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)

Substantial thrombomodulin activity could be detected in tissue thromboplastin preparations from placenta or from lung but not from brain. When the amount of these preparations was adjusted to contain 1 unit of tissue factor activity, up to 0.85 units of thrombomodulin activity could be measured, corresponding to the generation of 17 pmol/ml/min of activated protein C when 1.5 microM human protein C was activated by 20 nM human alpha-thrombin in the presence of 5 mM CaCl2. After treatment by phospholipase C, thrombomodulin activity was reduced in these samples. Addition of mixed brain procoagulant phospholipids partially restored thrombomodulin activity in the phospholipase C-treated samples. These results emphasize the role of phospholipids in the expression of optimal thrombomodulin activity in tissue thromboplastin preparations from placenta or from lung.
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PMID:Thrombomodulin activity is found in tissue thromboplastin preparations from placenta and from lung but not from brain. 303 9

Factors affecting the inhibition of tissue thromboplastin (TP)-mediated blood coagulation have been investigated. Human brain thromboplastin progressively loses procoagulant activity when incubated in the presence of defibrinated plasma and CaCl2. Inhibition is maximal at a CaCl2 concentration of 1.5 mM during incubation and involves the calcium dependent binding of a plasma component(s) to the TP-FVII complex, preventing the activation of FX. Chelation of calcium ions using EDTA releases active TP and FVII from the inhibited complex. No inhibition occurs during incubation of TP with Al (OH)3 adsorbed plasma and calcium ions unless a Factor VII concentrate (or purified FVII and FX) is also present. Incubation of TP with antithrombin III-deficient plasma and calcium ions also leads to inhibition. Moreover, purified AT III cannot substitute for adsorbed plasma in producing TP inhibition. The data are consistent with the presence in plasma of a potent AT III independent inhibitor of TP-mediated blood coagulation.
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PMID:Inhibition of tissue thromboplastin-mediated blood coagulation. 308 14

Thrombin can be formed in the tumor cell microenvironment following activation of the clotting cascade by procoagulants of cancer or host cells. We have tested here the effects of thrombin, either "endogenous" or "exogenous" (see below), on arachidonate mobilization from membrane phospholipids of mouse mammary tumor virus-induced (MMTV) carcinoma cells. These tumor cells exhibit in vitro a tissue type procoagulant activity (130 thromboplastin units/10(4) cells) and are therefore able to induce thrombin formation in a plasmatic milieu. To verify the effect of thrombin formation by tumor cell procoagulant ("endogenous thrombin"), either human or mouse platelet-free plasma (20% in DMEM) was added to the cell layer (prelabelled for 5 hr with a trace amount (0.013 microM) of 3H-arachidonate) and the system was recalcified (15 mM CaCl2). Thin-layer radiochromatography of the culture medium showed a significant release of 3H-labelled arachidonate products PGE2, PGF2 alpha and 6-ketoPGF1 alpha after 1 hr of incubation. To verify the effect of thrombin formation from host sources ("exogenous thrombin"), either bovine or purified human alpha-thrombin (0.1-10 U/ml) was added to the cells for different periods (from 5 min to 20 hr). Exogenous thrombin stimulated arachidonate release and metabolism in a dose-related manner. With short labelling periods (0.013 microM 3H-arachidonate for 30 min-1 hr) thrombin stimulated the release of unmetabolized 3H-arachidonate, but not of 3H-arachidonate metabolites. These processes were inhibited by a specific inhibitor of thrombin enzymatic activity (alpha-NAPAP, 140 microM) and by a cyclo-oxygenase inhibitor (ASA 4mM). Tumor-associated procoagulants may thus contribute not only to fibrin deposition but also to generation of multipotent mediators such as arachidonate metabolites.
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PMID:Thrombin stimulates arachidonate metabolism in murine tumor cells. 310 91

