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 effect of activated protein C on human coagulation factor VIII was evaluated by studying its effect on the intrinsic factor X activation using a system of purified coagulation factors (factor IXa, factor X, factor VIII, activated protein C). Activated protein C had no effect on the activation of factor X by factor IXa in the absence of factor VIII. In the presence of thrombin activated factor VIII the rate of factor X activation was decreased by activated protein C in a dose dependent way. The presence of factor IXa during the preincubation of factor VIII with activated protein C was found to protect the factor VIII against inactivation. The results suggest that activated protein C and factor IXa compete for the same part of the factor VIII molecule.
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PMID:Factor IXa protects activated factor VIII against inactivation by activated protein C. 643 1

Protein S is a vitamin K-dependent nonenzymatic anticoagulant protein that acts as a cofactor to activated protein C. Recently it was shown that protein S inhibits the prothrombinase reaction independent of activated protein C. In this study, we show that protein S can also inhibit the intrinsic factor X activation via a specific interaction with factor VIII. In the presence of endothelial cells, the intrinsic activation of factor X was inhibited by protein S with an IC50 value of 0.28 +/- 0.04 mumol/L corresponding to the plasma concentration of protein S. This inhibitory effect was even more pronounced when the intrinsic factor X activation was studied in the presence of activated platelets (IC50 = 0.15 +/- 0.02 mumol/L). When a nonlimiting concentration of phospholipid vesicles was used, the plasma concentration of protein S (300 nmol/L) inhibited the intrinsic factor X activation by 40%. Thrombin-cleaved protein S inhibited the endothelial cell-mediated factor X activation with an IC50 similar to that of native protein S (0.26 +/- 0.02 mumol/L). Protein S in complex with C4b-binding protein inhibited the endothelial cell-mediated factor X activation more potently than protein S alone (IC50 = 0.19 +/- 0.03 mumol/L). Using thrombin activated factor VIII, IC50 values of 0.53 +/- 0.09 mumol/L and 0.46 +/- 0.10 mumol/L were found for native protein S and thrombin-cleaved protein S, respectively. The possible interactions of protein S with factor IXa, phospholipids, and factor VIII were investigated. The enzymatic activity of factor IXa was not affected by protein S, and interaction of protein S with the phospholipid surface could not fully explain the inhibitory effect of protein S on the factor X activation. Using a solid-phase binding assay, we showed a specific, saturable, and reversible binding of protein S to factor VIII with a high affinity. The concentration of protein S where half-maximal binding was reached (B1/2max) was 0.41 +/- 0.06 mumol/L. A similar affinity was found for the interaction of thrombin-cleaved protein S with factor VIII (B1/2max = 0.40 +/- 0.04 mumol/L). The affinity of the complex protein S with C4B-binding protein appeared to be five times higher (B1/2max = 0.07 +/- 0.03 mumol/L). Because the affinities of the interaction of the different forms of protein S with factor VIII correspond to the IC50 values observed for the intrinsic factor X activating complex, the interaction of protein S with factor VIII may explain the inhibitory effect of protein S on the intrinsic factor X activating complex.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Inhibition of the intrinsic factor X activating complex by protein S: evidence for a specific binding of protein S to factor VIII. 762 Jan 60

