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:3.4.21.6 (thromboplastin)
13,278 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A recent study indicated that Tyr99 (chymotrypsin numbering) of factor Xa and Thr99 of activated protein C are S2 subsite residues that determine the P2 specificity of their substrates and inhibitors. To investigate the contribution of Leu99 to the P2 binding specificity of thrombin, three mutants of thrombin were prepared in which Leu99 was substituted with Tyr (L99Y), Thr (L99T), or Gly (L99G). Kinetic analysis indicated that antithrombin (AT with P2 Gly) inhibited thrombin L99Y, 14.1- and 5.5-fold slower than thrombin in the absence and presence of heparin, respectively. The L99Y mutation increased the stoichiometry of AT inhibition in the presence of heparin from approximately 1.6 to approximately 4.6, indicating that L99Y recognized AT as a substrate. The inhibition rates of L99T and L99G by AT, respectively, were 500.0- and 916.7-fold slower than thrombin in the absence of heparin but only 41.8- and 64.5-fold slower than thrombin in the presence of heparin. Resolution of the two-step reactions of AT with the mutant thrombins revealed that the impaired reactivities occurred in the second reaction step in which a non-covalent AT-thrombin encounter complex is converted to a stable, covalent complex. In reactions with protein C inhibitor (PCI with P2 Phe), L99Y was inhibited 3.5-fold slower than thrombin, L99T was inhibited at a similar or faster rate, and L99G was inhibited 23.9-fold faster than thrombin. The epidermal growth factor-like domains 4-6 of thrombomodulin (TM4-6) accelerated the PCI inhibition of wild-type and L99G thrombins 73.9- and 5.3-fold, respectively. Further studies indicated that the fibrinogen clotting and protein C activation rates by the mutants were impaired, but the cofactor function of TM was not affected as TM4-6 bound to wild-type [Kd(app) = 5.9 nM] and mutant thrombins with similar affinities [Kd(app) = 4.4-6.9 nM] and enhanced protein C activation rates by all mutants effectively. These results indicate that (1) Leu99 of thrombin is critical for determination of the P2 specificity of serpins, AT and PCI, (2) increasing the polarity of the S2 pocket of thrombin by introduction of a hydrophilic residue into this pocket is detrimental for reaction with AT, but it is tolerated in reaction with PCI, so that only the size of the S2 pocket of thrombin determines the P2 specificity of PCI, and (3) the thrombomodulin-induced conformational change that results in acceleration of thrombin inhibition by PCI involves Leu99.
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PMID:Role of Leu99 of thrombin in determining the P2 specificity of serpins. 920 Jun 92

In order to investigate the respective roles of prothrombinase and intrinsic tenase (IXa/VIIIa) in venous thrombosis, we compared the anti-thrombotic efficacy of inhibitors of these two coagulation complexes. The agents tested were dansyl-Glu-Gly-Arg chloromethyl ketone-inactivated bovine factor IXa(IXai) and Glu-Gly-Arg chloromethyl ketone-inactivated human factor Xa (Xai). In vitro formation of active complexes (prothrombinase or tenase) was inhibited by Xai and IXai resulting in IC50 values of 3 nM and 5 nM, respectively. Antithrombotic activity was measured by inhibition of clot accretion on cotton threads placed in the abdominal vena cava of anesthetized rabbits. Intravenous bolus dosing followed by infusion of Xai during the experimental protocol resulted in a dose dependent reduction of clot weight, a dosage of 16.0 micrograms/kg + 0.28 microgram/kg/min being sufficient to produce a 96% inhibition of thrombosis. A much higher dose of IXai (1.0 mg/kg + 17.3 micrograms/kg/min) resulted in a 39% reduction of clot weight. In a rabbit arterio-venous shunt model mimicking arterial thrombosis, the relative efficacy of the two agents was found to be more comparable. The doses required for optimum antithrombotic activity were 128.0 micrograms/kg + 2.2 micrograms/kg/min for Xai and 1.0 mg/kg + 17.3 micrograms/kg/min for IXai. We conclude that, in this study, prothrombinase rather than tenase inhibition was more effective in reducing venous thrombosis and that these effects can be achieved without disruption of extravascular hemostasis.
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PMID:Relative efficacy of active site-blocked factors IXa, Xa in models of rabbit venous and arterio-venous thrombosis. 924 47

