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)

Upon activation, factor X participates in the prothrombin activation complex. Similar to 4-carboxyglutamic acid (Gla)-domainless protein C, the Gla-domainless factor X (GDFX) contains a high affinity Ca(2+)-binding site critical for the function of these molecules. In the case of protein C, we recently demonstrated that the high affinity Ca(2+)-binding site critical for activation is outside the first epidermal growth factor (EGF) homology domain. To examine if this is also true for factor X, we have expressed in human 293 cells a deletion mutant of factor X (E2FX) which lacks the entire Gla region as well as the NH2-terminal EGF homology region of factor X. Direct binding studies by equilibrium dialysis indicate that E2FX contains a single Ca(2+)-binding site with a dissociation constant (Kd) of 154 +/- 48 microM. The functional properties of E2FX are equivalent or improved over those of GDFX. For instance, the factor X coagulant protein of Russell's viper venom activates E2FX three times faster than recombinant GDFX. Kinetic analysis of prothrombin activation in the absence of membranes indicates that activated GDFX and E2FX bind to factor Va with equal affinity (Kd = 4.1 microM). The Ca2+ concentration required for half-maximal prothrombin activation rates in the above activation system shifted from 721 +/- 113 microM for activated GDFX to 193 +/- 64 microM for activated E2FX. GDFX and E2FX activation rates with the soluble tissue factor-factor VIIa complex were identical as was the Ca2+ dependence of the reaction. We conclude that E2FX retains a high affinity Ca(2+)-binding site and that the first EGF homology domain does not appear to have a positive functional role in the GDFX molecule. However, Ca2+ occupancy of the Ca(2+)-binding site in the first EGF domain of intact factor X may be essential for optimal prothrombin activation.
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PMID:Analysis of the functions of the first epidermal growth factor-like domain of factor X. 846 28

In a previous report, we described the molecular cloning, expression, and partial characterization of a second human tissue factor pathway inhibitor (TFPI), which we designated as TFPI-2 [Sprecher, C. A., et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 3353-3357]. Recombinant TFPI-2 inhibited the amidolytic activity of trypsin as well as that of factor VIIa in complex with tissue factor. TFPI-2 recently has been shown to be identical to placental protein 5 (PP5), a glycoprotein originally isolated from placenta that exhibits serine protease inhibitory activity. In the present study, we have examined TFPI-2/PP5 for its ability to inhibit a number of serine proteases involved in blood coagulation and fibrinolysis, inasmuch as TFPI-2/PP5 prolonged the coagulation time of human plasma induced by either tissue factor or contact activation in a dose-dependent manner. In addition to its ability to inhibit the amidolytic and proteolytic activities of the factor VIIa-tissue factor complex, TFPI-2/PP5 strongly inhibited the amidolytic activities of human factor XIa, human plasma kallikrein, and human plasmin with Ki values of 15, 25, and 3 nM, respectively. TFPI-2/PP5 was also a weak inhibitor of the activation of factor X by a complex of human factor IXa and poly(lysine) with an apparent Ki of 410 nM. Heparin markedly enhanced the ability of TFPI-2/PP5 to inhibit factor VIIa-tissue factor both in the solution phase and on cell surfaces. In addition, heparin augmented the inhibition of human factor Xa amidolytic activity at relatively high levels (10-100 nM) of TFPI-2/PP5. No significant inhibition of glandular kallikrein, urinary plasminogen activator, tissue plasminogen activator, human activated protein C, human factor Xa, human thrombin, or leukocyte elastase was observed when these proteases were incubated with TFPI-2 in the absence of heparin.
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PMID:Inhibitory properties of a novel human Kunitz-type protease inhibitor homologous to tissue factor pathway inhibitor. 855 84

