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)

The inhibition mechanism of a polysaccharide anticoagulant, depolymerized holothurian glycosaminoglycan (DHG), was examined by analyzing its effects on the clotting time of human plasma depleted of antithrombin III (ATIII), of heparin cofactor II (HCII), or of both heparin cofactors. The effect exerted by this agent on the activation of prothrombin and factor X in purified human components were also examined and all effects were compared with those of other glycosaminoglycans (GAGs). The capacity of DHG to prolong activated partial thromboplastin time was not reduced in ATIII-depleted, HCII-depleted, HCII-depleted, or ATIII- and HCII-depleted plasma, whereas its capacity to prolong prothrombin time and thrombin clotting time was reduced in HCII-depleted plasma. DHG inhibited the amidolytic activity of thrombin in the presence of HCII with a second order rate constant of 1.2 x 10(8) (mol/L)-1 min-1. These results indicated that DHG has two different inhibitory activities, one being an HCII-dependent thrombin inhibition and the other an ATIII- and HCII-independent inhibition of the coagulation cascade. The heparin cofactors-independent inhibitory activity of DHG was investigated in the activation of prothrombin by factor Xa and in the activation of factor X by tissue factor-factor VIIa complex or by factor IXa. DHG significantly inhibited the activation of factor X by factor IXa in the presence of factor VIIIa, but not in the absence of factor VIIIa. The interaction between DHG and factors IXa, VIIIa, and X was investigated with a DHG-cellulofine column, on which DHG had strong affinity for factors IXa and VIIIa. These findings show that the heparin cofactors-independent inhibition exhibited by DHG was caused by inhibition of the interaction of factor X with the intrinsic factor Xase complex, probably by binding to the factor IXa-factor VIIIa complex.
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PMID:Depolymerized holothurian glycosaminoglycan with novel anticoagulant actions: antithrombin III- and heparin cofactor II-independent inhibition of factor X activation by factor IXa-factor VIIIa complex and heparin cofactor II-dependent inhibition of thrombin. 788 73

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 VIII is an important cofactor in the intrinsic activation of factor X. To function effectively as a cofactor, factor VIII must be activated. In plasma, factor VIII circulates in a complex with von Willebrand factor, and although thrombin can activate complexed factor VIII, the activation by activated factor X is inhibited by von Willebrand factor. In this study, the effect of von Willebrand factor on the generation of factor Xa by the factor IXa-VIII complex was investigated. Purified human factors VIII, IXa, and X were incubated on human umbilical vein endothelial cells or phospholipid vesicles in the presence of calcium ions, and the generation of factor Xa was followed. In the presence of von Willebrand factor, a prolonged lag-phase and a dose-dependent inhibition of factor X activation was observed. These effects were not observed when von Willebrand factor was preincubated with a monoclonal antibody directed against von Willebrand factor that blocks factor VIII binding. When factor VIII was activated with thrombin before the incubation, neither the monoclonal antibody nor von Willebrand factor had an effect on the rate of factor X activation. Preincubation of endothelial cells with the monoclonal antibody resulted in a somewhat higher rate of factor X activation. When endothelial cells from a patient with von Willebrand's disease type I were used, preincubation of the monoclonal antibody had no effect on the rate of factor X activation. We conclude that von Willebrand factor on the surface of endothelial cells can modulate the intrinsic factor X activation. This effect is greatly enhanced, however, by the addition of exogenous von Willebrand factor.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:von Willebrand factor as a regulator of intrinsic factor X activation. 814 7

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

The effects of a fucoidan (C-II), which was purified from the brown seaweed Ecklonia kurome, on the generation of thrombin and factor Xa have been investigated by measuring the amidolytic activities by using the respective specific chromogenic substrates in both plasma and purified systems. C-II inhibited significantly the generation of thrombin in both the intrinsic and the extrinsic pathways, although the intrinsic inhibitory effect by C-II was more remarkable than the extrinsic one. On the other hand, C-II was a good inhibitor of the factor Xa generation in the intrinsic pathway, while it was a poor one in the extrinsic pathway. In the purified systems C-II also inhibited the formation of prothrombin-activating complex (i.e., prothrombinase), but not its activity. The concentration of C-II required for 50% inhibition of thrombin generation was about one-tenth to one-seventh of that of the activity of the generated thrombin in plasma. These results indicate that C-II has an inhibitory effect on the generation of thrombin by blocking the formation of prothrombinase and by preventing the generation of intrinsic factor Xa in addition to its antithrombin activity, and also that the generation-inhibitory effect is more remarkable than C-II's enhancement effect on the antithrombin activity by heparin cofactor II in plasma.
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PMID:Inhibition of the generation of thrombin and factor Xa by a fucoidan from the brown seaweed Ecklonia kurome. 1055 83

