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)

The esterolytic and amidolytic properties of activated blood coagulation factor X (factor Xa) and the analogous decarboxy species were compared in order to find out if the gamma-carboxyglutamic acid residues influence the function of the active centre. It was found that the two proteins (1) showed similar kinetic parameters when titrated with p-nitrophenyl-p'-guanidinobenzoate hydrochloride (2) had a similar Km and kcat for various synthetic chromogenic tri- and tetrapeptides and (3) were inhibited in the same way by benzamidine. Further it was observed that (4) Ca2+ inactivates factor Xa, but has no influence on the amidase activity of decarbyxyfactor Xa (5) factor V prevents Ca2+-induced inactivation of factor Xa but does not influence the amidase activity of both factor Xa and decarboxyfactor Xa. We conclude that the interaction of the gamma-carboxyglutamic acid residues with Ca2+ in factor X has no measurable influence on the properties of the active site per se.
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PMID:The effect of gamma-carboxyglutamate residues on the enzymatic properties of the activated blood clotting factor X. I. Activity towards synthetic substrates. 64 13

The activation of bovine coagulation factor X has been studied by kinetic and spectrophotometric measurements. The pH dependence of the hydrolysis of specific ester substrates by activated factor Xa can be ascribed to two independently ionizing groups with pKa values of 6.9 and 8.8, respectively. The rates of reaction of factor X, before and after activation, with the active-site titrant methanesulfonyl fluoride, suggest that the reactivity of the active-site serine residue in factor X is similar to that in trypsinogen and in factor Xa similar to that in trypsin. Analogous comparisons using diisopropyl phosphofluoridate as the titrant suggest that a hydrophobic binding site is absent in both the enzyme and zymogen. This conclusion is consistent with the lack of change in circular dichroism when acyl derivatives of factor V are converted to their acyl enzyme counterparts.
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PMID:Activation of bovine factor X (Stuart factor)--analogy with pancreatic zymogen-enzyme systems. 67 35

Activation of coagulation factor X via the intrinsic pathway requires the assembly of factors IXa and VIII on lipid membranes. It is known that the platelet expresses membrane sites for assembly of factors IXa/VIII and promotes efficient factor X activation. We now show that human blood monocytes, but not lymphocytes or polymorphonuclear leukocytes, also express appropriate sites for factors IXa/VIII assembly. The maximal rate of factor X activation by factors IXa (0.75 nM) and VIII (1 unit/ml) assembled on monocytes is similar to the maximal rate on platelets. This rate, adjusted per micromole of lipid phosphorus, is 1636 +/- 358 nM factor Xa/min on monocyte, and 1569 +/- 54 nM factor Xa/min on platelets. At physiologic concentrations of factors X and VIII, the activation rate increases with factor IXa concentration asymptotically approaching a maximum. Half-maximal rate is achieved with 1.0 +/- 0.16 nM factor IXa. Monocytes and macrophages, but not platelets, can express membrane tissue factor and thus promote simultaneous assembly of two distinct factor X-activating protease complexes. In these studies, blood monocytes and alveolar macrophages are used as membrane sources in kinetic experiments comparing factor X activation by intrinsic (factor IXa/VIII) versus extrinsic (factor VII/tissue factor) protease complexes. At plasma concentration of factors VIII and VII, apparent Km on the monocyte is 14.6 +/- 1.4 nM for intrinsic and 117.0 +/- 10.1 nM for extrinsic activation. The apparent Km on alveolar macrophages is 12.1 +/- 1.9 and 90.6 +/- 10.2 nM for intrinsic and extrinsic activation, respectively. Maximal rates on monocytes at saturating concentration of factors IXa, VIII, and VII are 48.0 +/- 11.2 nM factor Xa/min, for intrinsic activation, and 16.5 +/- 5.5 nM factor Xa/min, for extrinsic activation. These data show that the monocyte/macrophage is the only blood-derived cell type with membrane sites for both intrinsic and extrinsic pathway assembly. We have exploited this characteristic of the monocyte/macrophage membrane to demonstrate that factor X activation by the intrinsic pathway protease is more efficient than activation via the extrinsic pathway protease complex.
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PMID:Functional difference between intrinsic and extrinsic coagulation pathways. Kinetics of factor X activation on human monocytes and alveolar macrophages. 190 70

