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)

Ecotin, a serine protease inhibitor found in the periplasm of Escherichia coli, has been characterized as an extremely potent anticoagulant and reversible tight-binding inhibitor of human factor Xa (FXa). The ecotin gene was cloned by PCR, highly expressed in E. coli, and purified from the E. coli periplasm. The binding of ecotin to FXa was stoichiometric with an equilibrium dissociation constant Ki of 54 pM. The association rate constant was 1.35 x 10(6) M-1 s-1, and the dissociation rate constant, measured in the presence of human leukocyte elastase (HLE) to prevent reassociation of ecotin with FXa, was 6.5 x 10(-5) s-1. Ecotin prolonged clotting time ca. 10-fold at 0.3 microM and at 2 microM in activated partial thromboplastin time and prothrombin time assays, respectively. Ecotin did not effectively inhibit the human plasma proteases thrombin, tissue factor.factor VIIa, factor XIa, activated protein C, plasmin, or tissue plasminogen activator (t-PA); however, it did potently inhibit factor XIIa, plasma kallikrein, HLE, and bovine trypsin and chymotrypsin. Coincubation of ecotin and FXa at 10 microM each resulted in a (ecotin)2.(FXa)2 complex as determined by gel filtration. Dimerization of ecotin alone was measured by fluorescence titration which yielded a Kd of ca. 390 nM. FXa cleaved ecotin slowly at pH 4.0 between M84 and M85. Replacement of the P1 Met84 residue with Arg and Lys led to FXa inhibitors with Ki values of 11 and 21 pM, respectively. The P1 Arg and Lys mutants also significantly inhibited thrombin, factor XIa, activated protein C, plasmin, factor XIIa, kallikrein, and bovine trypsin and chymotrypsin but did not inhibit tissue factor.factor VIIa, t-PA, or HLE.
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PMID:Ecotin is a potent anticoagulant and reversible tight-binding inhibitor of factor Xa. 814 99

Plasma from healthy individuals, pregnant women and patients on warfarin were distributed to 3 laboratories supporting major cardiovascular surveys (Northwick Park, Muenster and Houston) for assay of factor VII coagulant activity (VIIc) with their own bio-assays. The mean VIIc in 147 samples agreed to within 1% of standard in Northwick Park and Houston, but was 14% of standard lower in Muenster owing to its more potent standard. In samples with an increased VIIc the Northwick Park assay gave a higher result than the other assays owing to its increased responsiveness to activated factor VII (VIIa). Thus when VIIa concentrations were determined directly with a clotting assay which utilises a soluble recombinant tissue factor, the increase in VIIc with increase in VIIa was considerably greater with the Northwick Park assay than the Muenster assay. This feature of the Northwick Park assay was traced to the virtual absence of protein C in its substrate plasma. Factor Va appears rate-limiting for the coagulant expression of VIIa in test plasma. If the thrombotic response to release of tissue factor is determined by the circulating concentration of VIIa, then the Northwick Park factor VII bio-assay may be preferable to other bio-assays currently employed to estimate risk of acute coronary events.
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PMID:Factor VII-deficient substrate plasmas depleted of protein C raise the sensitivity of the factor VII bio-assay to activated factor VII: an international study. 816 45

This article has stressed the common hereditary and acquired blood protein defects associated with thrombosis. The commonest hereditary defects appear to be antithrombin, protein C, and protein S deficiency, and the commonest acquired defects are anticardiolipin antibodies and the lupus anticoagulant. Therefore these are the defects that should first be looked for in an individual with unexplained thrombosis. If these commoner defects are not found, the rarer defects, including HC-II, plasminogen or t-PA deficiency, dysfibrinogenemia, or elevated PAI-1, should next be sought. The incidence of activated protein C cofactor deficiency is not yet clear but may also represent a common defect. Likewise, PAI-1 defects may, with time, be shown to be quite common. The importance of finding these defects has significant implications for therapy of the individual patient and for institution of family studies to identify, inform, and possibly treat others at risk. It is expected that as knowledge of hemostasis expands, more hereditary and acquired defects, such as elevated lipoprotein (a) or defects of extrinsic (tissue factor) pathway inhibitor may be associated with enhanced risks of thrombosis.
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PMID:Hypercoagulability and thrombosis. 817 Feb 63

