-Asn-Thr-Val-... and the peptide corresponds to Asp-39 to Ile-66 of HC. A variety of synthetic peptides derived from this sequence were evaluated for leukocyte migration activity, and a dodecapeptide from Asp-49 to Tyr-60 (Asp-Trp-Ile-Pro-Glu-Gly-Glu-Glu-Asp-Asp-Asp-Tyr) was identified as the active site for leukocyte chemotactic action. The 12-mer synthetic peptide possesses significant neutrophil chemotactic action at 1 nM (60% of the maximal activity of formyl-Met-Leu-Phe), while a peptide with the reverse sequence has essentially no chemotactic activity. Cross-desensitization experiments also show that pretreatment of neutrophils with a 19-mer peptide (Asn-48 to Ile-66) greatly reduces subsequent chemotaxis to HC-neutrophil elastase proteolysis reaction products. When injected intraperitoneally in mice, the HC-neutrophil elastase digest elicits neutrophil migration. Our results demonstrate that not only does HC function as a thrombin inhibitor, but that limited proteolysis of HC near the amino terminus yields biologically active peptide(s) which might participate in inflammation and in wound healing and tissue repair processes.
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PMID:Leukocyte chemoattractant peptides from the serpin heparin cofactor II. 198 58


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