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)

Coagulation factor Va is an essential cofactor which combines with the serine protease factor Xa on a phospholipid surface to form the prothrombinase complex. In the present study, the structure of factor Va interacting with lipid surfaces containing phosphatidylserine was studied by electron microscopy. Two-dimensional crystals of factor Va were obtained on planar lipid films under quasi-physiological conditions. The two-dimensional projected structure of factor Va was calculated at a resolution of 2 nm, revealing dimers of factor Va arranged on the surface lattice with the symmetry of the plane group p2. Average unit cell dimensions are a = 14.4 nm, b = 8.8 nm, gamma = 107 degrees. Each factor Va molecule presents two distinct domains of protein density consisting of one small domain, of 3 nm in diameter, connected to a larger domain of about 6 nm x 4.5 nm. The projected structure of factor Va covers an area equivalent to about fifty phospholipid molecules. In addition, edge-on views of factor Va molecules bound to liposomes reveal a globular structure connected through a thin stem to the liposome surface. A three-dimensional model of membrane-bound factor Va is proposed.
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PMID:Structure of membrane-bound human factor Va. 808 90

Protein C is a vitamin K-dependent serine protease zymogen that upon activation inhibits the coagulation cascade by inactivating factors Va and VIIIa. In an attempt to improve the anticoagulant activity of activated protein C (APC), we have prepared a mutant of protein C in mammalian cells in which Glu at position 192 (chymotrypsin numbering system) has been replaced with Gln (PC E192Q). Our strategy is based on the observation that the same substitution in thrombin improves the catalytic activity toward natural and synthetic substrates that contain Asp residues at P3 and P3'. Since factor Va also has an Asp at position P3 in the APC cleavage site of the factor Va heavy chain, we hypothesized that APC E192Q would inactivate factor Va more rapidly than wild type APC. The mutant inactivated factor Va approximately 2-3-fold faster than wild type. In plasma the mutant exhibited slightly less anticoagulant activity than wild type enzyme. Further characterization revealed that APC E192Q is inhibited 280 times faster than APC by alpha 1-antitrypsin (K2 = 2.8 x 10(3) M-1S-1 versus 10 M-1 S-1), and unlike APC, APC E192Q is inhibited by antithrombin III in the presence of heparin (K2 = 1.17 x 10(3) M-1 S-1) M-1 S-1) and absence of heparin (K2 = 57 M-1 S-1). Ca2+ increased K2 more than 4-fold with or without heparin. Unlike wild type APC, APC E192Q was effectively inhibited by pancreatic trypsin inhibitor (Ki = 10.6 +/- 0.26 nM) and tissue factor pathway inhibitor (58 +/- 5 nM). Like factor Xa, APC E192Q rapidly processed factor IX to factor IX alpha. These observations suggest that even though Glu at position 192 is not an optimal residue for catalyzing factor Va inactivation, it is an evolutionary adaptation to slow inhibition by plasma protease inhibitors.
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PMID:Conversion of glutamic acid 192 to glutamine in activated protein C changes the substrate specificity and increases reactivity toward macromolecular inhibitors. 810 82

