Kinetics of Factor Xa Inhibition by Recombinant Tick Anticoagulant Peptide:  Both Active Site and Exosite Interactions Are Required for a Slow- and Tight-Binding Inhibition Mechanism†

Biochemistry ◽  
2004 ◽  
Vol 43 (12) ◽  
pp. 3368-3375 ◽  
Author(s):  
Alireza R. Rezaie
Keyword(s):  
Active Site ◽  
Tight Binding ◽  
Factor Xa ◽  
Inhibition Mechanism ◽  
Factor Xa Inhibition ◽  
Binding Inhibition ◽  
Kinetics Of ◽  
Tick Anticoagulant Peptide

scholarly journals Cabbage seed protease inhibitor: a slow, tight-binding inhibitor of trypsin with activity toward thrombin, activated Stuart factor (factor Xa), activated Hageman factor (factor XIIa), and plasmin

Blood ◽  
1990 ◽  
Vol 75 (1) ◽  
pp. 108-115 ◽  
Author(s):  
TH Carter ◽  
BA Everson ◽  
OD Ratnoff
Keyword(s):  
Tight Binding ◽  
Factor Xa ◽  
Clot Formation ◽  
Dot Blot ◽  
Induce Platelet Aggregation ◽  
Hageman Factor ◽  
Blot Technique ◽  
Binding Inhibition ◽  
Factor Xiia ◽  
Cabbage Seed

Abstract An inhibitor of procoagulant and fibrinolytic enzymes was derived from cabbage seeds by a procedure using acetone precipitation, ion-exchange chromatography, and gel filtration. The cabbage seed inhibitor was a 10- Kd monomeric protein with intrachain disulfide bonds. This preparation prevented clot formation in whole blood and blocked the ability of thrombin to induce clot formation in plasma and to induce platelet aggregation. A number of proteases were inhibited, as demonstrated by using purified enzymes in amidolytic assays. Tight-binding inhibition was observed for activated Stuart factor (factor Xa) and plasmin. Inhibition of thrombin and activated Hageman factor (factor XIIa) was observed with a molar excess of inhibitor. No inhibition was detected for activated plasma thromboplastin antecedent (factor XIa), plasma kallikrein, or C1 esterase. Reaction progress curves for trypsin indicated slow, tight-binding inhibition, with an apparent inhibition constant in the nanomolar range or less. The electrophoretic mobility of trypsin was altered by the inhibitor in nondenaturing polyacrylamide gel electrophoresis (PAGE) but not in sodium dodecyl sulfate (SDS)- PAGE, indicating noncovalent bonding. Only partial reversal of trypsin inhibition could be demonstrated by washing the inhibitor from enzyme immobilized on solid beads. A dot-blot technique with cabbage seed inhibitor was capable of detecting 10 ng nitrocellulose-bound trypsin. The dot-blot technique also appeared capable of detecting plasmin. These findings demonstrated the potential utility of this inhibitor as a probe for detection of tightly bound proteases. In summary, cabbage seed extracts contain an inhibitor with activity toward a broad range of proteases important to hemostasis. To our knowledge, this agent represents the first inhibitor isolated from a plant source that inhibits thrombin.

Functional characterisation of Vizottin, the first factor Xa inhibitor purified from the leech Haementeria vizottoi

2012 ◽  
Vol 108 (09) ◽  
pp. 570-578 ◽  
Author(s):  
Daniella Oliveira ◽  
Miryam Alvarez-Flores ◽  
Adriana Lopes ◽  
Ana Chudzinski-Tavassi
Keyword(s):  
Active Site ◽  
Factor Viia ◽  
Tight Binding ◽  
Gel Filtration ◽  
Factor Xa ◽  
Dependent Manner ◽  
Binding Model ◽  
New Anticoagulants ◽  
Functional Characterisation ◽  
High Concentrations

