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)

Blood coagulation studies showed there was a pronounced thrombocytopenia and hypofibrinogenemia in Holstein calves infected with Trypanosoma congolense TREU 112. There was also ineffective thrombopoiesis characterized by an increased megakaryocytic mass, reduced uptake of 35S-methionine into peripheral blood platelets and a normal platelet lifespan. There was an increased uptake of isotopic label into fibrinogen and a shortened half life indicating a consumptive error with increased peripheral use of fibrinogen. No consistent abnormalities were found in ethanol gelation, partial thromboplastin time, clot retraction and lysis or plasminogen assay. Fibrin split products were rarely detected. These findings suggest that in the chronic form of bovine trypanosomiasis there is a partially compensated consumption coagulopathy.
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PMID:The pathogenesis of Trypanosoma congolense infection in calves. IV. The kinetics of blood coagulation. 44 53

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

Secretion from recombinant yeast was used as a potential source of large quantities of the leech protein antistasin (ATS), a potent and highly specific inhibitor of the serine protease coagulation factor Xa. Mature recombinant ATS (r-ATS) is obtained after intracellular cleavage by the yscF protease of the mating factor alpha-1 pre-proleader from the fusion protein at the Lys-Arg sequence junction. Production levels are relatively low (ca. 1 mg/liter). Purification of the secreted product from a complex growth medium involved cell removal by microfiltration and diafiltration, cation-exchange capture and concentration on S-Sepharose Fast Flow, C-4 reverse-phase high-performance liquid chromatography (RP-HPLC), and HPLC cation-exchange chromatography step, and RP-HPLC concentration and desalting. The process was scaled up from the 16- to the 250-liter level with a corresponding increase in amount of r-ATS. From the 250-liter fermentation two major forms, r-ATS-I and r-ATS-II, distributed approximately 60:40, and a minor form, r-ATS-minor (ca. 1% of the purified r-ATS), were characterized. Limited N-terminal sequence analysis by Edman degradation indicated that r-ATS-I has the predicted mature N-terminus starting with Gln, that r-ATS-II is N-terminally blocked with pyroglutamate, and that r-ATS-minor is an incompletely processed form. RP-HPLC, hydrophilic-interaction HPLC, cation-exchange HPLC analysis, and electrophoresis results are consistent with the differences observed by sequencing. Preliminary in vitro characterization by intrinsic Ki determination for factor Xa inhibition indicated that the yeast r-ATS forms are indistinguishable from each other as well as from r-ATS expressed by the insect baculovirus host-vector system. Nevertheless, r-ATS-I and r-ATS-II appear less potent than insect-derived r-ATS in the activated partial thromboplastin time clotting assay. Further characterization indicated that C-terminal cleavage at Pro-116 had occurred in r-ATS-I and r-ATS-II as well as oxidation of methionine residues to methionine sulfoxide. The possible role of the C-terminus in inhibition of the prothrombinase complex is discussed.
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PMID:Characterization of recombinant antistasin secreted by Saccharomyces cerevisiae. 139 15

A variant strain of Rauscher leukemia virus (RLV-A) obtained from a transplantable murine monomyelocytic leukemia causes a disease characterized by frank anemia, wasting, hepatosplenomegaly and erythroblastosis. The involvement of platelets in this disease are reported here. The RLV-A induced a severe thrombocytopenia (25 percent of control level) at the terminal stage of disease. This thrombocytopenia was not associated with disseminated intravascular coagulopathy since the prothrombin times were always within normal limits. The partial thromboplastin time was elevated in the terminal stages of disease and was found to be associated with factor deficiencies, possibly owing to the presence of anti-factor antibodies, in the intrinsic coagulation pathway, especially factor VIII. Further, splenectomy did not abolish the thrombocytopenia, since splenectomized, virally infected animals also developed severe thrombocytopenia (29 percent of control levels). The ensuing splenomegaly during progression of disease was not the cause of the thrombocytopenia. A physiological response to the severe thrombocytopenia was the production of larger size platelets. At terminal stages of the disease, platelet volume increased to 4.2 mu 3 (normal is 3.0 mu 3). An increase in platelet volume was also observed in splenectomized, virally infected animals. Electron microscopy indicated that these circulating platelets contained c-type viral particles. Viral infection was associated with decreased life span of circulating platelets, as measured by 75Se-methionine at mid and terminal stages of the disease. Our results suggest that direct viral infection of platelets and/or megakaryocytes with subsequent cell lysis is a possible cause of the observed thrombocytopenia observed in RLVA-induced disease and may also occur in other retrovirally-induced diseases.
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PMID:Thrombocytopenia in a retrovirally-induced murine erythroleukemia. 145 28