Human factor X is a two-chain, 58-kDa, vitamin K-dependent blood coagulation zymogen. The light chain of factor X consists of an NH2-terminal gamma-carboxyglutamic acid (Gla) domain, followed by a few helical hydrophobic residues and the two epidermal growth factor-like domains, whereas the heavy chain contains the serine protease domain. In this study, native factor X was found to contain three classes of Ca2+-binding sites: two high affinity (Kd 100 +/- 30 microM), four intermediate affinity (Kd 450 +/- 70 microM), and five to six low affinity (Kd 2 +/- 0.2 mM). Decarboxylated factor X in which the Gla residues were converted to Glu retained the two high affinity sites (Kd 140 +/- 20 microM). In contrast, factor X lacking the Gla domain as well as a part of the helical hydrophobic residues (des-44-X) retained only one high affinity Ca2+-binding site (Kd 130 +/- 20 microM). Moreover, a synthetic peptide composed of residues 238-277 (58-97 in chymotrypsinogen numbering) from the protease domain of factor X bound one Ca2+ with high affinity (Kd 150 +/- 20 microM). From competitive inhibition assays for binding of active site-blocked factor Xa to factor Va in the prothrombinase complex, the Kd for peptide-Va interaction was calculated to be approximately 10 microM as compared with 30 pM for factor Xa and approximately 1.5 microM for decarboxylated factor Xa. A peptide containing residues 238-262(58-82) bound Ca2+ with reduced affinity (Kd approximately 600 microM) and did not inhibit Xa:Va interaction. In contrast, a peptide containing residues 253-277(73-97) inhibited Xa:Va interaction (Kd approximately 10 microM) but did not bind Ca2+. In additional studies, Ca2+ increased the amidolytic activity of native and des-44-Xa toward a tetrapeptide substrate (benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide) by approximately 1.6-fold. The half-maximal increase was observed at approximately 150 microM Ca2+ and the effect was primarily on the kcat. Ca2+ also significantly protected cleavage at Arg-332-Gln-333(150-151) in the protease domain autolysis loop. Des-44-Xa in which the autolysis loop was cleaved possessed </=5% of the amidolytic activity of the noncleaved form; however, the S1 binding site was not affected, as determined by the p-aminobenzamidine binding. Additionally, autolysis loop-cleaved, active site-blocked native factor Xa was calculated to have approximately 10-fold reduced affinity for factor Va as compared with that of the noncleaved form.
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PMID:Interaction of calcium with native and decarboxylated human factor X. Effect of proteolysis in the autolysis loop on catalytic efficiency and factor Va binding. 926 43

Tissue factor pathway inhibitor (TFPI) is a Kunitz-type protease inhibitor with three tandem inhibitory domains (K1, K2, and K3) that regulates the initial reactions of the extrinsic blood coagulation pathway through K1 and K2. In the present study, the effect of thrombin on TFPI in a purified system was first examined using recombinant TFPI from Chinese hamster ovary (CHO) cells. TFPI was inactivated by thrombin with cleavage of three peptide bonds, Lys 254-Thr 255 in the C-terminal basic region, Arg 107-Gly 108 (reactive site toward factor Xa in K2), and Lys 86-Thr 87 between K1 and K2. Then, degradation of radiolabeled TFPI by thrombin was examined in two systems: (1) mixed with plasma and then tissue factor (TF) and calcium ion, and (2) mixed with fibrinogen and then thrombin. TFPI degradation was detected in serum from normal plasma and more extensively from anti-thrombin (AT)-depleted plasma by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Significant radioactivity was found in the clot after coagulation of the plasma, which decreased after 20 hours' incubation. These changes were more prominent in AT-depleted plasma than in normal plasma. When TFPI lacking the C-terminal basic region was used instead of full-length TFPI, most of the radioactivity was found in serum rather than in fibrin clots. Incorporation of TFPI into the fibrin clot was prevented by a synthetic C-terminal peptide of TFPI. Similar results were obtained after mixing radiolabeled TFPI with fibrinogen and then thrombin in the presence of calcium ion or EDTA. These results demonstrate a novel degradation pathway of TFPI, ie, incorporation into fibrin via the C-terminal basic region and degradation by thrombin (possibly fibrin-bound thrombin).
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PMID:A novel degradation pathway of tissue factor pathway inhibitor: incorporation into fibrin clot and degradation by thrombin. 929 21