The complement protein C4b-binding protein plays an important role in the regulation of the protein C anticoagulant pathway. C4b-binding protein can bind to protein S, thereby inhibiting the cofactor activity of protein S for activated protein C. In this report, we describe a new role for C4b-binding protein in coagulation. We observed inhibition of the intrinsic factor X activating reaction by the complex of C4b-binding protein and protein S. At the plasma concentration of protein S, the factor X activation was inhibited for 50% and addition of C4b-binding protein led to a potentiation of the inhibition to almost 90%. Because C4b-binding protein alone had no effect on the activation of factor X, we hypothesized that binding of C4b-binding protein to protein S was a prerequisite for optimal inhibition of factor X activation. C4b-binding protein lacking the beta-chain, which is unable to bind to protein S, did not potentiate the inhibitory effect of protein S. In an earlier study, we observed that C4b-binding protein increased the binding affinity of protein S for factor VIII. Therefore, a possible interaction of C4b-binding protein with factor VIII was investigated. C4b-binding protein bound to factor VIII and to thrombin activated factor VIII in a saturable and specific way. Also, factor VIII in complex with von Willebrand factor was able to bind C4b-binding protein. The beta-chain of C4b-binding protein was not required for the interaction with factor VIII because C4b-binding protein lacking the beta-chain also bound to factor VIII. Monoclonal antibodies directed against the alpha-chain of C4b-binding protein inhibited the binding to factor VIII, whereas monoclonal antibodies directed against the beta-chain had no effect on the binding to factor VIII. This finding indicates that the binding site for factor VIII on C4b-binding protein is localized on the alpha-chains of C4b-binding protein. The potentiation by C4b-binding protein of the inhibition of the factor X activation by protein S was blocked by a monoclonal antibody directed against the alpha-chain of C4b-binding protein. This finding indicates that the potentiation of the inhibitory effect of protein S was mediated via an interaction of C4b-binding protein with factor VIII. C4b-binding protein did not bind to factor V and was not able to potentiate the inhibitory effect of protein S on prothrombinase activity.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Synergistic inhibition of the intrinsic factor X activation by protein S and C4b-binding protein. 767 Jan 8

Factor VIIIa is a non-covalent heterotrimer of A1, A2, and A3-C1-C2 subunits. Previously, we speculated that the central portion of the A2 subunit, in and around the activated protein C-sensitive bond at Arg562-Gly (Fay, P. J., Smudzin, T.M., and Walker, F.J. (1991) J. Biol. Chem. 266, 20139-20145), is important for macromolecular interactions within the factor Xase enzyme complex. A peptide corresponding to factor VIII residues 558-565, SVDQRGNQ and designated FVIII558-565, was chemically synthesized and inhibited factor Xa generation in a purified system with an apparent KI of 105 microM. Tryptic cleavage of FVIII558-565 eliminated its inhibitory activity, whereas a scrambled sequence version of the peptide possessed < 30% the inhibitory activity of the native version. Overlapping peptides FVIII556-564 and FVIII561-569 were also inhibitory and confirmed the importance of residues in and around the scissile bond for functional factor Xase. Kinetic analysis revealed that peptide-mediated inhibition was non-competitive with respect to factor X. However, increasing factor IXa concentration overcame the observed inhibition. Furthermore, the peptide inhibited the factor IXa-dependent enhancement of factor VIIIa reconstituted from isolated A1/A3-C1-C2 dimer plus A2 subunit. Isolated factor VIII heavy chain (contiguous A1-A2 domains) was cleaved at Arg336 by an equimolar concentration of factor IXa in a reaction that was phospholipid-independent. No proteolysis of the isolated A1 subunit was observed in a similar reaction. These results indicate that the A2 subunit sequence delineated by residues 558-565 contributes to the interaction of cofactor with protease and that this interaction is essential for intrinsic factor Xase activity. Furthermore, that this peptide blocks both factor Xase activity and the capacity of factor IXa to stabilize the labile factor VIIIa heterotrimer suggest that this latter property is of physiologic significance.
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PMID:Factor VIIIa A2 subunit residues 558-565 represent a factor IXa interactive site. 805 Nov 50