This review presents the rationale for and main results of coagulation inhibitor substitution during experimental and human sepsis. Activation of the contact system induces activation of the classical complement pathway with generation of anaphylatoxins, of the kinins pathway and of fibrinolysis. Physiologic inhibition depends on the C1-inhibitor (C1-Inh.). Septic patients exhibit a relative deficiency of biologically active C1-Inh. Substitution with concentrations of C1-Inh has been safely performed and preliminary results are consistent with a possible beneficial effect on hypotension and vasopressor requirement in septic shock. The extrinsic pathway is the main initial coagulation process involved in sepsis-induced DIC. Endothelial and monocyte generation of tissue factor (TF) is activated by bacterial products and endotoxin. Activation of TF is counteracted by a specific tissue factor pathway inhibitor (TFPI). The potential for TFPI substitution to inhibit the activation of the coagulation cascade in sepsis requires further study. Thrombin generation is inhibited by antithrombin III (AT III) and the protein C-protein S system. During sepsis, AT III is consumed and degraded by elastase. Animal studies have shown that DIC and death were prevented by high doses of AT III concentrates. Although a significant reduction in the duration of biological symptoms of DIC has been reported in most human studies, the usefulness of AT III substitution in human sepsis is still debated. None of the studies was able to document a statistically significant reduction in mortality. Protein C is activated by thrombomodulin and, with its cofactor protein S, inhibits factors Va and VIIIa. The free level of protein S depends on the level of the C4b binding protein (C4bBP), an acute-phase complement regulatory protein. During sepsis, protein C activity is significantly reduced, either by acute consumption or by thrombomodulin down-regulation, and increased levels of plasma C4bBP inhibit protein S. Infusion of activated protein C and protein S substitution both protect animals from the lethal effects of bacteria. Combining these different coagulation inhibitors should be carefully studied before its use in septic patients is recommended.
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PMID:Coagulation inhibitor substitution during sepsis. 863 34

Growing evidence suggests that moderately elevated levels of homocysteine are associated not only with arterial thrombosis and atherosclerosis but also with venous thrombosis as well. We have reviewed recent studies that indicate that homocysteine inhibits several different anticoagulant mechanisms that are mediated by the vascular endothelium. The protein C enzyme system appears to be one of the most important anticoagulant pathways in the blood. Homocysteine inhibits the expression and activity of endothelial cell surface thrombomodulin, the thrombin cofactor responsible for protein C activation. Homocysteine inhibits the antithrombin III binding activity of endothelial heparan sulfate proteoglycan, thereby suppressing the anticoagulant effect of antithrombin III. Homocysteine also inhibits the ecto-ADPase activity of human umbilical vein endothelial cells (HUVECS). Because ADP is a potent platelet aggregatory agent, this action of homocysteine is prothrombotic. Homocysteine also interferes with the fibrinolytic properties of the endothelial surface because it inhibits the binding of tissue plasminogen activator. Homocysteine stimulates HUVEC tissue factor activity. We have found that lipoprotein(a) [Lp(a)] also stimulates HUVEC tissue factor activity. The combination of Lp(a) plus homocysteine induced more tissue factor activity than either agent alone. These disruptions in several different vessel wall-related anticoagulant functions provide plausable mechanisms for the occurrence of thrombosis in hyperhomocysteinemia.
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PMID:Homocysteine and hemostasis: pathogenic mechanisms predisposing to thrombosis. 864 72

Factor VIIa is a vitamin K-dependent enzyme whose gamma-carboxyglutamic acid (Gla)-containing domain is important for calcium ion-dependent binding to the cofactor tissue factor and membrane surfaces. This domain contains 10 Gla residues, the individual roles and importance of which are not known. Comparisons with the homologous protein C, factor IX and prothrombin may provide functional information on the first nine Gla residues, whereas no data can be extrapolated to Gla-35 in factor VIIa. Therefore, the effects of posttranslational gamma-carboxylation and site-directed mutagenesis of Glu-35 were investigated. Mutations to Asp, Gln or Val all lead to a lower affinity for tissue factor by decreasing the rate of association, in the case of the Val mutant by a factor of 200, as measured by surface plasmon resonance. In contrast, Glu or Gla side chains at position 35 appear to fulfil the functional roles equally well.
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PMID:Site-directed mutagenesis but not gamma-carboxylation of Glu-35 in factor VIIa affects the association with tissue factor. 864 60