Factor IX consists of a gamma-carboxyglutamic acid-rich domain followed by two epidermal growth factor (EGF)-like domains and the C-terminal protease domain. To delineate the function of EGF1 domain in factor IX, we constructed three mutants: an EGF1 domain-deleted mutant (IX delta EGF1), a point mutant (IXQ50P) with a Gln-50-->Pro change, and a replacement mutant (IXPCEGF1) in which the EGF1 domain of factor IX was replaced by that of protein C. These mutants and wild-type (WT) factor IX (IXWT) were expressed in 293 kidney cells by using pRc/CMV vector. The purified proteins had the same gamma-carboxyglutamic acid content as the normal plasma factor IX (IXNP) and were activated normally by factor XIa-Ca2+. In contrast, IX delta EGF1 could not be activated by factor VIIa-tissue factor-Ca2+, and the activation of IXPCEGF1 in this system was markedly slow; however, IXQ50P was activated at a normal rate. In additional studies, both IXWT and IX delta EGF1 were rapidly converted to their respective IX alpha forms by factor Xa-phospholipid-Ca2+. Since this reaction has an absolute requirement for phospholipid, it indicates that the mutants under study are not impaired in their interactions with phospholipid. Relative coagulant activities of factor XIa-activated proteins were IXNP, 100%; IXWT, 75-85%; IX delta EGF1, < or = 1%; IXPCEGF1, < or = 2%; and IXQ50P, 6-10%. We conclude that the EGF1 domain of factor IX is required for its activation by factor VIIa-tissue factor and that the Gln-50 residue is not critical for this activation. Further, the EGF1 domain of factor IX is not essential for phospholipid binding and for its activation by factor XIa. In addition, the low coagulant activities of the activated mutants indicate that the EGF1 domain is also important in factor X activation by factor IXa-factor VIIIa-Ca(2+)-phospholipid complex.
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PMID:First epidermal growth factor-like domain of human blood coagulation factor IX is required for its activation by factor VIIa/tissue factor but not by factor XIa. 817 Sep 49

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

The plasminogen activator systems in the blood, the coagulation system, and the complement pathways are reviewed. The review describes the role of the vascular intima in activation of coagulation and fibrinolysis and the interrelations between the complement system and haemostatic mechanisms. Physiological activation of fibrinolysis may be triggered by and limited to fibrin because of a special affinity of plasminogen and plasminogen activators. The binding of plasminogen to fibrin is regulated by histidine-rich glycoprotein, and the primary physiological inhibitor of generated plasmin is alpha 2-antiplasmin and especially the plasminogen-binding form of this immediate plasmin inhibitor. Plasminogen activator inhibitors in the blood, that is, notably plasminogen activator inhibitor type 1 (PAI-1), bind circulating tissue-type plasminogen activator (t-PA). However, local fibrinolysis in vivo mediated by t-PA may be independent of complex formation between plasminogen activator inhibitors and t-PA in the fluid phase. Circulating plasminogen activator inhibitors might regulate fibrinolysis by increasing the clearance of t-PA from the blood. The urokinase-type and factor XII-dependent fibrinolytic proactivator system can be activated following t-PA-mediated generation of plasmin, and could thus serve as an amplification system of t-PA-induced fibrinolysis. It is claimed that the as yet uncharacterized proactivator is essential for optimal generation of plasminogen activator activity by the factor XII-dependent fibrinolytic system. The normal antithrombotic condition of the vascular intima probably results from lack of tissue factor activity and the presence of significant antithrombotic components comprising, among others, antithrombin III and the protein C-protein S system. A number of pathophysiologic stimuli, notably mediators of the acute phase response such as the cytokines interleukin-1 and tumour necrosis factor-alpha (cachectin), have the potential to induce the vascular endothelium to express procoagulant activity. Vascular endothelium promoting coagulant activity releases increased amounts of t-PA antigen and PAI-1 antigen into the circulation, and elevated levels in the blood of both may be regarded as a marker of a generalized procoagulant condition involving the vascular endothelium. In a prospective study in patients with unstable angina pectoris, patients in whom disease progresses and acute myocardial infarction develops, have increased amounts of t-PA antigen and PAI-1 antigen in the blood. This suggests that the procoagulant potential and atherosclerotic process of the vascular intima is more pronounced in the risk group.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Fibrinolysis in patients with acute ischaemic heart disease. With particular reference to systemic effects of tissue-type plasminogen activator treatment on fibrinolysis, coagulation and complement pathways. 822 63

