Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.69 (APC)
 16,337 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The previously unassigned gene coding for the anti-coagulatory protein C has been mapped on chromosome 2 using a cDNA probe and genomic blots from a human-hamster somatic cell hybrid panel. The assignments of the genes coding for the coagulation factor X to chromosome 13, and for alpha 1-acid glycoprotein to chromosome 9 have been confirmed using a similar direct approach.
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PMID:Mapping through somatic cell hybrids and cDNA probes of protein C to chromosome 2, factor X to chromosome 13, and alpha 1-acid glycoprotein to chromosome 9. 346 31

A protease from the venom of the Southern Copperhead snake (Agkistrodon contortrix contortrix) that activates protein C was purified to homogeneity by a combination of sulfopropyl (SP)-Sephadex C-50, Sephadex G-150 and Mono-S column chromatography. The purified enzyme is a glycoprotein, and migrated as a single band in sodium dodecylsulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 37,000 under non-reducing conditions. Upon reduction with 2-mercaptoethanol, the enzyme exhibited a Mr of 40,000. The purified enzyme prolonged the clotting time of human plasma in a dose- and temperature-dependent manner. Purified bovine protein C was completely activated within 10 minutes upon incubation with the purified protease at a 1:500 enzyme: substrate ratio. This reaction was markedly inhibited by calcium ions. The purified venom protein C activator had no effect on human fibrinogen.
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PMID:Isolation of a protein C activator from southern copperhead venom. 356 62

The protease from Southern Copperhead venom that activates protein C was purified to homogeneity by sulfopropyl (SP)-Sephadex C-50 ion-exchange chromatography, Sephadex G-150 gel filtration, and Mono-S fast protein liquid chromatography. The purified enzyme is a glycoprotein containing 16% carbohydrate, and migrated as a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular mass of 40,000 kDa. The enzyme is composed of a single polypeptide chain possessing an NH2-terminal sequence of Val-Ile-Gly-Gly-Asp-Glu-Cys-Asn-Ile-Asn-Glu-His. The purified venom protein C activator hydrolyzed several tripeptide p-nitroanilides. The amidolytic and proteolytic activities of the enzyme were readily inhibited by phenylmethanesulfonyl fluoride, p-amidinophenylmethanesulfonyl fluoride, chloromethyl ketones, and human antithrombin III. Covalent binding of diisopropyl fluorophosphate to the enzyme was confirmed using a tritium-labeled preparation of the inhibitor. The venom protease readily activated human and bovine protein C at 1:1000 enzyme:substrate weight ratio. The protease also cleaved human prothrombin, factor X, factor IX, factor VII, and fibrinogen. Prothrombin coagulant activity decreased upon incubation with the venom protease, and the rate of this reaction was reduced in the presence of calcium. Factor X and factor IX coagulant activity increased upon incubation with the venom protease in the presence of calcium, and decreased in the absence of calcium. Human factor VII clotting activity decreased slightly upon incubation with the venom protease. Although the venom protease did not clot human fibrinogen, it nonetheless cleaved the A alpha chain of fibrinogen, and this cleavage appeared to be associated with a measurable increase in the clottability of the protease-treated fibrinogen by thrombin. These data demonstrate that the protein C activator from Southern Copperhead venom is a typical serine protease with a relatively broad specificity.
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PMID:Characterization of a protein C activator from Agkistrodon contortrix contortrix venom. 362 72

Human activated protein C (APC) is a plasma serine protease that possesses amidolytic and anticoagulant activity. The rate at which the amidolytic and anticoagulant activity of APC was neutralized in normal plasma was essentially identical to that observed in plasma obtained from four individuals with combined Factor V/VIII deficiency disease. Incubation of radioiodinated APC with either normal human plasma or the combined Factor V/VIII-deficient plasmas resulted in the formation of a stable complex (Mr = 96,000) of the enzyme and a plasma protein as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Pretreatment of the radiolabeled APC with diisopropyl fluorophosphate prevented the formation of the enzyme-protein complex. On the basis of its ability to form a complex with radiolabeled APC, the APC-binding protein was purified to homogeneity from normal human plasma by ammonium sulfate fractionation, heparin-agarose chromatography, and QAE-Sephadex A-50 chromatography. The APC-binding protein (Mr = 54,000) is a glycoprotein, and possesses an amino-terminal sequence of Gly-Arg-Thr-Cys-Pro-Lys-Pro-Asp. The amino-terminal sequence of the APC-binding protein exhibited considerable homology with bovine colostrum inhibitor and pancreatic trypsin inhibitor, but no apparent sequence homology with the plasma serine protease inhibitors. Affinity-purified antibody against APC-binding protein immunoprecipitated a complex of radiolabeled APC and native APC-binding protein from normal human plasma. Complex formation was virtually eliminated in plasma immunodepleted of the APC-binding protein. Quantitative electroimmunoassay indicated essentially equal levels of APC-binding protein antigen in normal plasma compared with plasma from four patients with combined Factor V/VIII deficiency disease.
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PMID:Evidence of normal functional levels of activated protein C inhibitor in combined Factor V/VIII deficiency disease. 629 39

