Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0272170 (SDS)
 50,377 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Tissue factor pathway inhibitor (TFPI) is a plasma-derived protein which inhibits two of the active serine proteases present during normal blood coagulation. Inhibition of both of these proteases, factors VIIa and Xa, is thought to require a factor Xa-TFPI complex. To begin to investigate the interactions between factor Xa and TFPI, amino acids 94-155, which encode for the second Kunitz domain (K2) of TFPI, were expressed, purified, and partially characterized. Expression of the recombinant peptide was accomplished using an E. coli expression system which produced the peptide at an expression level of approximately 2-5% of total cell protein. The peptide was localized to disulfide-linked refractile bodies which were solubilized by reduction in the presence of denaturant and the soluble protein refolded. Oxidized K2 was purified from the refold mixture using a two-step procedure employing gel filtration chromatography and reverse-phase HPLC. The unprocessed form of the recombinant peptide, Met-Ala-K2 (rMA-K2), was characterized. This peptide was purified to apparent homogeneity as determined by SDS-PAGE, quantitative amino acid, Edman degradation, and electrospray mass spectrometry analyses (> 95% pure). The product bound to factor Xa covalently coupled to a solid support in the presence of 2M sodium chloride demonstrating its affinity for this enzyme. Preincubation of rMA-K2 peptide with factor Xa neutralized, with 1.1:1 stoichiometry, the ability of factor Xa to hydrolyze a small chromogenic substrate. Additionally, rMA-K2 prolonged the time to clot formation in a plasma-based assay dependent on factor Xa concentration. Finally, this peptide mildly prolonged the prothrombin and modified prothrombin times of normal pooled plasma. Taken together this data demonstrates that this region of TFPI inhibits factor Xa activity and allows for further characterization of this enzyme-inhibitor complex.
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PMID:Bacterial expression, purification, and partial characterization of amino acids 94-155 of human tissue factor pathway inhibitor (TFPI) as an inhibitor of blood coagulation factor Xa. 129 Jan 65

An elastomeric polypeptide was produced, with the sequence G-(VPGVG)19-VPGV, as a fusion to glutathione S-transferase using the vector pGEX-3X. The fusion protein was expressed to high levels in Escherichia coli as indicated by SDS-PAGE analysis of induced cells. The fusion protein was affinity purified and cleaved with protease factor Xa, and the elastomeric polypeptide was recovered to a high degree of purity as indicated by SDS-PAGE followed by staining with CuCl2. The physical characterizations of carbon-13 and proton nuclear magnetic resonance and of the temperature profile for turbidity formation for the inverse temperature transition of hydrophobic folding and assembly attest to the successful microbial synthesis of the polypentapeptide of elastin. The results of these studies provide the initial progress toward achieving a more economical and practical means of producing material for elastic protein-based polymer research and applications.
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PMID:Production and purification of a recombinant elastomeric polypeptide, G-(VPGVG)19-VPGV, from Escherichia coli. 136 56

Coagulation factor X is activated by the extrinsic Xase complex composed of factor VIIa associated with the integral membrane protein tissue factor. The kinetics of human factor X activation was studied following reconstitution of this reaction system using purified human proteins and synthetic phospholipid vesicles composed of phosphatidylcholine and phosphatidylserine (PCPS) or phosphatidylcholine alone (PC). Factor X activation was evaluated by discontinuous measurements of the amidolytic activity of the product, factor Xa, or continuously monitored using the fluorescent serine protease inhibitor 4-aminobenzamidine. The results of both techniques were verified by direct physical measurements of zymogen activation using SDS-polyacrylamide gel electrophoresis. The rate of factor X activation with PC vesicles was less than 5% of that observed with PCPS vesicles. Since factor X does not bind to vesicles containing only PC, these data suggested an important role for the substrate-membrane interaction in the catalytic cycle. The importance of the substrate-membrane interaction in the activation process was investigated by using membrane-binding proteins to compete with the substrate for combining sites on PCPS vesicles. Prothrombin fragment 1 was an inhibitor of factor X activation. The dependence of inhibition by fragment 1 on PCPS and factor X was consistent with a significant reduction in initial velocity due to the displacement of factor X from the membrane surface. The inhibition data also suggested that the membrane-bound pool of factor X was the preferred substrate for the human extrinsic Xase complex. The influence of PCPS concentrations on the rate of factor X activation was systematically investigated. Increasing concentrations of PCPS resulted in a modest change in the Km,app and a dramatic change in the Vmax,app for the reaction. The initial velocity data could be globally analyzed according to the preferential utilization of membrane-bound factor X with the intrinsic kinetic constants: Km approximately equal to 1 microM and kcat = 37 s-1 at saturating PCPS. In addition, the equilibrium parameters for the factor X-membrane interaction inferred from these studies were in excellent agreement with the directly determined values. Collectively, the data suggest that the substrate-membrane interaction must precede catalysis for the efficient activation of human factor X by the extrinsic Xase complex.
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PMID:Role of the membrane surface in the activation of human coagulation factor X. 146 22