Thrombomodulin (TM) is a cofactor for the thrombin-catalyzed activation of anticoagulant protein C. However, we have no evidence that thrombomodulin actually activates protein C during blood coagulation processing, nor do we know whether this activated protein C acts as an anticoagulant. We studied the inhibitory action of recombinant human soluble TM (rhs-TM) on thrombin generation in whole plasma. Human plasma was activated with small amounts of tissue factor using phospholipid vesicles in place of activated platelets. Thrombin generation was observed. The addition of only 2 nM of rhs-TM prevented rapid generation of thrombin and reduced the total amount of thrombin generated. In order to study the influence of the protein C activation pathway on this inhibitory action of rhs-TM, protein C-depleted plasma was used. rhs-TM had little inhibitory effect on protein C-depleted plasma. However, the addition of protein C caused a delay in thrombin generation and a reduction of the maximum thrombin concentration. We concluded that the anticoagulant activity of rhs-TM was amplified by the protein C activation pathway.
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PMID:Evidence that the protein C activation pathway amplifies the inhibition of thrombin generation by recombinant human thrombomodulin in plasma. 825 42

Blood coagulation-fibrinolysis factors are conventionally measured using fibrinogen or fibrin as a natural substrate. With recent advances in the technology to biochemically separate and purify, individual coagulation-fibrinolysis factors were isolated and purified, and their biochemical properties, function, and primary structure were clarified. Measurement techniques for blood coagulation-fibrinolysis factors were also developed. At present, enzyme activity for each factor is measured by the synthetic substrate method, on the other hand trace proteins and complex proteins are measured using monoclonal antibody. We present an outline of blood coagulation-fibrinolysis factors and synthetic substrates, and describe the characteristics of the synthetic substrate method using measurement of plasma tissue factor and protein C activity as examples.
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PMID:[Blood coagulation-fibrinolysis tests by a biochemical method]. 836 Oct 43

Endothelial cell regulation of protein C activation and fibrinolysis are important components of the hemostatic response to vascular injury or perturbation. Procoagulant albumin (P-A1), a constituent of normal human plasma has been purified and identified as an inducer of endothelial cell tissue factor activity. The purpose of the studies reported herein was to investigate the effects of P-A1 on human endothelial cell protein C activation and fibrinolysis. P-A1 suppressed protein C activation, enhanced release of plasminogen activator inhibitor-1, but had no effect on tissue-plasminogen activator release. Plasminogen activator inhibitor-1 released by P-A1 was functional as evidenced by the capacity to form a covalent complex with 125I-urokinase. Inactive albumin (isolated during the same purification procedure as P-A1, but without tissue factor-inducing activity) did not suppress protein C activation or increase plasminogen activator inhibitor-1 release. These results indicate that P-A1, a component of human plasma, can modulate multiple vascular hemostatic properties, and support the hypothesis that P-A1 is involved in normal or pathologic hemostasis.
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PMID:Regulation of endothelial cell protein C activation and fibrinolysis by procoagulant albumin. 836 71

Clinical observations have added to the understanding of basic mechanisms of blood coagulation and its alterations in certain hemorrhagic and thrombotic states. Much clinical evidence exists for concluding that the exposure of blood to tissue factor (thromboplastin) on tissue cells represents the key event initiating fibrin clot formation after tissue injury. This then results in the formation of activated factor VII (VIIa)-tissue factor complexes, which must activate both factor X and factor IX for normal hemostasis. I describe the possible clinical consequences of an aberrant function of the natural anticoagulants regulating blood coagulation--antithrombin, protein C, and tissue factor pathway inhibitor. Understanding the physiologic function of tissue factor pathway inhibitor can illuminate why hemophilic patients bleed, but many other questions remain. I briefly review the four causes for acquired disorders of the blood coagulation reactions--vitamin K deficiency, hepatocellular disease, antibodies to clotting factors, and disseminated intravascular coagulation--but limit my comments to the mechanisms that trigger the formation of antibodies to clotting factors and how these antibodies can deplete the blood of clotting factor activities. Finally, heparin is able to potentiate tissue factor pathway inhibitor function, which is a possible reason why the use of heparin but not warfarin can prevent the numerous thrombotic episodes of the Trousseau's syndrome.
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PMID:Blood coagulation and its alterations in hemorrhagic and thrombotic disorders. 843 80


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