Factor VIIIa, the cofactor for the factor IXa-dependent conversion of factor X to factor Xa, is proteolytically inactivated by activated protein C (APC). APC cleaves at two sites in factor VIIIa, Arg336, near the C terminus of the A1 subunit; and Arg562, bisecting the A2 subunit (Fay, P., Smudzin, T., and Walker, F. (1991) J. Biol. Chem. 266, 20139-20145). Factor VIIIa increased the fluorescence anisotropy of fluorescein-Phe-Phe-Arg factor IXa (Fl-FFR-FIXa; Kd = 42.4 nM), whereas cleavage of factor VIIIa by APC eliminated this property. Isolation of the APC-cleaved A1/A3-C1-C2 dimer (A1336/A3-C1-C2), and the fragments derived from cleaved A2 subunit (A2N/A2C), permitted dissection of the roles of individual cleavages in cofactor inactivation. Intact A1/A3-C1-C2 dimer increased Fl-FFR-FIXa anisotropy and bound factor X in a solid phase assay, while these activities were absent in the A1336/A3-C1-C2. However, the residues removed by this cleavage, Met337 Arg372, did not directly participate in these functions since neither a synthetic peptide to this sequence nor an anti-peptide polyclonal antibody blocked these activities using intact dimer. CD spectral analysis of the intact and truncated dimers indicated reduced alpha and/or beta content in the latter. The A1/A3-C1-C2 dimer plus A2 subunit reconstitutes cofactor activity and produced a factor VIIIa-like effect on the anisotropy of Fl-FFR-FIXa. However, when A2 was replaced by the A2N/A2C fragments, the resulting fluorescence signal was equivalent to that observed with the dimer alone. These results indicate that APC inactivates the cofactor at two levels within the intrinsic factor Xase complex. Cleavage of either subunit modulates the factor IXa active site, suggesting an essential synergy of interactive sites in factor VIIIa. Furthermore, cleavage of the A1 site alters the conformation of a factor X binding site within that subunit, thereby reducing the affinity of cofactor for substrate.
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PMID:Activated protein C-catalyzed proteolysis of factor VIIIa alters its interactions within factor Xase. 862 29

Intrinsic factor X activation is accelerated >10(7)-fold by assembly of the entire complex on the activated platelet surface. We have now observed that increasing the concentration of zymogen factor IX to physiologic levels ( approximately 100 nM) potentiates factor IXa-catalyzed activation of factor X on both activated platelets and on negatively charged phospholipid vesicles. In the presence and absence of factor VIIIa, factor IX (100 nM) lowered the K(d,appFIXa) approximately 4-fold on platelets and 2-10-fold on lipid vesicles. Treatment of two factor IX preparations with active-site inhibitors did not affect these observations. Autoradiographs of PAGE-separated reactions containing either (125)I-labeled factor IX or (125)I-labeled factor X showed that the increased factor X activation was not due to factor Xa-mediated feedback activation of factor IX and that there was increased cleavage of factor X heavy chain in the presence of factor IX in comparison with control reactions but only in the presence of both the enzyme and the surface. Since plasma concentrations of prothrombin, factor VII, protein C, or protein S did not by themselves potentiate factor Xa generation and did not interfere with the potentiation of the reaction of factor IX, the effect is specific for factor IX and is not attributable to the Gla domain of all vitamin K-dependent proteins. These observations indicate that under physiologic conditions, plasma levels of the zymogen factor IX specifically increase the affinity of factor IXa for the intrinsic factor X activation complex.
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PMID:Zymogen factor IX potentiates factor IXa-catalyzed factor X activation. 1093 3

Factor VIII, a metal ion-dependent heterodimer, circulates in complex with von Willebrand factor. At sites of vessel wall damage, this procofactor is activated to factor VIIIa by limited proteolysis and assembles onto an anionic phospholipid surface in complex with factor IXa to form the intrinsic factor Xase; an enzyme complex that efficiently converts factor X to factor Xa during the propagation phase of coagulation. Factor Xase activity is down-regulated by mechanisms that include self-dampening by dissociation of a critical factor VIIIa subunit and proteolytic inactivation by the activated protein C pathway. Recent studies identify putative metal ion coordination sites as well as ligands involved in the catabolism of the activated and procofactor forms of the protein. Our knowledge of these multiple intra- and inter-molecular interactions has been facilitated by the application of naturally occurring and site-directed mutations to study factor VIII structure and function. In this review, we document important and novel contributions following this line of investigation.
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PMID:Mutating factor VIII: lessons from structure to function. 1557 14