Blood coagulation factors IX and X are two serine proteases with a similar modular structure. The non-catalytic part of each protein consists of a gamma-carboxyglutamic acid (Gla)-containing module and two modules homologous to the epidermal growth factor (EGF) precursor. We have now found that the NH2-terminal EGF-like module of both factors IX and X inhibits factor Xa formation in a Gla-independent manner, both in the presence and absence of phospholipid and the cofactor, factor VIIIa. In contrast, the COOH-terminal EGF-like module has no such effect. Our data indicate that the NH2-terminal EGF-like module of factor IXa beta interacts either with the corresponding module or with the serine protease module in the substrate, factor X, without affecting the hydrolysis of low molecular weight substrates. Using antibodies as structural probes, we found that Ca2+ binding to the Gla module of factor IXa beta induces a conformational transition in the serine protease module. No evidence was found for a direct interaction between the Gla module and factor VIIIa. We therefore propose that the Gla module in factor IXa beta is indirectly involved in the cofactor interaction, in that Ca2+ binding to sites in this module induces a conformation in the serine protease module that is commensurate with factor VIIIa interaction. In addition, the immunochemical approach revealed a Gla-independent Ca2+ binding site in the serine protease module (apparent Kd of approximately 120 microM) that also might influence its conformation. Antibodies against the EGF-like modules of factor IX were used to probe Ca2+ binding to these modules in intact and in Gla-domainless factor IXa beta. The data indicate a Ca2+ binding site with an apparent Kd of approximately 50 microM in the NH2-terminal EGF-like module of both factor IX species.
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PMID:The gamma-carboxyglutamic acid and epidermal growth factor-like modules of factor IXa beta. Effects on the serine protease module and factor X activation. 810 13

The coagulation enzyme thrombin, a serine protease like all other coagulation factors, plays a central role in the hemostatic processes engaged after injurious events. It induces, with particular efficacy, the aggregation of blood platelets (primary hemostasis) and accounts, via splitting of fibrinogen to fibrin, for the event actually responsible for the coagulation of blood (secondary hemostasis). As is well-known, thrombin itself is generated by a cascade of activation events involving various coagulation factors (F). In this respect the "tissue factor" (TF, formerly known as thromboplastin), in combination with F VIIa, attains decisive significance, not only in the extrinsic pathway of coagulation (activation of F X-->Xa), but also in the intrinsic pathway (activation of F IX-->IXa). Under physiological circumstances, platelet aggregation and coagulation are restricted to the area of the vascular lesion, since the surrounding intact endothelium inhibits an intraluminal spreading of both processes. These "antithrombotic" features of the endothelium encompass antiaggregatory mechanisms (formation and release of prostacyclin [PGI2], adenosine, EDRF [NO], degradation of ADP and other nucleotides mediated by ecto-nucleotidases) as well as anti-coagulatory properties (formation and release of "tissue factor pathway inhibitor" [TFPI], which blocks the coagulation cascade by joining F Xa, TF and F VIIa into an inactive complex, thrombomodulin--thrombin induced activation of protein C, which, together with protein S, inactivates F Va and F VIIIa, thereby attenuating further generation of thrombin, and the heparan sulfate-enhanced activation of antithrombin III and heparin-cofactor II).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:[Interaction of blood and the vascular wall: hemostatic aspects]. 815 53

Recent advances in determining anti-thrombogenic functions of vascular endothelial cells are reviewed. The following anticoagulant and fibrinolytic systems of endothelial cells are physiologically important; (1) Endothelial cell-derived metabolites including prostacyclin and nitric oxide (NO) support platelet inactivity. (2) Antithrombin III and tissue factor pathway inhibitor (TFPI) bound to heparin-like proteoglycans on endothelial cell membrane inhibit activated serine protease coagulation factors such as thrombin, factor Xa and factor VIIa-tissue factor complex. (3) Thrombomodulin converts thrombin from procoagulant into anticoagulant. Thrombin associated to thrombomodulin on endothelial cells activates protein C. Activated protein C in concert with protein S bound to endothelial cell membrane inactivates factors Va and VIIIa. (4) A receptor for both tissue plasminogen activator and plasminogen on endothelial cells provides an efficient plasmin generating system. Perturbation of these anti-thrombogenic systems of endothelial cells is caused by endotoxin (LPS), cytokines such as interleukin-1 and tumor necrosis factor (TNF), and risk factors for atherogenesis including lipoprotein(a) and homocysteine may result in arterial or venous thrombosis with subsequent development of atherosclerosis.
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PMID:[Anticoagulant and fibrinolytic systems of the injured vascular endothelial cells]. 817 40