A low molecular weight platelet inhibitor of factor XIa (PIXI) has been purified 250-fold from releasates of washed and stimulated human platelets. Molecular weight estimates of 8400 and 8500 were determined by gel filtration and SDS-polyacrylamide gel electrophoresis, respectively, although a second band of Mr 5000 was present upon electrophoresis. The inhibitor does not appear to be one of the platelet-specific, heparin-binding proteins, since it neither bound to nor was affected by heparin. An amount of PIXI which inhibited by 50% factor XIa cleavage of the chromogenic substrate S2366 (Pyr-Glu-Pro-Arg-pNA-2H2O) only slightly inhibited (5-9%) factor XIIa, plasma kallikrein, plasmin, and activated protein C and did not inhibit factor Xa, thrombin, tPA, or trypsin, suggesting specificity for factor XIa. Kinetic analyses of the effect of PIXI on factor XIa activity demonstrated mixed-type, noncompetitive inhibition of S2366 cleavage and of factor IX activation with Ki's of 7 x 10(-8) and 3.8 x 10(-9) M, respectively. Immunoblot analysis showed that PIXI is not the inhibitory domain of protease nexin II, a potent inhibitor of factor XIa also secreted from platelets. Amino acid analysis showed that PIXI has no cysteine residues and, therefore, is not a Kunitz-type inhibitor. PIXI can prevent stable complex formation between alpha 1-protease inhibitor and factor XIa light chain as demonstrated by SDS-polyacrylamide gel electrophoresis. The inhibition by PIXI of factor XIa-catalyzed activation of factor IX and its capacity to prevent factor XIa inactivation by alpha 1-protease inhibitor, combined with the specificity of PIXI for factor XIa among serine proteases found in blood, suggest a role for PIXI in the regulation of intrinsic coagulation.
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PMID:A low molecular weight platelet inhibitor of factor XIa: purification, characterization, and possible role in blood coagulation. 173 24

An abnormal prothrombin has been detected in a 26-year-old female, who had no history of excessive bleeding. Prothrombin activity was approximately 10% when measured using either the classical one-stage assay or the assay with Echis carinatus venom, whereas prothrombin antigen level was normal. In keeping with current nomenclature practices, the abnormal prothrombin was designated "Prothrombin Himi". The electrophoretic behavior and calcium binding properties of Prothrombin Himi did not differ significantly from normal. Prothrombin Himi was isolated by chromatography on Q-Sepharose. Electrophoretic migration of the purified abnormal prothrombin on SDS-PAGE was normal. Upon prothrombin activation by Echis carinatus venom, the clotting activity produced from Prothrombin Himi was only 37% of the normal level after 90 minutes of the activation time, where as the amidolytic activity was almost the same as normal. The cleavage patterns of Prothrombin Himi by factor Xa or Echis carinatus venom investigated by SDS-PAGE, were found to be normal. These results indicate that Prothrombin Himi was characterized by a defective thrombin enzymatic activity.
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PMID:Prothrombin Himi; an abnormal prothrombin characterized by a defective thrombin activity. 192 60