Factor VIII, a non-covalent heterodimer comprised of a heavy chain (A1-A2-B domains) and light chain (A3-C1-C2 domains), circulates as an inactive procofactor in complex with von Willebrand factor. Metal ions are critical to the integrity of factor VIII, with Cu and Ca ions stabilizing the heterodimer and generating the active conformation, respectively. Activation of factor VIII catalyzed by thrombin appears dependent upon interactions with both anion-binding exosites I and II, and converts the heterodimer to the active cofactor, factor VIIIa. This protein, comprised of A1, A2, and A3-C1-C2 subunits, is labile due to weak affinity of the A2 subunit. Association of factor VIIIa with factor IXa to form the intrinsic factor Xase complex is membrane-dependent and involves multiple inter-protein contacts that remain poorly characterized. This complex catalyzes the conversion of factor X to factor Xa, a reaction that is essential for the propagation phase of coagulation. The role of factor VIIIa in this complex is to increase the catalytic efficiency for factor Xa generation by several orders of magnitude. Mechanisms for the down-regulation of factor Xase focus upon inactivation of the cofactor and include dissociation of the A2 subunit as well as activated protein C-catalyzed proteolysis.
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PMID:Factor VIII structure and function. 1651 27

Protein S functions as an activated protein C (APC)-independent anticoagulant in the inhibition of intrinsic factor X activation, although the precise mechanisms remain to be fully investigated. In the present study, protein S diminished factor VIIIa/factor IXa-dependent factor X activation, independent of APC, in a functional Xa generation assay. The presence of protein S resulted in an c. 17-fold increase in K(m) for factor IXa with factor VIIIa in the factor Xase complex, but an c. twofold decrease in K(m) for factor X. Surface plasmon resonance-based assays showed that factor VIII, particularly the A2 and A3 domains, bound to immobilized protein S (K(d); c. 10 nmol/l). Competition binding assays using Glu-Gly-Arg-active-site modified factor IXa showed that factor IXa inhibited the reaction between protein S and both the A2 and A3 domains. Furthermore, Sodium dodecyl sulphate polyacrylamide gel electrophoresis revealed that the cleavage rate of factor VIIIa at Arg(336) by factor IXa was c. 1.8-fold lower in the presence of protein S than in its absence. These data indicate that protein S not only down-regulates factor VIIIa activity as a cofactor of APC, but also directly impairs the assembly of the factor Xase complex, independent of APC, in a competitive interaction between factor IXa and factor VIIIa.
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PMID:Protein S down-regulates factor Xase activity independent of activated protein C: specific binding of factor VIII(a) to protein S inhibits interactions with factor IXa. 1875 61

Dabigatran etexilate is a new oral anticoagulant that functions as a direct thrombin inhibitor. An inhibitor of thrombin has the potential to interfere with essentially all clot-based coagulation assays and select chromogenic assays, whereas the drug would not be expected to interfere in antigen-based assays. The purpose of this study was to evaluate the effect of dabigatran on various specialized coagulation assays using normal plasma specimens with varying concentrations of dabigatran (the active form of dabigatran etexilate). We have demonstrated that samples containing therapeutic levels of dabigatran may lead to underestimation of intrinsic factor activities with abnormal activated partial thromboplastin time (aPTT) mixing study results and a false-positive factor VIII Bethesda titer; overestimation of protein C and protein S activity and activated protein C resistance ratio when determined using aPTT-based methods; and overestimation of results based on chromogenic anti-IIa assays but no effect on antigen assays and select chromogenic assays.
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PMID:The effect of dabigatran on select specialty coagulation assays. 2327 Sep 5


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