A recombinant gene for BPTI (bovine pancreatic trypsin inhibitor) is expressed in Escherichia coli using a MBP (maltose-binding protein) fusion vector. BPTI is fused through an FXa (blood coagulation factor Xa protease) target sequence (Ile-Glu-Gly-Arg) to the C-terminus of MBP. The MBP moiety of the hybrid protein enables purification in one step utilizing MBP's affinity to cross-linked amylose, and the FXa target sequence allows specific cleavage of the hybrid protein. Effective FXa cleavage is achieved by spacing the FXa target sequence and Arg-1 of the BPTI sequence with four residues (Met-Glu-Ala-Glu). The resulting N-terminal extended BPTI is readily converted to the wild-type sequence by trimming with cathepsin C exopeptidase, for the activity of which the spacing tetrapeptide is optimized. FXa cleavage is prohibited when the target sequence is placed next to Arg-1. In this construction, off-target cleavage at a somewhat homologous sequence (Val-Pro-Gly-Arg) results in five- or six-residue extended BPTI, indicating new details of the FXa specificity. The yield of highly purified recombinant BPTI is 3-6 mg/liter of culture, making the MBP-BPTI expression system convenient for the production of sufficient amounts of protein for NMR studies. 1H NMR is used to analyze the N-extended BPTI analogues.
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PMID:BPTI and N-terminal extended analogues generated by factor Xa cleavage and cathepsin C trimming of a fusion protein expressed in Escherichia coli. 182 11

A gene which codes for the 66-residue polypeptide of kappa-bungarotoxin has been chemically synthesized by linking together 3 synthetic double-stranded oligonucleotides in a bacterial plasmid. The synthesis incorporated six unique silent restriction sites spaced throughout the gene for use in cassette mutagenesis. Direct expression of the kappa-bungarotoxin polypeptide by itself in Escherichia coli failed to result in a stable product. The toxin polypeptide was stabilized and expressed in E. coli as part of a fusion protein with rat intestinal fatty acid binding protein under control of the nalidixic acid inducible recA promoter. Two fusion protein constructs were prepared that differed only in the cleavage site between the fatty acid binding protein and the toxin polypeptide. One contained a factor Xa cleavage site, and the other, since the toxin itself is devoid of methionine, contained a methionyl residue that served as a cyanogen bromide cleavage site. The fusion proteins were isolated by ion-exchange chromatography and reverse-phase HPLC. The construct containing the factor Xa cleavage site could not be cleaved under nondenaturing conditions. On the other hand, kappa-bungarotoxin was efficiently cleaved from the methionyl fusion protein with CNBr. The toxin polypeptide was isolated by reverse-phase HPLC and ion-exchange chromatography and produced a complete and specific blockade of neuronal nicotinic acetylcholine receptors in chick ciliary ganglia which was indistinguishable from that produced by a comparable amount of venom-purified kappa-bungarotoxin.
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PMID:Synthesis and expression in Escherichia coli of a gene for kappa-bungarotoxin. 193 58

Immunotherapy with Interleukin-2 (IL-2) and LAK cells has shown antitumoral activity in metastatic cancer patients. So far, thrombocytopenia is the major side effect reported in hemostasis. We have studied coagulation parameters in 6 patients treated with r-Met Hu IL-2 [ala-125]. In each case, we have observed a significant fall in prothrombin time, fibrinogen, protein C, anti-thrombin III, plasminogen, alpha 2-antiplasmin and all of the clotting factors except factor VIII. There was a significant increase in the activated thromboplastin time. No significant modifications of the D-Dimer test, fibrin-fibrinogen degradation products (FDP) and thrombin time were observed. Our data suggest that r-Met Hu IL-2 [ala-125] could interfere with the hepatic synthesis of the clotting factors and their inhibitors.
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PMID:Blood coagulation abnormalities during adoptive immunotherapy with interleukin-2 (r-Met Hu IL-2 [ala 125]). 200 36