The binding of 125I-factor Xa to human aortic smooth muscle cell (SMC) monolayers was studied. At 4 degreesC, 125I-factor Xa bound to a single class of binding sites with a dissociation constant value of 3.6+/-0.7 nM and a binding site density of 11,720+/-1,240 sites/cell (n = 9). 125I-factor Xa binding was not affected by factor X, thrombin, or by DX9065, a direct inhibitor of factor Xa, but was inhibited by factor Xa (IC50 = 5.4+/-0.2 nM; n = 9) and by antibodies specific for the effector cell protease receptor 1 (EPR-1), a well-known receptor of factor Xa on various cell types. A factor X peptide duplicating the inter-EGF sequence Leu83-Leu88-(Gly) blocked the binding of 125I-factor Xa to these cells in a dose-dependent manner (IC50 = 110+/-21 nM). Factor Xa increased phosphoinositide turnover in SMCs and when added to SMCs in culture was a potent mitogen. These effects were inhibited by DX9065 and by antibodies directed against EPR-1 and PDGF. Increased expression of EPR-1 was identified immunohistochemically on SMCs growing in culture and in SMCs from the rabbit carotid artery after vascular injury. When applied locally to air-injured rabbit carotid arteries, antibodies directed against EPR-1 (100 mug/ artery) strongly reduced myointimal proliferation 14 d after vascular injury (65-71% inhibition, P < 0.01). DX9065 (10 mg/kg, subcutaneous) inhibited myointimal proliferation significantly (43% inhibition, P < 0.05). These findings indicate that SMCs express functional high affinity receptors for factor Xa related to EPR-1, which may be of importance in the regulation of homeostasis of the vascular wall and after vascular injury.
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PMID:Effector protease receptor 1 mediates the mitogenic activity of factor Xa for vascular smooth muscle cells in vitro and in vivo. 948 69

A 64-year-old white male was referred for evaluation of prolonged prothrombin time (PT) and activated partial thromboplastin time (aPTT) obtained before elective surgery with initial PT and PTT results of 14.9 and 38.4 seconds, respectively, which corrected to normal in 1:1 mixes with normal plasma. Functional prothrombin assay indicated a level of 51% with thromboplastin as an activator. The prothrombin antigen was 102%. This discordance in the functional and immunologic prothrombin levels was evidence for dysprothrombinemia. Western blotting showed that thrombin was formed at a normal rate in diluted plasma consistent with a mutation within the thrombin portion of prothrombin. DNA was isolated from leukocytes and the thrombin exons were amplified by polymerase chain reaction, cloned, and sequenced. For exon 13, eight clones were sequenced with four clones showing a point mutation in the codon for Arg517, which would result in substitution by Gln. Arg517 is part of the Arg-Gly-Asp(RGD) sequence in thrombin and contributes to an ion cluster with aspartic acid residues 552 and 554. Mutation at this residue most probably distorts the structure of the Na+ binding site in thrombin. This is the first report indicating the critical role of Arg517 in the normal physiological interaction of thrombin with fibrinogen. This dysprothrombin is designated Prothrombin Greenville.
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PMID:Prothrombin Greenville, Arg517-->Gln, identified in an individual heterozygous for dysprothrombinemia. 949 Jun 87

The structure of two selective inhibitors, Ac-Tyr-Ile-Arg-Ile-Pro-NH2 and Ac-(4-Amino-Phe)-(Cyclohexyl-Gly)-Arg-NH2, in the active site of the blood clotting enzyme factor Xa was determined by using transferred nuclear Overhauser effect nuclear magnetic resonance (NMR) spectroscopy. They represent a family of peptidic inhibitors obtained by the screening of a vast combinatorial library. Each structure was first calculated by using standard computational procedures (distance geometry, simulated annealing, energy minimization) and then further refined by systematic search of the conformation of the inhibitor docked in the active site and repeating the simulated annealing and energy minimization. The final structure was optimized by molecular dynamics simulations of the inhibitor-complex in water. The NMR restraints were kept throughout the refinement. The inhibitors assume a compact, very well defined conformation, embedded into the substrate binding site not in the same way as a substrate, blocking thus the catalysis. The model allows to explain the mode of action, affinity, and specificity of the peptides and to map the active site.
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PMID:Mapping the active site of factor Xa by selective inhibitors: an NMR and MD study. 951 42