Factor VIII (antihemophilic factor) is the protein that is deficient or defective in patients with classical hemophilia and Von Willebrand syndrome. Factor VIII in plasma is thought to be associated in a complex with the highest molecular weight multimers of another glycoprotein, Von Willebrand protein. Highly purified human factor VIII appears to have an Mr of between 200,000 and 300,000 and to consist of several polypeptide chains. The concentration of factor VIII in plasma is around 100-200 ng/ml, equivalent to around 1 nM. The purified proteins retain one or more of the known properties of factor VIII, including the acceleration of factor IXa-mediated activation of factor X, ability to be activated by thrombin and factor Xa, inactivation by activated protein C, and by human antibodies to factor VIII. Among the known clotting factors, factors VIII and V are exceptional in not possessing enzymatic activity. Factors IXa and VIII and X appear to form a functional complex, all of which need to be present and active simultaneously for optimal activation of factor X. The mechanism by which factor VIII promotes activation of factor X by factor IXa is not known, but the major effect is to increase the rate of the reaction. Following treatment of factor VIII with thrombin, a new and smaller polypeptide Mr around 70,000 +/- 5,000 is produced. Factors IXa and Xa also have been reported to activate factor VIII. It is not known whether limited proteolytic cleavage is required absolutely for the expression of factor VIII activity or if it only increases an activity already expressed by the uncleaved protein. Factor VIII is inactivated by thrombin and by activated protein C. Thus, factor VIII can be modulated by at least four of the serine proteases in the clotting system. A major goal for future research is to increase our understanding of the role in blood clotting played by factor VIII, and to apply this information to clinical problems which result from inherited abnormalities of factor VIII.
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PMID:Factor VIII: structure and function in blood clotting. 642 37

Thrombomodulin (TM) on endothelial cells is a glycoprotein that functions as a cofactor for thrombin-catalyzed activation of protein C. The structural requirement for thrombin binding and cofactor activity were investigated using monoclonal antibodies (moAbs) against TM and site-directed mutagenesis of recombinant human soluble TM (rsTM). Results showed that moAb 2A2 inhibited thrombin binding to rsTM and also abolished its functions as a cofactor in thrombin-catalyzed activation of protein C and as an anticoagulant by modifying thrombin-induced fibrinogen clotting and platelet aggregation, moAb 1F2 did not affect its activity as an anticoagulant, but inhibited its cofactor activity, and moAb 10A3 did not inhibit either activity. Epitope analysis was carried out by site directed mutagenesis of rsTM expressed in CHO cells. Some proteins with mutations within the second disulfide loop of the fourth EGF-like domain showed reduced affinity for moAb 1F2, but retained cofactor activity. These results suggest that the epitope of moAb 1F2 includes the second disulfide loop of the fourth EGF-like domain, which is close to a region required for cofactor activity. Mutant proteins of the third disulfide loop of the fifth EGF-like domain showed loss of interaction with moAb 2A2. Thus the epitope of moAb 2A2 may include the third disulfide loop of the fifth EGF-like domain. Furthermore, replacement of Asn-439 by Gln decreased the cofactor activity and anticoagulant activity, and resulted in low affinity for either moAb 1F2 or 2A2, suggesting that Asn-439, which is located in the second disulfide loop of the sixth EGF-like domain, is critical for determining the functional conformation of the EGF-like domains 4-6.
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PMID:Monoclonal antibodies against human thrombomodulin whose epitope is located in epidermal growth factor-like domains. 751 85

Two classes of antiphospholipid antibodies (APA) are associated with adverse pregnancy outcomes. Those APA identified by immunoassays using phospholipid-coated surfaces (e.g., anticardiolipin antibodies) seem to bind to the 57 kD anticoagulant protein, beta 2-glycoprotein-I, when complexed with anionic phospholipid bilayers. Such APA may or may not prolong phospholipid-dependent clotting assays. A second class of APA are identified by their interference with phospholipid-dependent clotting assays (i.e., lupus anticoagulants). The latter bind to phospholipids present in a unique hexagonal phase either alone or complexed with prothrombin or beta 2-glycoprotein-I. There is evidence that both classes of APA are directly responsible for adverse pregnancy outcomes including spontaneous abortions, stillbirths, fetal growth retardation, thrombosis, thrombocytopenia, and preeclampsia. Putative APA-mediated pathogenic mechanisms include intervillous thrombosis, intravillous infarctions and decidual vasculopathy. The thrombogenicity of APA may result from their interference with endothelial phospholipids required for antithrombin III and protein C and S anticoagulant activity and prostacyclin synthesis and/or increased endothelial expression of the procoagulants: tissue factor, von Willebrand factor, platelet-activating factor, and plasminogen activator inhibitor type-1. Other prothrombotic properties seem to include: increased platelet aggregation, and reduced beta 2-glycoprotein-1 and annexin V anticoagulant activity. Rigorous diagnostic criteria must be applied to the detection of both classes of APA because the prevention of adverse pregnancy outcomes requires potentially hazardous anticoagulant therapy.
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PMID:The immunobiology and obstetrical consequences of antiphospholipid antibodies. 752 11

