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 molecular defect responsible for a dramatic prolongation of all standard clotting tests discovered in a 15-yr-old boy has been identified. Initial investigations revealed the presence of an activated Factor X (Factor Xa) and thrombin inhibitor which copurified with alpha 1-antitrypsin (alpha 1-AT), thereby suggesting the occurrence of an alpha 1-AT variant similar to alpha 1-AT Pittsburgh. This was confirmed by dot-blot analysis and direct sequencing after amplification by the polymerase chain reaction. A G to T transition at nucleotide 10038 results in the substitution of Met to an Arg, converting alpha 1-AT into an Arg-Ser protease inhibitor (serpin) that inhibited thrombin and Factor Xa more effectively than antithrombin III. Surprisingly, there was no bleeding history in the proband. The common mutation Z, which may explain a reduced expression of the allele bearing the Arg 358 Met mutation, was not observed in the propositus' DNA. To exclude the presence of another mutation, the coding regions and intron/exon junctions were sequenced. No other mutation was found. Recently, the patient experienced his first hemorrhagic episode at the age of 17. The level of the abnormal inhibitor had increased twofold 2 mo before. The large decrease in protein C concentration may account for the mild bleeding tendency in this case, despite the presence of the alpha 1-AT Pittsburgh mutation. An abnormal protein C pattern was observed in patient's plasma, suggesting that the circulating deficiency might be due to a deleterious effect of the abnormal inhibitor on both intracellular processing and catabolism of protein C.
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PMID:Met 358 to Arg mutation of alpha 1-antitrypsin associated with protein C deficiency in a patient with mild bleeding tendency. 156 92

Antibody inhibitors against human thrombin are rare and have remained poorly characterized. We report the case of a 40-yr-old patient who developed a potent thrombin inhibitor revealed by mild bleeding symptoms and marked prolongation of most laboratory clotting times. After two years of evolution, he died from cerebral hemorrhage. The inhibitor, a polyclonal IgG, was associated with hematological and immunological criteria of autoimmune disorder. Antithrombin IgG was isolated from the patient's plasma by protein A- and thrombin-affinity chromatography. Fab fragments inhibited amidolytic activity of alpha thrombin, and thrombin-thrombomodulin catalyzed protein C activation with a Ki of approximately 10(-8) M in a noncompetitive manner. Alpha to gamma conversion of thrombin resulted in a moderate loss of affinity for the inhibitor. Upon complex formation of thrombin with staphylocoagulase or alpha 2-macroglobulin (alpha 2M), inhibition was decreased by two orders of magnitude and acquired an apparent competitive character. In Western blot experiments, the antibody reacted with active alpha-thrombin, did not react with chloromethylketone-inhibited thrombin and reacted with a lower affinity with iPr2P-thrombin. The inhibitor did not block thrombin binding to benzamidine-, heparin-, or fibrin-Sepharose, but displaced proflavin from its complex with thrombin. Taken together, these results indicate that the patient's autoantibody recognized a conformational structure which includes, at least in part, the apolar binding site adjacent to the catalytic site of thrombin.
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PMID:An acquired antithrombin autoantibody directed toward the catalytic center of the enzyme. 171 42

The levels of prothrombin fragment F1 + 2 were measured by a double antibody radioimmunoassay in blood samples collected into different anticoagulant solutions. We evaluated healthy males between the ages of 42 and 77, asymptomatic patients with hereditary deficiencies of protein C or protein S, and persons receiving tumor necrosis factor infusions. The results in specimens collected in an anticoagulant containing ACD, EDTA, adenosine, and 25 U/ml of heparin (a) were highly correlated with those collected in an anticoagulant containing a synthetic thrombin inhibitor, EDTA, and aprotinin (b). However, in asymptomatic patients with congenital antithrombin III deficiency, we found that the plasma levels of F1 + 2 in blood collected in anticoagulant (a) were usually substantially higher than those collected in anticoagulant (b). We determined that this phenomenon was not attributable to the venipuncture procedure itself, but rather appears to be due to the action of low concentrations of heparin in the presence of reduced blood levels of antithrombin III. Our data show that the previously documented elevations in plasma F1 + 2 levels in patients with congenital antithrombin III deficiency appear to be caused by the above in vitro anticoagulant effect, and that this population does not exhibit evidence of a prethrombotic state as defined by the F1 + 2 assay.
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PMID:Influence of anticoagulants used for blood collection on plasma prothrombin fragment F1 + 2 measurements. 178 Aug 6