Macrophages were harvested from home cage control (HCC) mice, and from mice which had been stressed by repeated brief exposures (3-8 min) to cold water at 10-15 degrees C twice daily for 8 or 14 days. Macrophages obtained from mice stressed 8 or 14 days compared to macrophages from HCC mice showed in vitro increased amounts of membrane-bound prothrombinase activity, whereas the thrombin degradation activity was unchanged. Furthermore, macrophages of mice stressed 8 days showed increased release of coagulation factor X/Xa to supernatant in vitro. These findings suggest an increased amount of prothrombinase complex enzymes on the surface of macrophages from mice stressed 8 days, and increased activity of the prothrombinase enzyme in macrophages from mice stressed 14 days. The synthesis of proteoglycans (PG) and glycosaminoglycans (GAG) was increased in macrophages from mice stressed 8 days compared to macrophages from HCC mice and mice stressed 14 days. When macrophages from mice stressed 8 days or HCC mice were stimulated in vitro with phorbol myristate acetate (PMA) and IL-1 or PMA and IL-2, a changed PG/GAG synthesis was observed only in macrophages from the HCC animals. Furthermore, both the tumour cytotoxicity and the released tumour necrosis factor (TNF) were decreased from macrophages from mice stressed 14 days compared to HCC mice. The results suggest that the macrophages of stressed mice have an altered mode of function more complex than a simple general suppression or activation.
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PMID:The effect of stress in vivo on the function of mouse macrophages in vitro. 204 61

Low molecular weight heparin (LMH) acts as an anticoagulation agent mainly through its anti-activated coagulation factor X (Xa) activity. Thrombelastography (TEG) is expected to be useful to monitor the dosage of LMH during hemodialysis because reaction time on TEG (TEG-r) is considered to reflect blood thromboplastin formation time, which depends on the formation of Xa. To test this possibility, we compared the usefulness of TEG, activated coagulation time (ACT), activated partial thromboplastin time (APTT), and anti-Xa activity in 28 hemodialysis patients using both conventional unfractionated heparin (UFH) and LMH on separate dialysis procedures. Anti-Xa activity of LMH was comparable to that of UFH when it was measured using both LMH and UFH as standards. Anti-Xa activity, which theoretically depended on the heparin concentration in blood samples, did not correlate with the degree of dialyzer clotting. The APTT correlated well with anti-Xa activity in patients using LMH (r = 0.686, p less than 0.01) and UFH (r = 0.906, p less than 0.01), but not with the degree of dialyzer clotting. The TEG-r correlated well with the degree of dialyzer clotting both in patients using LMH and those using UFH (measurements of samples obtained from the venous side of the extracorporeal circuit) and weakly correlated with anti-Xa activity in patients using LMH (r = 0.402, p less than 0.05). The ACT did not correlate with the degree of dialyzer clotting or anti-Xa activity. These results suggest that TEG-r reflects the efficacy of heparin in the extra-corporeal blood circuit, whereas APTT mainly reflects heparin concentration of the blood samples.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Usefulness of thrombelastography for dosage monitoring of low molecular weight heparin and unfractionated heparin during hemodialysis. 217 70

Synthesis and secretion of blood coagulation factor X was studied during incubations of hepatocytes prepared by perfusion of rat livers with collagenase. The apparent molecular weight of factor X isolated from the incubation medium was about 14,000 less than factor X isolated from rat plasma. The extracellular form of factor X was a two-chain polypeptide and the observed difference in molecular weight was reflected in the heavy chain. Since these properties were more characteristic of factor Xa than factor X, experiments were designed to determine if factor X activation occurred during the incubations. Clotting factor assays indicated that factor X secreted by hepatocytes was present as factor Xa. Also, when purified plasma factor X was added to incubations of hepatocytes the added factor X was converted to factor Xa. Plasma membranes prepared from isolated hepatocytes or from liver homogenates contained an enzyme that converted factor X to factor Xa in a calcium-dependent reaction. The results suggest that the activity is due to the presence of thromboplastin (tissue factor) and factor VII in the membrane preparations.
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PMID:The activation of factor X by hepatocyte plasma membranes. 261 30

Using affinity chromatography on a column of factor X-Cellulofine, we have isolated a novel blood coagulation factor X-binding protein with anticoagulant activity from the venom of Trimeresurus flavoviridis (Habu snake). This anticoagulant protein was also purified by chromatography on Sephadex G-75 and S-Sepharose Fast Flow. The yield of the purified protein was approximately 16 mg from 400 mg of crude venom. The purified protein gave a single band on both analytical alkaline disc-gel electrophoresis and SDS-PAGE. This protein had a relative molecular weight (Mr) after SDS-PAGE of 27,000 before reduction of disulfide bonds and 14,000 after reduction of disulfide bonds. The protein prolonged the clotting time induced by kaolin or factor Xa. In the presence of Ca2+, it formed a complex with factor X, the molar ratio being 1 to 1. Similar complex formation was observed with factor Xa and factor IX/factor IXa, but not with other vitamin K-dependent coagulation factors, i.e., prothrombin, factor VII, protein C, protein S, and protein Z. The interaction of this anticoagulant protein with factor IX/factor X was dependent on gamma-carboxyglutamic acid (Gla) domains, since Gla-domainless derivatives of factor X and factor IXa beta' did not interact with this anticoagulant protein.
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PMID:A novel blood coagulation factor IX/factor X-binding protein with anticoagulant activity from the venom of Trimeresurus flavoviridis (Habu snake): isolation and characterization. 261 88