We have identified, in two unrelated patients, factor X deficiency that we have designated factor X Nagoya 1 and Nagoya 2, respectively. The proband with factor X Nagoya 1 showed factor X activity level of 3% and factor X antigen level < 10% of the normal control value. All the exons and intron/exon junctions of the factor X gene were studied using a strategy combining polymerase chain reaction (PCR) amplification and nonradioactive single-strand conformational polymorphism (SSCP) analysis. Exon 8 containing DNA fragment of the proband with factor X Nagoya 1 showed aberrant migration on SSCP analysis. All exon-containing DNA fragments amplified by PCR were sequenced, and we identified a C-to-T substitution in exon 8 in the human factor X gene of the proband, which results in the replacement of Arg306 by Cys. This genetic defect has been transmitted from her father, and her sister also carried the same mutation; both showed almost half the normal levels of both factor X activity and antigen. The coordinates of human factor Xa indicated that Arg306 in the catalytic domain is positioned at the beginning of the alpha-helix near the second EFG-like domain. The substitution for Arg of Cys has been supposed to cause the destruction of local alpha-helix formation, possibly leading to the secretion problem. The proband with dysfunctional factor X Nagoya 2 was characterized by factor X activity level of 34% with normal factor X antigen level of 80%. We identified one substitution of G for A in exon 8 in the human factor X gene of the proband, which results in the replacement of Gly366 by Ser. As the Gly366 is positioned at the primary substrate binding pocket. the replacement of Gly with Ser would cause a defect of substrate binding, leading to the loss of enzymatic activity.
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PMID:Factor X Nagoya 1 and Nagoya 2: a CRM- factor X deficiency and a dysfunctional CRM+ factor X deficiency characterized by substitution of Arg306 by Cys and of Gly366 by Ser, respectively. 953 Oct 27

The importance of thrombin in arterial and venous thrombosis renders thrombin inhibition an important therapeutic target. Identification of novel inhibitors requires an appropriate animal model. We modified a previously reported rat arterial thrombosis model to allow simultaneous assessment of the arterial and venous antithrombotic efficacies of heparin, hirudin, hirulog, a novel thrombin inhibitor H-(N-Me-D-Phe)-Pro-L-trans-4-aminocyclohexyl-Gly-[CO-CO]-NHCH3+ ++ (L-370,518) and the factor Xa inhibitor tick anticoagulant peptide in rabbits. Thrombosis was induced through application of 70% ferric chloride to the femoral artery and jugular vein. Incidence of occlusion, thrombus weight, aPTT and plasma inhibitor concentrations were determined. Heparin was efficacious in preventing arterial and venous occlusive thrombosis but at a dose that profoundly elevated aPTT. On a molar dosing basis, the approximate order of potency of the thrombin and factor Xa inhibitors was similar in artery and vein: hirudin>tick anticoagulant peptide>hirulog> or =L-370,518. Data suggested that compounds tended to be more potent in preventing venous thrombosis than arterial. This thrombin-dependent model is an economical and efficient approach to arterial and venous antithrombotic efficacy screening that eliminates variabilities encountered when multiple model/multiple animal strategies are employed.
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PMID:Assessment of thrombin inhibitor efficacy in a novel rabbit model of simultaneous arterial and venous thrombosis. 953 Oct 58

The signaling pathway initiated by factor Xa on vascular endothelial cells was investigated. Factor Xa stimulated a 5- to 10-fold increased release of nitric oxide (NO) in a dose-dependent reaction (0.1-2.5 microG/ml) unaffected by the thrombin inhibitor hirudin but abolished by active site inhibitors, tick anticoagulant peptide, or Glu-Gly-Arg-chloromethyl ketone. In contrast, the homologous clotting protease factor IXa or another endothelial cell ligand, fibrinogen, was ineffective. A factor Xa inter-epidermal growth factor synthetic peptide L (83)FTRKL(88) (G) blocking ligand binding to effector cell protease receptor-1 inhibited NO release by factor Xa in a dose-dependent manner, whereas a control scrambled peptide KFTGRLL was ineffective. Catalytically active factor Xa induced hypotension in rats and vasorelaxation in the isolated rat mesentery, which was blocked by the NO synthase inhibitor L-N(G)-nitroarginine methyl ester (L-NAME) but not by D-NAME. Factor Xa/NO signaling also produced a dose-dependent endothelial cell release of interleukin 6 (range 0.55-3.1 ng/ml) in a reaction inhibited by L-NAME and by the inter-epidermal growth factor peptide Leu(83)-Leu(88) but unaffected by hirudin. Maximal induction of interleukin 6 mRNA required a brief, 30-min stimulation with factor Xa, unaffected by subsequent addition of tissue factor pathway inhibitor. These data suggest that factor Xa-induced NO release modulates endothelial cell-dependent vasorelaxation and cytokine gene expression. This pathway requiring factor Xa binding to effector cell protease receptor-1 and a secondary step of ligand-dependent proteolysis may preserve an anti-thrombotic phenotype of endothelium but also trigger acute phase responses during activation of coagulation in vivo.
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PMID:Hypotension and inflammatory cytokine gene expression triggered by factor Xa-nitric oxide signaling. 953 8


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