Markers of endothelial cell activation were measured in 28 patients presenting with various forms of limited or focal type cutaneous vasculitis. Plasma levels of tissue plasminogen activator antigen (t-PA:Ag), plasminogen activator inhibitor type 1 antigen (PAI-1:Ag) and PAI-1 activity, fibrin plate, von Willebrand factor antigen (vWF:Ag), tissue factor (TF) and soluble thrombomodulin (sTM) were measured. In comparison with the control group (n = 20) there was a significant increase in t-PA:Ag, vWF:Ag and TF (P < 0.05, Mann-Whitney U-test) in the cutaneous vasculitis group. This study confirms that measurable degrees of endothelial activation occur in cutaneous vasculitis. Cutaneous vasculitis includes a diverse group of clinical conditions, which are associated with inflammatory changes in cutaneous blood vessels with local fibrin deposition. The aetiology and pathogenesis of the majority of these entities remain unknown. Causative mediators are thought to include immune complexes, anti-endothelial cell antibodies, cytotoxic lymphocytes and viruses. Histologically, immune complexes and complement are frequently detected on the vessel wall, and serologically anti-endothelial antibodies are often detected in patients with vasculitis and in systemic lupus erythematosus (SLE) which correlate with the severity of cutaneous vasculitis, arthritis and nephritis. Lymphocyte-mediated toxicity to endothelial cells has been reported in a small number of patients with giant cell arteritis and Takayasu's arteritis. The vascular endothelium plays a central part in the control of haemostasis. Under physiological conditions endothelial cells present an anticoagulant surface to blood constituents, partially due to surface expression of heparan sulphate and thrombomodulin (TM). Heparan sulphate binds antithrombin III (ATIII), thereby accelerating inactivation of intrinsic coagulation enzymes. Thrombomodulin is an endothelial cell surface glycoprotein which promotes anticoagulation by forming a complex with thrombin which then activates protein C. Activated protein C together with a cofactor, protein S, inactivates FVa and FVIIIa. von Willebrand factor (vWF) is synthesized by endothelial cells, stored in Weibel-Palade bodies and released into the circulation upon endothelial stimulation. vWF mediates the binding of platelets to the subendothelium and is the carrier molecule for FVIIIC. The endothelium controls fibrinolysis by producing t-PA and its inhibitor PAI-1. Inflammatory cytokines such as interleukin-1 (IL-1) and tumour necrosis factor (TNF) activate endothelial cells, causing a shift from an antithrombotic to prothrombotic state, including expression of tissue factor, increased synthesis of PAI-1 and decreased expression of TM. Fibrin deposition and intravascular thrombosis are seen in cutaneous vasculitis syndromes, suggesting local endothelial cell activation. The aim of this pilot study was to assess whether perturbation of the endothelium in cutaneous vasculitis could be detected in the patients' plasma samples. If so, further studies to assess any correlation in levels of these markers with disease activity might prove useful in the future.
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PMID:Endothelial cell activation in cutaneous vasculitis. 868 65

Previous studies have shown that antithrombin III-heparin effectively inhibited the factor VIIa-tissue factor complex. Herein, we show that the neutralization of factor VIIa in complex with the cell surface tissue factor by antithrombin III-heparin was markedly enhanced by plasma levels of factor X. Active site-mutated factor X (S376A factor X) and factor Xa previously inactivated with dansyl-Glu-Gly-Arg-chloromethyl ketone were as effective as plasma-derived factor X in this reaction, indicating that the active site serine residue of factor Xa was not involved in this mechanism. Furthermore, Gla-domainless factor X had no effect in this system, emphasizing the importance of the factor X Gladomain in this reaction. Antibody experiments revealed that this effect was not due to trace levels of a tissue factor pathway inhibitor contaminating either the factor X or antithrombin III preparations. The presence of heparin in this system was essential, as deletion of heparin resulted in a factor VIIa-tissue factor neutralization rate essentially identical to that observed for antithrombin III alone. Plasma levels of factor IX also accelerated the inhibition of factor VIIa-tissue factor by antithrombin III-heparin, although its effect was not as pronounced as that of factor X. Other vitamin K-dependent plasma proteins including protein S, protein C and prothrombin failed to augment the inhibition of factor VIIa-tissue factor by antithrombin III-heparin. Factor X did not enhance the neutralization rate of factor VIIa-tissue factor by antithrombin III-heparin when a carboxyl-terminal truncated tissue factor construct (TF1-219) was used, even in the presence of mixed phospholipids. Our collective finding suggest that antithrombin III and factor X bind to heparin at distinct sites on the heparin molecule resulting in a transient ternary complex of antithrombin III-heparin-factor X that represents the anticoagulant species. Factor X conceivably guides complex to a phosphatidylserine-rich site on the cell surface in close proximity to the factor VIIa-tissue factor complex and facilitates rapid neutralization of factor VIIa. Our findings also suggest that the effect of heparin on the regulation of the extrinsic pathway of blood coagulation may be more profound than previously recognized.
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PMID:The inhibition of human factor VIIa-tissue factor by antithrombin III-heparin is enhanced by factor X on a human bladder carcinoma cell line. 871 77