Antithrombin is a serine protease inhibitor that participates in the inactivation and removal from the circulation of thrombin and a variety of other procoagulant serine proteases. Antithrombin is also the major plasma cofactor of heparin which exerts its therapeutic effect primarily through its ability to substantially increase the rate of inactivation by antithrombin of the procoagulant serine proteases. Binding of heparin to antithrombin is thus believed to be a prerequisite for this rate enhancement effect. Heparin binding to antithrombin is mediated by a well-defined unique heparin pentasaccharide sequence. Interaction between this pentasaccharide sequence and antithrombin induces a conformational change in antithrombin, an alteration that appears to be sufficient to explain the enhanced ability of antithrombin to inhibit factor Xa and related serine proteases, but not thrombin. Heparin species with longer polysaccharide chains appear to be required in order to enhance the inhibition of thrombin by antithrombin. This may be because the enhancement of this reaction requires that heparin interacts simultaneously with both the antithrombin and the thrombin molecules. This review describes the interactions between heparin and antithrombin, focusing on the antithrombin residues which are involved in the binding of heparin. The role of the heparin-induced conformational change in enhancing serine protease inhibition by antithrombin is also explored. Then, based on available data, an hypothesis is proposed to explain the mechanisms by which heparin accelerates the rate of inactivation by antithrombin of the various serine proteases.
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PMID:Defining the heparin-binding domain of antithrombin. 818 Mar 43

Factor X circulates as a serine protease which is converted to the active form at the point of convergence of the intrinsic and extrinsic coagulation pathways. Subsequently, the enzymatic species, factor Xa, is involved in macromolecular complex formation with its cofactor factor Va, a phospholipid surface and calcium to convert prothrombin into thrombin. The gene encoding factor X shares a number of structural and organisational features in common with the other vitamin K-dependent coagulation proteins, suggesting that they have evolved from a common ancestral gene. Each of the exons encoding these proteins can be considered as a module coding for a homologous domain in each protein. These structural domains in factor X are responsible for specific functional properties including gamma-carboxylase recognition, calcium binding, phospholipid surface interaction, as well as cofactor and substrate binding. Studies of recombinant proteins and proteolytic fragments continue to provide significant insight into structure-function relationships of the protein modules within factor X.
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PMID:Biochemistry of factor X. 820 10

An integral membrane protease was solubilized and purified to homogeneity from rat submaxillary mitochondria. The purified enzyme could coagulate rabbit plasma. The molecular mass of the enzyme is 22 kDa on SDS-polyacrylamide gel electrophoresis under reducing conditions and 24 kDa on gel filtration on a Sephadex G-100 column. Its isoelectric point is 4.2-4.25. Enzyme activity is strongly inhibited by diisopropyl fluorophosphate, soybean trypsin inhibitor, benzamidine, aprotinin, and antipain, suggesting the enzyme as a serine protease. Its pH optimum for activity is 8.5. Zn2+ is strongly inhibitory; at 1 mM concentration it produced 72% inhibition. The enzyme is active toward different synthetic substrates (p-nitroanilide derivatives) containing Arg at the P1 position with blocked NH2 terminus. Kcat/Km was highest with the substrate N-Bz-Pro-Arg-pNa (where Bz is benzoyl and pNA is paranitroanilide). The purified enzyme coagulates rabbit plasma in a dose-dependent manner. Plasma coagulation by the enzyme is completely blocked in the presence of aprotinin or soybean trypsin inhibitor, suggesting that protease activity is required for this coagulation reaction. Antibody raised against the purified enzyme inhibits the plasma coagulation initiated by the enzyme. The enzyme can correct the prolonged clotting time of factor X-deficient human plasma but is unable to convert purified fibrinogen to fibrin clots, indicating factor Xa-like activity of the enzyme. The enzyme has the ability to activate prothrombin. Several properties of the enzyme distinguish it from other reported submaxillary proteases.
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PMID:A new blood-coagulating protease in mitochondrial membranes of rat submaxillary glands. Purification and characterization of protease and its blood-coagulating activity. 820 26