Anticoagulant as well as anti-platelet drugs are important medicines for the prophylaxis in various kinds of thrombotic diseases. However, the conventional anticoagulant drugs, heparin and coumarin congeners, have some disadvantages and limitations in clinical usage. Recently newly anticoagulants, both synthetic and recombinant, have been developing. They include synthetic thrombin inhibitor, recombinant hirudin, protein C and thrombomodulin. Here we reviewed synthetic thrombin inhibitor, Argipidine (MD805) in clinical trial and investigated its effect on thrombin catalyzed protein C activation on endothelial cells. Argipidine inhibited the protein C activating activity of thrombin on endothelium in a dose response manner. Next we examined the effect of Argipidine on thrombin-induced endothelin release from cultured endothelial cells. The augmentation of endothelin release from endothelial cells by thrombin was also inhibited by Argipidin. The effect was considered one of the advantage of this drug in the treatment of thrombosis. Recombinant thrombomodulin had potent antithrombotic effect on thrombin-induced acute thromboembolism in mice, suggesting that this may be expectant anticoagulant for DIC or thromboses in human.
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PMID:[Synthetic anticoagulant]. 217 Jul 4

A complex composed of factor Xa and phospholipid vesicles assembled in the presence of calcium ions catalyzes a discrete cleavage of the heavy chain of bovine protein C that is indistinguishable from that produced by thrombin as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This cleavage generates an active site capable of hydrolyzing small substrates and inactivating factor Va function in the prothrombinase complex. Activation of protein C by factor Xa requires both calcium ions and phospholipid vesicles and proceeds at a rate an order of magnitude greater than that observed for alpha-thrombin in solution. gamma-Carboxyglutamic acid-domainless protein C is not activated by factor Xa, consistent with the requirement for phospholipid and distinguishing this reaction from protein C activation by thrombin. Thrombomodulin serves as a cofactor for the factor Xa-catalyzed reaction, forming a 1:1 complex with factor Xa (apparent Kd = 5.7 X 10(-10) M) and stimulating the saturated rate of protein C activation by factor Xa (kcat = 149 min-1) to levels comparable with the thrombin-thrombomodulin complex. Protein C activation by factor Xa is not inhibited by the specific thrombin inhibitor dansyl-N-(3-ethyl-1,5-pentanediyl)amide but is inhibited by antithrombin III, tripeptide-chloromethyl ketones, and the monoclonal antibody alpha-BFX-2b that is highly specific for factor Xa. These data indicate that thrombomodulin is promiscuous in its role as a cofactor and suggest the existence of an alternative pathway for protein C activation in vivo.
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PMID:The activation of bovine protein C by factor Xa. 255 Apr 35

Utilizing site-directed mutagenesis, 77 charged and polar residues that are highly exposed on the surface of human thrombin were systematically substituted with alanine. Functional assays using thrombin mutants identified residues that were required for the recognition and cleavage of the procoagulant substrate fibrinogen (Lys21, Trp50, Lys52, Asn53 + Thr55, Lys65, His66, Arg68, Tyr71, Arg73, Lys77, Lys106 + Lys107, Asp193 + Lys196, Glu202, Glu229, Arg233, Asp234) and the anticoagulant substrate protein C (Lys21, Trp50, Lys65, His66, Arg68, Tyr71, Arg73, Lys77, Lys106 + Lys107, Glu229, Arg233), interactions with the cofactor thrombomodulin (Gln24, Arg70) and inhibition by the thrombin aptamer, an oligonucleotide-based thrombin inhibitor (Lys65, His66, Arg70, Tyr71, Arg73). Although there is considerable overlap between the functional epitopes, distinct and specific residues with unique functions were identified. When the functional residues were mapped on the surface of thrombin, they were located on a single hemisphere of thrombin that included both the active site cleft and the highly basic exosite 1. No functional residues were located on the opposite face of thrombin. Residues with procoagulant or anticoagulant functions were not spatially separated but interdigitated with residues of opposite or shared function. Thus thrombin utilizes the same general surface for substrate recognition regardless of substrate function although the critical contact residues may vary.
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PMID:Functional mapping of the surface residues of human thrombin. 762 1