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

Poly-L-lysine has been demonstrated to partially replace biological cofactors in the activation of prothrombin by factor Xa. The present study was initiated to determine if poly-L-lysine has an effect on the enzymatic activity of factor Xa in the absence of prothrombin. At low ionic strength (50 mM Tris-Cl, pH 8.0, ambient temperature), poly-L-lysine inhibits amidase activity (S-2222) of bovine factor Xa with high affinity (Ki = 7 nM). The inhibition was readily reversed by 100 mM NaCl. The inhibition was also markedly reduced by the addition of 1.0 mM CaCl2 but not by MnCl2 or MgCl2. All three metal ions enhance amidase activity in the absence of poly-L-lysine. Poly-L-lysine also inhibits the amidase activity of factor Xa from which the gamma-carboxyglutamic acid domain has been removed by limited proteolysis with chymotrypsin (factor Xa-GD) but with somewhat lower avidity (Ki = 35 nM). As with native factor Xa, calcium ions reduce the observed inhibition while either manganese or magnesium ions are much less effective. The amidase activity of factor Xa-GD is enhanced with any one of the three divalent cations. These results provide additional support for the existence of a functionally significant binding site for calcium ions outside of the gamma-carboxyglutamic domain of factor Xa.
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PMID:Interaction of polylysine with bovine factor Xa: effect of divalent cations. 348 86

Hexadimethrine bromide was evaluated as a heparin-neutralizing agent in a simple modification of the activated partial thromboplastin time test in canine plasma. Addition of various amounts of heparin in vitro to canine plasma indicated that heparin could be neutralized by adding 0.5 micrograms of hexadimethrine bromide 15 s before CaCl2 was added to the reaction mixture of the activated partial thromboplastin time test. In 8 dogs given (subcutaneous injection) 500 USP units of sodium heparin/kg, marked individual variations in clotting time prolongations were observed over the 12-hour period of study. The hexadimethrine bromide modification effectively neutralized the heparin-related clotting time prolongations to values that were not significantly different from base-line (preheparin) activated partial thromboplastin time values. The modification seems to be useful in confirming the presence of heparin and in monitoring heparin therapy in dogs.
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PMID:Use of hexadimethrine bromide as a heparin-neutralizing agent in canine plasma. 356 7

Human hepatic triglyceride lipase (HTGL), purified from plasma obtained after heparin injection, markedly enhanced the anti-Xa clotting activity of normal plasma. This was shown to be caused by direct inhibition of factor Xa clotting activity by HTGL, although the amidolytic activity of factor Xa was unaffected. Preincubation of factor Xa with CaCl2 and phospholipid reduced the rate of inhibition of HTGL, indicating that phospholipid-binding sites may be involved. Heparin, and low-affinity heparin, reduced the anti-Xa activity of HTGL, suggesting that heparin and factor Xa compete for the same binding sites on the lipase molecule. These results suggest that at least part of the enhanced anti-Xa clotting activity observed after injection of heparin and heparin analogues is caused by the release of HTGL. This release could contribute toward the anticoagulant and antithrombotic actions of these drugs.
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PMID:Anti-Xa activity of human hepatic triglyceride lipase. 358 39

A chromogenic substrate, H-D-Phe-Pip-Arg-pNA (S-2238) is a highly specific substrate to thrombin and releases p-nitroaniline (pNA) by the action of thrombin. We describe new modified APTT and PT methods using S-2238 in combination with the diazotization of pNA. In the modified APTT method, 100 microliter citrated plasma (diluted to 10-fold), 90 microliter 1 mM S-2238, 100 microliter 20 mM CaCl2 and 100 microliter Actin were mixed in an ice-bath, then incubated for 8 min at 37 degrees C. The reaction was stopped, and the generated pNA was diazotized by adding the following solutions sequentially: 975 microliter 0.04% sodium nitrite, 975 microliter 0.3% ammonium sulfamate and 975 microliter 0.07% N-(l-naphthyl)-ethylenediamine dihydrochloride. Diazotization changed pNA from yellow to pink. Then, absorbance at 545 nm was read, and values were expressed as thrombin units/ml plasma. In the modified PT method, 100 microliter citrated plasma (diluted to 20-fold), 90 microliter 1 mM S-2238 and 200 microliter tissue thromboplastin-C solution were mixed and processed as above. Correlations of the present modified APTT and APTT methods, and of modified PT and PT methods were significant (r = 0.426, p less than 0.01 and r = 0.561, p less than 0.01, respectively).
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PMID:New modified activated partial thromboplastin time and prothrombin time methods using a synthetic chromogenic substrate in combination with diazotization. 360 22