SummaryThe strategic position of factor Xa (FXa) in blood coagulation makes it a compelling target for the development of new anticoagulants. Bloodsucking animals have in their salivary glands mixtures of anticoagulants, which could be used for designing novel antithrombotic compounds. Herein, we describe Vizottin, the first FXa inhibitor from the salivary complex of the leech Haementeria vizottoi. Vizottin was purified by gel filtration and reverse-phase chromatography, and shown to have anticoagulant effects in human plasma, prolonging the recalcification time in a dose-dependent manner (IC50 40 nM). Vizottin induced blood incoagulability in FX-deficient plasma, whereas in normal and reconstituted plasma, Vizottin doubled the prothrombin time at 160 nM. This peptide competitively inhibited human FXa (Ki 2 nM) like FXa inhibitors from other leeches, albeit via a distinct mechanism of action. At high concentrations, vizottin inhibited the amidolytic activity of factor VIIa/tissue factor (IC50 96.4 nM). Vizottin inhibited FXa in the prothrombinase complex and Gla-domainless FXa. Moreover, vizottin did not interfere with FX activation induced by RVV-X, a known enzyme that requires the Gla-domain of FX for activation. Competition experiments in the presence of FXa and GGACK-FXa (active site blocked) demonstrated that the inhibition of FXa by vizottin is through binding to the active site rather than an exosite. This novel inhibitor appears to exert its inhibitory effects through direct binding to the active site of FXa in a time-dependent manner, but not involving a tight-binding model. In this context, vizottin is a promising model for designing novel anticoagulants for the treatment of thrombotic diseases.

Substrate-dependent, non-hyperbolic kinetics of pig brain prolyl oligopeptidase and its tight binding inhibition by JTP-4819

2002 ◽  
Vol 64 (3) ◽  
pp. 463-471 ◽  
Author(s):  
Jarkko I. Venäläinen ◽  
Risto O. Juvonen ◽  
Markus M. Forsberg ◽  
Arturo Garcia-Horsman ◽  
Antti Poso ◽  
...  
Keyword(s):  
Tight Binding ◽  
Prolyl Oligopeptidase ◽  
Pig Brain ◽  
Binding Inhibition ◽  
Kinetics Of

scholarly journals The kinetics of slow-binding and slow, tight-binding inhibition: the effects of substrate depletion

1993 ◽  
Vol 294 (1) ◽  
pp. 195-200 ◽  
Author(s):  
S G Waley
Keyword(s):  
Substrate Concentration ◽  
Dissociation Constants ◽  
Tight Binding ◽  
Slow Step ◽  
First Order ◽  
Initial Substrate ◽  
Binding Inhibition ◽  
Kinetics Of ◽  
Nanomolar Range ◽  
Step Mechanism

Inhibitors with dissociation constants in the micromolar to nanomolar range are important, but hard to characterize kinetically, especially when the substrate concentration in the assay is less than Km. When inhibition increases during the course of the assay (slow-binding inhibition) the concentration of substrate may decrease appreciably. Methods that take substrate depletion into account are described for analysing experiments in which the initial substrate concentration is below Km. Fitting progress curves gives the rate constants for the second (slow) step in a two-step mechanism. An approximate value for the overall dissociation constant may be determined from measurements of rates when the reaction is treated as a first-order process. When the concentrations of inhibitor and enzyme are comparable numerical methods are required. Procedures, suitable for implementation on a microcomputer, for the solution of the differential equations and the fitting of progress curves are described.

Fibrin-targeted direct factor Xa inhibition: construction and characterization of a recombinant factor Xa inhibitor composed of an anti-fibrin single-chain antibody and tick anticoagulant peptide

2004 ◽  
Vol 92 (07) ◽  
pp. 47-53 ◽  
Author(s):  
Uta Weirich ◽  
Nicole Bassler ◽  
Meike Schwarz ◽  
Marschall Runge ◽  
Christoph Bode ◽  
...  
Keyword(s):  
Factor Xa ◽  
Coagulation Cascade ◽  
Bleeding Complications ◽  
Scale Production ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Factor Xa Inhibition ◽  
N Terminus ◽  
Fxa Inhibitor ◽  
Tick Anticoagulant Peptide