Previous studies demonstrated proteolytic activation of human blood coagulation factor VII by an unidentified protease following complex formation with tissue factor expressed on the surface of a human bladder carcinoma cell line (J82). In the present study, an active-site mutant human factor VII cDNA (Ser344----Ala) has been constructed, subcloned, and expressed in baby hamster kidney cells. Mutant factor VII was purified to homogeneity in a single step from serum-free culture supernatants by immunoaffinity column chromatography. Mutant factor VII was fully carboxylated, possessed no apparent clotting activity, and was indistinguishable from plasma factor VII by SDS-PAGE. Cell binding studies indicated that mutant factor VII bound to J82 tissue factor with essentially the same affinity as plasma factor VII and was cleaved by factor Xa at the same rate as plasma factor VII. In contrast to radiolabeled single-chain plasma factor VII that was progressively converted to two-chain factor VIIa on J82 monolayers, mutant factor VII was not cleaved following complex formation with J82 tissue factor. Incubation of radiolabeled mutant factor VII with J82 cells in the presence of recombinant factor VIIa resulted in the time-dependent and tissue factor dependent conversion of single-chain mutant factor VII to two-chain mutant factor VIIa. Plasma levels of antithrombin III had no discernible effect on the factor VIIa catalyzed activation of factor VII on J82 cell-surface tissue factor but completely blocked this reaction catalyzed by factor Xa. These results are consistent with an autocatalytic mechanism of factor VII activation following complex formation with cell-surface tissue factor, which may play an important role in the initiation of extrinsic coagulation in normal hemostasis.
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PMID:Initiation of the extrinsic pathway of blood coagulation: evidence for the tissue factor dependent autoactivation of human coagulation factor VII. 193 2

Kinetic analyses of antithrombin III (AT-III)-thrombin or heparin cofactor II (HC-II)-thrombin or AT-III-factor Xa interactions were carried out in the absence or in the presence of one of the sulfated xylans or unfractionated heparin or low molecular weight (LMW) heparin utilizing chromogenic substrates. These studies demonstrated that under pseudo first order conditions the inhibitions were proportional to the AT-III or HC-II concentrations used and the apparent second order rate constants determined from the slopes of the pseudo first order plots of log of thrombin or Xa remaining as a function of time were significantly elevated in presence of the sulfated compounds. On a molar basis oat spelts xylan sulfate was the most effective compound in accelerating the rate of thrombin-AT-III interaction followed by commercial heparin while the latter was most effective in accelerating the rate of thrombin-HC-II interaction. Heparin and LMW heparin were more effective in that order in accelerating the rate of Xa-AT-III interaction while oat spelts xylan sulfate, corn cob xylan sulfate, SP-54 were less effective than the heparins in that order. Studies were also conducted on the concentrations of the sulfated compounds required to inhibit by 50% the thrombin activity by AT-III or HC-II or that required to inhibit by 50% the factor Xa activity by AT-III. The results showed an inverse relationship between the increase in the rate of acceleration by the sulfated compound with the decrease in the amount required for 50% inhibition. SDS-polyacrylamide gel study of the reaction mixture containing thrombin, AT-III or HC-II along with heparin or oat spelts xylan sulfate showed that like heparin, oat spelts xylan sulfate potentiated the formation of thrombin-AT-III or thrombin-HC-II complexes which were stable in presence of denaturing or reducing agents. Chemical modification of arginine or lysine of AT-III significantly lowered its potentiation of thrombin or Xa inhibition by oat spelts xylan sulfate.
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PMID:Mechanism of potentiation of antithrombin III and heparin cofactor II inhibition by sulfated xylans. 197 2