A prothrombin activator from the venom of Bothrops neuwiedi was purified by gel filtration on Sephadex G-100, ion-exchange chromatography on DEAE-Sephacel and affinity chromatography on a Zn2+-chelate column. The overall purification was about 200-fold, which indicates that the prothrombin activator comprises about 0.5% of the crude venom. The venom activator is a single-chain protein with an apparent molecular weight of 60 kDa. It readily activated bovine prothrombin with a Km of 38 microM and a Vmax of 120 mumol prothrombin activated per min per mg of venom activator. Venom-catalyzed prothrombin activation was not accelerated by the so-called accessory components of the prothrombinase complex, phospholipids plus Ca2+ and Factor Va. Gel-electrophoretic analysis of prothrombin activation indicated that the venom activator only cleaved the Arg-323-Ile-324 bond of bovine prothrombin, since meizothrombin was the only product of prothrombin activation. The activator did not hydrolyze commercially available p-nitroanilide substrates and its prothrombin-converting activity was not inhibited by benzamidine, phenylmethylsulfonyl fluoride, dansyl-Glu-Gly-Arg-chloromethyl ketone and soy-bean trypsin inhibitor. However, chelating agents such as EDTA, EGTA and o-phenanthroline rapidly destroyed the enzymatic activity of the venom activator. The activity of chelator-treated venom activator could be partially restored by the addition of an excess CaCl2. These results indicate that the venom activator remarkably differs from Factor Xa and that the enzyme is not a serine proteinase, but likely belongs to the metalloproteinases. The structural and functional properties of the venom prothrombin activator from B. neuwiedi are similar to those reported for the venom activator from Echis carinatus.
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PMID:Structural and functional characterization of a prothrombin activator from the venom of Bothrops neuwiedi. 368 99

The effect of calcium chloride (CaCl2) on the activated partial thromboplastin time (aPTT) of heparinized plasma was studied. The aPTT ratio (heparinized plasma:control plasma) increased as the CaCl2 concentration to recalcify the plasma was increased from 15 to 35 mmol/L CaCl2. Platelet-poor plasma from patients receiving intravenous heparin, and in vitro heparinized plasmas from either coumarinized patients or plasma depleted of the vitamin K-dependent factors, displayed the calcium-dependent increase in the aPTT ratio. The magnitude of the calcium-dependent change in the aPTT ratio was similar for the three partial thromboplastins studied. Heparinized blood collected in 3.2% and 3.8% citrate demonstrated the calcium-dependent increase in the aPTT ratio. The authors have also studied the effect of the divalent cations (Ca+2, Mg+2, Zn+2, and Sr+2) on the anti-Factor Xa activity of heparin to determine whether the calcium-dependent increase in the aPTT was due to an increase in the anti-Factor Xa activity. The anti-Factor Xa activity of heparin was measured using chromogenic substrate S-2251, purified Factor Xa, and excess antithrombin III. The anti-Factor Xa activity of heparinized plasma increased 2.4-2.8-fold as the divalent cation concentration was increased from 0-5 mmol/L. Similar results were obtained using purified bovine Factor Xa, antithrombin III, and heparin in the absence of plasma. These results suggest that divalent cations play an important role in modulating heparin's anticoagulant activity in vitro. In addition, the CaCl2 concentration used to recalcify plasma is an important variable that modifies heparin sensitivity of the aPTT. Furthermore, divalent cations play an important role in regulating the anti-Factor Xa activity of heparin in vitro.
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PMID:The effect of calcium ions on the activated partial thromboplastin time of heparinized plasma. 376 61


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