Protein C inhibitor (PCI) is a heparin-binding serine proteinase inhibitor (serpin) which is thought to be a physiological regulator of activated protein C (APC). The residues F353-R354-S355 (P2-P1-P1') constitute part of the reactive site loop of PCI with the R-S peptide bond being cleaved by the proteinase. Changing the reactive site P1 and P2 residues to those of either proteinase nexin-1, alpha 1-proteinase inhibitor or heparin cofactor II resulted in a decrease in inhibitory activity towards thrombin and APC. Changing the P2 residue F353-->P generated a rPCI which was a better thrombin inhibitor, but was 10-fold less active with APC. While these results support the concept that the P1 and P2 residues are important in the specificity of PCI, they suggest that the reactive site residues are not the only determinant of serpin specificity. Kinetic analysis of the rPCI variants was consistent with PCI operating by a mechanism similar to that proposed for other serpins. In this model an intermediary complex forms between inhibitor and proteinase that can proceed to either cleavage of the inhibitor as substrate or formation of an inactive complex.
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PMID:Reactive site mutants of recombinant protein C inhibitor. 781 27

Platelet-dependent thrombosis and subsequent embolization are major causes of cerebral ischaemia. Beside aspirin which irreversibly blocks platelet cyclo-oxygenase, several other substances interfere in different platelet metabolic pathways and block platelet adhesion and aggregation. We found in an experimental model using non-human primates that a specific peptide inhibitor blocking GP IIb/IIIa platelet receptor which binds fibrinogen completely, prevents the retention of embolized platelet aggregates in the cerebral circulation. As thrombin may play a key role for platelet activation in vivo leech-derived hirudin, a direct thrombin inhibitor as well as activated protein C which limits thrombin production and also prevents platelet dependent thrombus formation very effective. We demonstrated in the same non-human primate model of platelet embolization that the amount of retention of platelet emboli in the vascular bed depends on the nature of the vasculature. For example, platelet emboli were cleared very quickly from brain microcirculation, whereas platelet embolization into the lower limb via the femoral artery caused a significantly longer retention of the embolized material. Such specific mechanisms may be caused by different levels of local vasodilators as PGI2 or EDRF.
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PMID:Platelet thromboembolism. 801 31

In this study, we evaluated the effects of anticoagulants used in blood sampling on the measurements of coagulation activation markers F1 + 2, TAT, D-Dimers by Elisa methods. The study was carried out on normal subjects and patients with inherited deficiency of coagulation inhibitors, antithrombin III (ATIII) protein C (PC) and protein S (PS). Three different anticoagulant solutions were compared: 1) ACD/EDTA/adenosine/heparin, 2) EDTA/aprotinin/a synthetic thrombin inhibitor and 3) sodium citrate. The results showed that sodium citrate, commonly used in coagulation laboratories, is a suitable anticoagulant for the study of coagulation activation markers. In addition, the type of tubes (plastic tubes vs glass Vacutainer R tubes) used for blood sampling as well as the order of sampling (early or late after the phlebotomy procedure) did not influence the results. We concluded that assays of coagulation activation markers F1 + 2 and D-Dimers can be performed in samples collected routinely by haemostasis laboratory staff using Vacutainer R tubes with sodium citrate. Further investigations are needed to understand why TAT measurements gave a pattern of results quite different from F1 + 2 or D-Di measurements.
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PMID:Influence of conditions of blood sampling on coagulation activation markers (prothrombin fragment 1 + 2, thrombin-antithrombin complexes and D-dimers) measurements. 808 41

A new thrombin inhibitor, bothrojaracin, has been identified and purified to homogeneity from the venom of Bothrops jararaca, the most common venomous snake of South America. Bothrojaracin has an isoelectric point of 4.2 and a molecular mass of 27 kDa and is made of two distinct polypeptide chains of 15 and 13 kDa, linked by disulfide bridges. Purified bothrojaracin is devoid of phospholipase A2, amidolytic, or fibrino (geno)lytic activity. Bothrojaracin forms a noncovalent complex with alpha-thrombin, without changing its catalytic activity on small peptide substrates. Bothrojaracin behaves as a potent and specific antagonist of thrombin-induced platelet aggregation and secretion, characterized by an IC50 ranging from 1 to 20 nM depending on the alpha-thrombin concentration. Bothrojaracin prolongs fibrinogen clotting time, and this effect is related to a competitive inhibition of the binding of alpha-thrombin to fibrin(ogen) (Ki 15 nM). Binding of alpha-thrombin to thrombomodulin is inhibited up to 87% by bothrojaracin, and the rate of protein C activation by alpha-thrombin is also decreased. Bothrojaracin antagonizes the inhibition of thrombin amidolytic activity by hirudin. These results indicate that bothrojaracin acts as a very potent ligand of the exosite of alpha-thrombin.
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PMID:Bothrojaracin, a new thrombin inhibitor isolated from Bothrops jararaca venom: characterization and mechanism of thrombin inhibition. 839 28


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