Colburn and Buonassisi (In Vitro Cell Dev. Biol. 24, 1133-1136, 1988) have isolated a single chain sulfated glycoprotein inhibitor of factor VIIa/tissue factor-catalyzed activation of factor X from conditioned media of an established rabbit endothelial cell line. We report herein that their endothelial cell-derived inhibitor and extrinsic pathway inhibitor (EPI) isolated from rabbit plasma have identical functional properties with respect to their interactions with factor Xa and with factor VIIa/tissue factor. In addition, the endothelial cell inhibitor and rabbit plasma EPI migrate with the same apparent molecular weights on non-reduced SDS-PAGE and contain similar amounts of N-linked carbohydrate. Like the endothelial cell inhibitor the EPI of rabbit plasma exists as a single chain molecule. Furthermore, the endothelial cell inhibitor is recognized and neutralized by a polyclonal antibody raised against rabbit plasma EPI. We therefore conclude that cultured rabbit endothelial cells produce an inhibitor of factor VIIa/tissue factor activity that is functionally and immunologically identical to rabbit plasma EPI.
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PMID:A sulfated rabbit endothelial cell glycoprotein that inhibits factor VIIa/tissue factor is functionally and immunologically identical to rabbit extrinsic pathway inhibitor (EPI). 202 53

Antithrombin-III-Hamilton has been shown to be a structural variant of antithrombin-III (AT-III) with normal heparin affinity but impaired protease inhibitory activity. The molecular defect of AT-III-Hamilton is the substitution of Thr for Ala at amino acid residue 382. The plasma of affected individuals contains approximately equal quantities of normal AT-III and AT-III-Hamilton. When AT-III was isolated from the plasma of the propositus by heparin-Sepharose chromatography, it had identical mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to normal plasma-derived AT-III, under both reducing and nonreducing conditions. However, the AT-III-Hamilton species, separated from the propositus' normal AT-III by a combination of heparin-Sepharose and thrombin-Sepharose chromatography, had increased mobility on reductive SDS-PAGE compared with AT-III from the propositus isolated by heparin-Sepharose chromatography alone. Under nonreducing conditions this AT-III-Hamilton species had decreased mobility compared with AT-III from the propositus (or normal AT-III) isolated only by heparin-Sepharose chromatography. When incubated with either human alpha-thrombin or human factor Xa, this AT-III-Hamilton species was unreactive. Approximately 50% of the AT-III from the propositus isolated by heparin-Sepharose chromatography, when incubated with either human alpha-thrombin or factor Xa, did not form complex but was cleaved, presumably at the reactive center Arg393-Ser394. To further substantiate the biological behavior of this variant, AT-III-Hamilton polypeptides were synthesized in a cell-free system. This recombinantly produced AT-III-Hamilton, when incubated with either human alpha-thrombin or factor Xa, was cleaved by both these proteases, but did not show any complex formation. The results indicate that AT-III-Hamilton does not form a stable covalent inhibitory complex with these serine proteases but can be cleaved at the reactive center. Thus, the inhibition of serine proteases by their natural inhibitors (the serpins) involves at least two separate, but interrelated events; hydrolysis at the reactive center followed by complex formation. AT-III-Hamilton is capable of only the first of these events.
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PMID:Antithrombin-III-Hamilton, Ala 382 to Thr: an antithrombin-III variant that acts as a substrate but not an inhibitor of alpha-thrombin and factor Xa. 202 79

We have discovered and characterized a novel coagulation factor Xa inhibitor from the salivary gland of the black fly, Simulium vittatum. Salivary glands were surgically dissected from the flies and a crude salivary gland extract was tested for inhibition of a number of coagulation assays. The gland extract inhibited both thrombin and factor Xa. To purify further the factor Xa inhibitor, a factor Xa affinity column was utilized. Final purification of the black fly factor Xa inhibitor was achieved by reverse-phase C8 microbore high pressure liquid chromatography. Inhibition of factor Xa was nearly stoichiometric by the purified inhibitor with no inhibitor of thrombin detected. SDS-polyacrylamide gel electrophoresis indicated the inhibitor had a molecular weight of 18,000 and sequence analysis of the inhibitor revealed a blocked amino terminus. These data indicate that the blood-sucking black fly has evolved a highly potent inhibitor of mammalian coagulation factor Xa to disrupt its host normal hemostatic clotting mechanisms.
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PMID:Isolation and characterization of a coagulation factor Xa inhibitor from black fly salivary glands. 227 May 32


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