Monocytes initiate coagulation through regulated surface expression of tissue factor and local assembly of a proteolytic enzymatic complex formed by tissue factor and factor VII/activated factor VII. We now show that, in the absence of these initiating molecules, monocytes and cell lines of monocytic/myeloid differentiation can alternatively initiate coagulation after exposure to ADP. The molecular basis for this procoagulant response consists of two distinct events. First, cell stimulation with ADP induces high-affinity binding of coagulation factor X to the surface-adhesive receptor Mac-1. Locally, Mac-1-concentrated factor X is then rapidly proteolytically cleaved to an active protease with size and activity characteristics of activated factor X, which supports the cell-associated formation of thrombin and the procoagulant response. We conclude that the monocytic/myeloid adhesive receptor Mac-1 has the unexpected, specifically inducible property to organize a molecular assembly culminating in rapid fibrin formation that is independently regulated from tissue factor and factor VII/activated factor VII.
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PMID:Adhesive receptor Mac-1 coordinates the activation of factor X on stimulated cells of monocytic and myeloid differentiation: an alternative initiation of the coagulation protease cascade. 297 72

The ability of malignant tissue from 50 patients with colorectal carcinoma to activate blood coagulation factor X directly was compared with samples of adjacent, macroscopically normal colonic mucosa from the same patients, and tissue from four patients with non-malignant bowel disease. The resected tissue was homogenized and incubated with purified factor X and calcium ions. The subsequent generation of activated factor X was measured spectrophotometrically with a chromogenic substrate. Results were expressed as absorbance units, and as the ratio of tumour and normal activities. Factor X-activating activity (FXAA) was present in all normal and malignant tissues tested. FXAA was significantly greater (P less than 0.001) in the tumour homogenates than in the uninvolved tissue. The tumour:normal ratio was significantly (greater than 1.2) elevated in 38 patients (76 per cent). FXAA was not correlated with the degree of differentiation of the tumour, the Dukes' classification of the disease or the exact site of the tumour. There was no difference between the FXAA content of non-involved tissue from the colorectal cancer group and colonic mucosa from patients with non-malignant bowel disease. It is concluded that colorectal carcinomas contain significantly more FXAA than adjacent, non-malignant colonic mucosa from the same subject, but there is no direct evidence for a relationship between procoagulant levels and the extent of malignancy in these patients.
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PMID:Factor X-activating activity in patients with colorectal carcinoma. 342 59

Tissue factor, a known initiator of blood coagulation, was found to be active in Triton X-100. A system consisting of tissue factor, factor VIIa, calcium ions, and coagulation factor X generated activated factor X at an appreciable rate. Based on this observation, we coupled human and bovine factor VII to a solid support. Each column bound tissue factor, solubilized in Triton X-100, in a species-specific manner. These interactions required calcium ions; when the columns were washed with detergent containing calcium ions, no tissue factor was eluted. When calcium ions were omitted from the eluant, tissue factor emerged as a sharp peak. Human tissue factor was extracted from an acetone brain powder into 2% Triton X-100. This extract, made 10 mM in CaCl2, was passed over a factor VII column. Human factor VII (1.2 mg) was coupled to 30 ml of Affi-Gel 15. This column bound approximately equal to 15 micrograms of human tissue factor. The eluted material was approximately equal to 25% pure. Final purification was achieved by gel filtration after chymotryptic digestion of contaminants. The tissue factor activity was stable to this treatment. The molecular weight determined by sodium dodecyl sulfate/PAGE (approximately equal to 46,000) was also unchanged by chymotrypsin. The final material was a single band on PAGE, demonstrated similar resistance to tryptic and chymotryptic digestion as bovine tissue factor, and had approximately the same specific coagulant activity as the previously purified bovine material. Tissue factor was also purified from human placenta, yielding a similar protein. A partial 28-residue sequence of the latter has been obtained.
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PMID:Affinity purification of human tissue factor: interaction of factor VII and tissue factor in detergent micelles. 345 66


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