Hepatitis C virus (HCV) is the major cause of transfusion-acquired non-A, non-B hepatitis. HCV is an enveloped positive-sense RNA virus which has been classified as a new genus in the flavivirus family. Like the other two genera in this family, the flaviviruses and the pestiviruses, HCV polypeptides appear to be produced by translation of a long open reading frame and subsequent proteolytic processing of this polyprotein. In this study, a cDNA clone encompassing the long open reading frame of the HCV H strain (3,011 amino acid residues) has been assembled and sequenced. This clone and various truncated derivatives were used in vaccinia virus transient-expression assays to map HCV-encoded polypeptides and to study HCV polyprotein processing. HCV polyproteins and cleavage products were identified by using convalescent human sera and a panel of region-specific polyclonal rabbit antisera. Similar results were obtained for several mammalian cell lines examined, including the human HepG2 hepatoma line. The data indicate that at least nine polypeptides are produced by cleavage of the HCV H strain polyprotein. Putative structural proteins, located in the N-terminal one-fourth of the polyprotein, include the capsid protein C (21 kDa) followed by two possible virion envelope proteins, E1 (31 kDa) and E2 (70 kDa), which are heavily modified by N-linked glycosylation. The remainder of the polyprotein probably encodes nonstructural proteins including NS2 (23 kDa), NS3 (70 kDa), NS4A (8 kDa), NS4B (27 kDa), NS5A (58 kDa), and NS5B (68 kDa). An 82- to 88-kDa glycoprotein which reacted with both E2 and NS2-specific HCV antisera was also identified (called E2-NS2). Preliminary results suggest that a fraction of E1 is associated with E2 and E2-NS2 via disulfide linkages.
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PMID:Expression and identification of hepatitis C virus polyprotein cleavage products. 767 46

A novel plasminogen activator from Trimeresurus stejnegeri venom (TSV-PA) has been identified and purified to homogeneity. It is a single chain glycoprotein with an apparent molecular weight of 33,000 and an isoelectric point of pH 5.2. It specifically activates plasminogen through an enzymatic reaction. The activation of human native Glu-plasminogen by TSV-PA is due to a single cleavage of the molecule at the peptide bond Arg561-Val562. Purified TSV-PA, which catalyzes the hydrolysis of several tripeptide p-nitroanilide substrates, does not activate nor degrade prothrombin, factor X, or protein C and does not clot fibrinogen nor show fibrino(geno)lytic activity in the absence of plasminogen. The activity of TSV-PA was readily inhibited by phenylmethanesulfonyl fluoride and by p-nitrophenyl-p-guanidinobenzoate. Oligonucleotide primers designed on the basis of the N-terminal and the internal peptide sequences of TSV-PA were used for the amplification of cDNA fragments by polymerase chain reaction. This allowed the cloning of a full-length cDNA encoding TSV-PA from a cDNA library prepared from the venom glands. The deduced complete amino acid sequence of TSV-PA indicates that the mature TSV-PA protein is composed of 234 amino acids and contains a single potential N-glycosylation site at Asn161. The sequence of TSV-PA exhibits a high degree of sequence identity with other snake venom proteases: 66% with the protein C activator from Agkistrodon contortrix contortrix venom, 63% with batroxobin, and 60% with the factor V activator from Russell's viper venom. On the other hand, TSV-PA shows only 21-23% sequence similarity with the catalytic domains of u-PA and t-PA. Furthermore, TSV-PA lacks the sequence site that has been demonstrated to be responsible for the interaction of t-PA (KHRR) and u-PA (RRHR) with plasminogen activator inhibitor type 1.
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PMID:A novel plasminogen activator from snake venom. Purification, characterization, and molecular cloning. 773 Mar 29

The mouse model described in this study offers a unique method of determining the characteristics and mechanism(s) of action of aCL antibodies in thrombosis in vivo. In addition, this animal model enables the study of the kinetics of formation and dissolution of thrombus, as well as clot area, to be studied in a dynamic fashion. Other models for evaluation of thrombus formation rely on measurements of thrombus size and weight in ligated vessel segments where flow may be interrupted artificially. In addition, two important findings can be extracted from the study. (1) The size of the thrombi were significantly larger in mice that were passively immunized with IgG-APS (four patient samples examined) and with IgM-APS (two patient samples examined) compared with mice injected with saline or with immunoglobulin from control patients. (2) The clot persisted significantly for longer periods of time (total time) in animals injected with IgG-APS or IgM-APS when compared with control animals. Based on in vitro experiments, it is possible that these antibodies may inhibit protein C activation, neutralize the inhibitory activity action of beta 2 glycoprotein I (beta 2GPI), or activate platelets at the site of the femoral vein injury. Because this model enables to study the dynamics of thrombus formation, it is possible that these hypotheses and other mechanisms by which aPL antibodies are thrombogenic be investigated. Future studies will also include the effects of different levels of antibodies, as well as effects of affinity purified and monoclonal aPL antibodies on thrombus formation.
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PMID:Antiphospholipid antibodies in an in vivo thrombosis model in mice. 780 10


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