Human coagulation factor V is a protein cofactor that is an essential component of the prothrombinase complex. A full-length factor V cDNA has been subcloned into the mammalian expression vector pDX and used to transfect COS cells. Approximately 95 +/- 4% of the recombinant human factor V (rHFV) synthesized in COS cells is secreted into the culture medium. Forty-eight hours after transfection rHFV antigen levels in the conditioned medium were 70 +/- 15 ng/mL. Factor V activity determined by fibrometer assay increased approximately 5-fold from 0.027 +/- 0.012 to 0.124 +/- 0.044 unit/mL following activation by the factor V activating enzyme from Russell's viper venom (RVV-V). A chromogenic assay specific for factor Va indicated that recombinant factor V had 3.8 +/- 1.3% of the activity of the activated protein. The estimated specific activity of the recombinant factor Va was approximately 1800 +/- 500 units/mg, which is similar to the specific activity of purified plasma factor Va of 1700-2000 units/mg. Immunoprecipitation of [35S]methionine-labeled rHFV revealed a single high molecular mass component (approximately 330 kDa). Treatment of rHFV with thrombin or RVV-V resulted in the formation of proteolytic products that were similar to those seen with plasma factor V. We have also expressed a mutant, rHFV-des-B811-1441, that lacks a large portion of the highly glycosylated connecting region that is present in factor V. Immunoprecipitation of [35S]methionine-labeled rHFV-des-B811-1441 revealed a single-chain polypeptide with Mr approximately 230 kDa. This mutant constitutively expressed 38 +/- 7% of the activity of the RVV-V-activated protein.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Expression and characterization of recombinant human factor V and a mutant lacking a major portion of the connecting region. 239 12

The effect of ONO-3307 (4-sulfamoyl phenyl-4-guanidinobenzoate methanesulfonate), a new protease inhibitor, was studied on various proteases in vitro and in an experimental thrombosis model in vivo. ONO-3307 competitively inhibited trypsin, thrombin, plasma kallikrein, plasmin, pancreatic kallikrein and chymotrypsin; and their Ki values were 0.048 microM, 0.18 microM, 0.29 microM, 0.31 microM, 3.6 microM and 47 microM, respectively. In addition, ONO-3307 inhibited both elastase release from N-formyl-Met-Leu-Phe (fMLP)-stimulated leukocytes and tissue thromboplastin release from endotoxin-stimulated leukocytes. To examine the effects of ONO-3307 on disseminated intravascular coagulation (DIC), we developed an experimental thrombosis model. ONO-3307 (10 mg/kg/hr) completely inhibited the deposition of radioactive fibrin in kidney and lung. Gabexate mesilate (50 mg/kg/hr) was also effective in this model, but the effect of nafamostat mesilate was unclear. These results indicate that ONO-3307 exhibits a wide range of inhibitory effects on various proteases, and ONO-3307 may be useful for the treatment of protease-mediated diseases such as thrombosis and DIC.
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PMID:Inhibitory effects of ONO-3307 on various proteases and tissue thromboplastin in vitro and on experimental thrombosis in vivo. 251 29

Expression of cellular procoagulant activity may be one of the more important responses to vascular injury. Because factor V, a coagulation cofactor in the prothrombinase complex, catalyzes the conversion of prothrombin to thrombin, it may be a key to understanding this response. Therefore, we have investigated the synthesis, secretion and expression of factor V by vascular smooth muscle cells, which proliferate at sites of vascular injury. Cultured aortic vascular smooth muscle cells constitutively secreted Factor V activity, as determined by a functional assay. Labeled factor V was immunoprecipitated from conditioned medium of [35S]methionine-labeled cells, indicating that the secreted factor V was synthesized by vascular smooth muscle cells. Treatment of vascular smooth muscle cells with tunicamycin prevented secretion of factor V, suggesting that its secretion was dependent on the presence of N-linked carbohydrate. Factor V activity was also expressed on the vascular smooth muscle cell surface, as indicated by the ability of cultured cells to promote factor Xa-catalyzed prothrombin activation. These data suggest that the proliferation of smooth muscle cells in response to vascular injury may be one mechanism that links vascular disease with thrombosis.
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PMID:Vascular smooth muscle cells synthesize, secrete and express coagulation factor V. 333 43


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