The procoagulant activity of mononuclear cells (MNCs) may play an important role in the disseminated intravascular coagulation seen in septic shock. This study compares the capacity of Escherichia coli (E. coli) and recombinant human TNF-alpha (rhTNF-alpha) to induce procoagulant activity by baboon MNCs. In vivo studies showed that MNC procoagulant activity was significantly increased at T + 120 min after LD100 E. coli infusion into baboons. Most of this procoagulant activity was attributable to tissue factor. In contrast, a bolus infusion of rhTNF-alpha (150 micrograms/kg) and a monoclonal antibody to activated protein C (2 mg/kg) did not induce any increase of MNC procoagulant activity at T + 120 min even though the plasma TNF-alpha level was 10 times higher than that seen following infusion of E. coli. In vitro studies showed that E. coli at concentrations comparable to that observed in the vivo study and LPS at a concentration of 2.5 ng/mL induced more intense tissue factor expression by both human and baboon monocytes than rhTNF-alpha in the concentrations ranging from 10 to 1,000 ng/mL. These results suggest that TNF-alpha alone is not sufficient to induced noticeable MNC procoagulant activity, at least, in the early stage of this septic shock model.
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PMID:Comparison of the capacity of rhTNF-alpha and Escherichia coli to induce procoagulant activity by baboon mononuclear cells in vivo and in vitro. 872 87

Vascular endothelium regulates multiple aspects of platelet function through secretion of a variety of substances, including von Willebrand factor, nitric oxide, and prostacyclin (PGI2). The objective of this study was to determine whether procoagulant albumin (P-A1), a modified form of albumin present in normal human plasma could modulate endothelial cell secretion of these substances. P-A1 did not affect constitutive secretion of von Willebrand factor or nitric oxide, but did increase PGI2 secretion in a time- and concentration-dependent manner. Pre-treatment of endothelial cells with aspirin, or use of suramin, a broad-specificity inhibitor, prevented the response to P-A1. Prostaglandin H synthase-2 contributed to the P-A1-induced PGI2 secretion. These results indicate that in addition to inducing tissue factor activity and reducing protein C activation and fibrinolysis, P-A1 also modulates vascular endothelial cell PGI2 secretion, and potentially, platelet function.
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PMID:Procoagulant albumin increases vascular endothelial cell prostacyclin secretion. 877 39

Barley serpin BSZx is a potent inhibitor of trypsin and chymotrypsin at overlapping reactive sites (Dahl, S.W., Rasmussen, S.K. and Hejgaard, J. (1996) J. Biol. Chem., in press). We have now investigated the interactions of BSZx with a range of serine proteinases from human plasma, pancreas and leukocytes, a fungal trypsin and three subtilisins. Thrombin, plasma kallikrein, factor VIIa/tissue factor and factor Xa were inhibited by BSZx at heparin independent association rates (k(ass)) of 4.5 X 10(3)-1.3 x 10(5) M(-1) s(-1) at 22 degrees C. Only factor Xa turned a significant fraction of BSZx over as substrate. Complexes of these proteinase with BSZx resisted boiling in SDS, and amino acid sequencing showed that cleavage in the reactive center loop only occurred after P1 Arg. Activated protein C and leukocyte elastase were slowly inhibited by BSZx (k(ass)=1-2 x 10(2) M(-1) s(-1)) whereas factor XIIa, urokinase and tissue type plasminogen activator, plasmin and pancreas kallikrein and elastase were not or only weakly affected. The inhibition pattern with mammalian proteinases reveal a specificity of BSZx similar to that of antithrombin III. Trypsin from Fusarium was not inhibited while interaction with subtilisin Carlsberg and Novo was rapid but most BSZx was cleaved as a substrate. Identification of a monoclonal antibody specific for native BSZx indicate that complex formation and loop cleavage result in similar conformational changes.
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PMID:Inhibition of coagulation factors by recombinant barley serpin BSZx. 884 56


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