The assembly of macromolecular complexes containing factors Xa and Va on suitable phospholipid surfaces is crucial for rapid activation of prothrombin. We have used quantitative affinity chromatography to characterize the interaction between factor Va and intact factor Xa on the one hand and between factor Va and factor Xa lacking the gamma-carboxyglutamic acid (Gla)-containing module on the other. The dissociation constants were found to be 1.0 +/- 0.1 and 9.5 +/- 1.8 microM, respectively. There was good agreement between these dissociation constants and the concentrations of active site-inhibited factor Xa and Gla-domainless factor Xa that caused half-maximal inhibition of prothrombin activation. To investigate whether the noncatalytic modules of factor Xa interacted directly with factor Va, intact modules were isolated from proteolytic digests of factor X and used as inhibitors of prothrombin activation. The inhibitory effect observed with the isolated Gla module in the absence of phospholipid was due to inhibition of the amidolytic activity of factor Xa rather than to an interaction with factor Va. The epidermal growth factor-like modules did not inhibit prothrombin activation. Using antibodies specific for calcium-dependent epitopes in the serine protease module of factor Xa we demonstrated that Ca2+ binding to the Gla module alters the conformation of the catalytic module. Half-maximal binding was observed at approximately 0.8 mM Ca2+. Evidence was also obtained for the presence of two Gla-independent Ca(2+)-binding sites in factor Xa. One of these sites, located in the NH2-terminal epidermal growth factor-like module, was half-saturated at approximately 60 microM Ca2+ in intact factor Xa and at approximately 1.2 mM Ca2+ in Gla-domainless factor Xa. This site appeared not to influence the conformation of the protease module. The second site, which was half-saturated at approximately 0.16 mM Ca2+, appeared to reside in the serine protease module and to alter its conformation as judged by binding of antibodies specific for calcium-dependent epitopes.
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PMID:Effects of Ca2+ binding on the protease module of factor Xa and its interaction with factor Va. Evidence for two Gla-independent Ca(2+)-binding sites in factor Xa. 822 63

A Limulus intracellular coagulation inhibitor, designated LICI, was isolated from hemocytes of the Japanese horseshoe crab (Tachypleus tridentatus), using three steps of chromatography, including dextran sulfate-Sepharose CL-6B, Sephacryl S-200, and Mono S. LICI is a single-chain glycoprotein with an apparent M(r) = 48,000 estimated by SDS-polyacrylamide gel electrophoresis. It blocks the amidolytic activities of Limulus lipopolysaccharide-sensitive serine protease, factor C, by forming a covalent 1:1 complex with the protease. The second-order rate constant for inhibition of factor C was 2.5 x 10(6) M-1 s-1 at 37 degrees C. LICI also inhibited human alpha-thrombin, rat salivary kallikrein, bovine plasmin, and trypsin but not Limulus clotting enzyme, Limulus factor B, bovine factor Xa, human factor XIa, human tissue plasminogen activator, human urokinase, chymotrypsin, elastase, and papain. Glycosaminoglycans such as heparin and heparan sulfate had no effect on the inhibitory activity. A cDNA coding for LICI was isolated from a hemocyte cDNA library. The open reading frame of the 1,257-base pair cDNA codes for the mature protein of 394 amino acids, of which 223 residues were confirmed by amino acid sequence analysis. LICI shows significant sequence identities to members of the serpin superfamily, such as human plasminogen activator inhibitor type 2 (40%) and human monocyte/neutrophil elastase inhibitor (39%). LICI contains a putative reactive site, -Arg-Ser-, at the corresponding position present in several inhibitors of the serpin superfamily. The subcellular localization, determined using an anti-LICI polyclonal antibody, indicated that LICI colocates with the Limulus serine protease zymogens in large granules in the hemocyte.
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PMID:A Limulus intracellular coagulation inhibitor with characteristics of the serpin superfamily. Purification, characterization, and cDNA cloning. 827 48


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