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

In buffer systems, heparin and low molecular weight heparin (LMWH) directly inhibit the intrinsic factor X-activating complex (intrinsic tenase) but have no effect on the prothrombin-activating complex (prothrombinase). Although chemical modification of LMWH, to lower its affinity for antithrombin (LA-LMWH) has no effect on its ability to inhibit intrinsic tenase, N-desulfation of LMWH reduces its activity 12-fold. To further explore the role of sulfation, hypersulfated LA-LMWH was synthesized (sLA-LMWH). sLA-LMWH is not only a 32-fold more potent inhibitor of intrinsic tenase than LA-LMWH; it also acquires prothrombinase inhibitory activity. A direct correlation between the extent of sulfation of LA-LMWH and its inhibitory activity against intrinsic tenase and prothrombinase is observed. In plasma-based assays of tenase and prothrombinase, sLA-LMWH produces similar prolongation of clotting times in plasma depleted of antithrombin and/or heparin cofactor II as it does in control plasma. In contrast, heparin has no effect in antithrombin-depleted plasma. When the effect of sLA-LMWH on various components of tenase and prothrombinase was examined, its inhibitory activity was found to be cofactor-dependent (factors Va and VIIIa) and phospholipid-independent. These studies reveal that sLA-LMWH acts as a potent antithrombin-independent inhibitor of coagulation by attenuating intrinsic tenase and prothrombinase.
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PMID:Hypersulfated low molecular weight heparin with reduced affinity for antithrombin acts as an anticoagulant by inhibiting intrinsic tenase and prothrombinase. 1113 31

Thrombosis is the major mechanism underlying acute complications of atherosclerosis. Although thrombogenicity of atherosclerotic plaques has been ascribed to activation of the extrinsic pathway of blood coagulation, in the present study we investigated contribution of the intrinsic factor VIII (fVIII)-dependent pathway. We found that in vitro exposure of human macrophages and smooth muscle cells (SMCs) to atherogenic oxidized low-density lipoprotein (oxLDL) enhances their ability to support activity of 2 major complexes of the intrinsic pathway, Xase and prothrombinase, leading to a 20- and 10-fold increase in thrombin formation, respectively. In contrast, human aortic endothelial cells were less responsive to oxLDL. The increase in the intrinsic procoagulant activity was related to formation of additional fVIII binding sites due to enhanced translocation of phosphatidylserine to the outer surface of oxLDL-treated cells and a 5-fold higher affinity of interaction between components of the Xase complex, activated factors VIII and IX. Processes occurring at early apoptotic stages, including changes in the cell membrane induced by free radicals, may be related to activation of the intrinsic pathway as suggested by effects of inhibitors of early apoptosis on thrombin formation. Immunohistochemical studies on human atherectomy specimens revealed the presence of fVIII in the vicinity of macrophages and SMCs in atheromatous regions with massive deposits of oxLDL, supporting the possible involvement of the intrinsic pathway in thrombus formation in vivo. Our data predict that the intrinsic pathway significantly enhances thrombogenicity of atherosclerotic lesions after removal of the endothelial layer and exposure of SMCs and macrophages to blood flow.
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PMID:Intrinsic pathway of blood coagulation contributes to thrombogenicity of atherosclerotic plaque. 1203 78

Low-molecular-weight heparin (LMWH) inhibits the activity of the intrinsic factor X activation complex, a property that persists when LMWH is rendered low affinity (LA) for antithrombin, but is reduced when it is N-desulfated. When LA-LMWH is hypersulfated (sLA-LMWH), its potency against intrinsic tenase is increased and it acquires inhibitory activity against prothrombinase. sLA-LMWH functions by interfering with the association of enzyme and cofactor in both activation complexes. In a rabbit carotid artery thrombosis prevention model, sLA-LMWH is superior to LMWH. Because of its low affinity for antithrombin and multiple sites of action, sLA-LMWH may prove to be safer and more effective than other anticoagulants.
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PMID:Antithrombin-independent anticoagulation by hypersulfated low-molecular-weight heparin. 1245 89

Activated platelets promote intrinsic factor X-activating complex assembly by presenting high affinity, saturable binding sites for factor IXa mediated by two disulfide-constrained loop structures (loop 1, Cys88-Cys99; loop 2, Cys95-Cys109) within the second epidermal growth factor (EGF2) domain. To identify amino acids essential for factor X activation complex assembly, recombinant factor IXa point mutants in loop 1 (N89A, I90A, K91A, and R94A) and loop 2 (D104A, N105A, and V107A) were prepared. All seven mutants were similar to the native factor IXa by SDS-PAGE, active site titration, and content of gamma-carboxyglutamic acid residues. Kinetic constants obtained by either titrating factor X or factor VIIIa on SFLLRN-activated platelets or phospholipid vesicles revealed near normal values of Km(app) and Kd(app)FVIIIa for all mutants, indicating normal substrate and cofactor binding. In a factor Xa generation assay in the presence of activated platelets and cofactor factor VIIIa, compared with native factor IXa (Kd(app)FIXa approximately 1.1 nm, Vmax approximately 12 nm min(-1)), N89A displayed an increase of approximately 20-fold in Kd(app)FIXa and a decrease of approximately 20-fold in Vmax; I90A had an increase of approximately 5-fold in Kd(app)FIXa and approximately 10-fold decrease in Vmax; and V107A had an increase of approximately 3-fold in Kd(app)FIXa and approximately 4-fold decrease in Vmax. We conclude that residues Asn89, Ile90, and Val107 within loops 1 and 2 (Cys88-Cys109) of the EGF2 domain of factor IXa are essential for normal interactions with the platelet surface and for the assembly of the factor X-activating complex on activated platelets.
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PMID:Identification of residues Asn89, Ile90, and Val107 of the factor IXa second epidermal growth factor domain that are essential for the assembly of the factor X-activating complex on activated platelets. 1532 60


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