SummaryWe investigated whether the direct fXa inhibitor tick anticoagulant peptide (TAP) can be N-terminally coupled to a clot-targeting, single-chain antibody specific for fibrin (scFv59D8). Due to its unique position at the convergence point of the intrinsic and extrinsic pathways early in the coagulation cascade, factor Xa (fXa) represents an attractive therapeutic target. In contrast to indirect inhibitors, direct fXa inhibitors effectively inhibit clotbound and prothrombinase-associated fXa. Targeting of direct fXa inhibitors to clots promises to enhance local anticoagulative potency and to reduce systemic anticoagulation which potentially results in less bleeding complications. TAP is a highly potent fXa inhibitor. Since its N-terminus is essential for antifXa activity, it was a challenging question, whether TAP will be active as a N-terminally coupled fusion molecule. Two step affinity chromatography with Ni2+ and β15-22-peptide of human fibrin results in a pure 36 kDa protein, which was tested for its targeting function and anti-fXa activity. The recombinant fusion did not destroy the function of the fusion partners. Antibody binding function was on a par with the parent molecule. TAP activity was partially reduced, arguing that a free N-terminus is not required for anti-fXa activity, but is important for maximal potency. In human whole blood clots, scFv59D8-TAP revealed anticoagulative properties at concentrations (200 to 500 nM) where non-targeted TAP did not reveal anticoagulative activity at all. In summary, scFv59D8-TAP constitutes a promising new anticoagulant with fibrin-targeted factor Xa inhibition. The production in E. coli and the established purification methods are a solid basis for a modern, large scale production at low cost and reproducible activity.

scholarly journals Cabbage seed protease inhibitor: a slow, tight-binding inhibitor of trypsin with activity toward thrombin, activated Stuart factor (factor Xa), activated Hageman factor (factor XIIa), and plasmin

Blood ◽  
1990 ◽  
Vol 75 (1) ◽  
pp. 108-115
Author(s):  
TH Carter ◽  
BA Everson ◽  
OD Ratnoff
Keyword(s):  
Tight Binding ◽  
Factor Xa ◽  
Clot Formation ◽  
Dot Blot ◽  
Induce Platelet Aggregation ◽  
Hageman Factor ◽  
Blot Technique ◽  
Binding Inhibition ◽  
Factor Xiia ◽  
Cabbage Seed

An inhibitor of procoagulant and fibrinolytic enzymes was derived from cabbage seeds by a procedure using acetone precipitation, ion-exchange chromatography, and gel filtration. The cabbage seed inhibitor was a 10- Kd monomeric protein with intrachain disulfide bonds. This preparation prevented clot formation in whole blood and blocked the ability of thrombin to induce clot formation in plasma and to induce platelet aggregation. A number of proteases were inhibited, as demonstrated by using purified enzymes in amidolytic assays. Tight-binding inhibition was observed for activated Stuart factor (factor Xa) and plasmin. Inhibition of thrombin and activated Hageman factor (factor XIIa) was observed with a molar excess of inhibitor. No inhibition was detected for activated plasma thromboplastin antecedent (factor XIa), plasma kallikrein, or C1 esterase. Reaction progress curves for trypsin indicated slow, tight-binding inhibition, with an apparent inhibition constant in the nanomolar range or less. The electrophoretic mobility of trypsin was altered by the inhibitor in nondenaturing polyacrylamide gel electrophoresis (PAGE) but not in sodium dodecyl sulfate (SDS)- PAGE, indicating noncovalent bonding. Only partial reversal of trypsin inhibition could be demonstrated by washing the inhibitor from enzyme immobilized on solid beads. A dot-blot technique with cabbage seed inhibitor was capable of detecting 10 ng nitrocellulose-bound trypsin. The dot-blot technique also appeared capable of detecting plasmin. These findings demonstrated the potential utility of this inhibitor as a probe for detection of tightly bound proteases. In summary, cabbage seed extracts contain an inhibitor with activity toward a broad range of proteases important to hemostasis. To our knowledge, this agent represents the first inhibitor isolated from a plant source that inhibits thrombin.

Kinetics of the Reversible Tight-Binding Inhibition of Pig Liver Catechol-O-Methyltransferase by [2-(3,4-Dihydroxy-2-Nitrophenyl) Vinyl] Phenyl Ketone

1994 ◽  
Vol 8 (2) ◽  
pp. 123-131 ◽  
Author(s):  
Rosa A. Perez ◽  
Eldiberto Fernandez-Alvarez ◽  
Ofelia Nieto ◽  
F. Javier Piedrafita
Keyword(s):  
Tight Binding ◽  
Pig Liver ◽  
Binding Inhibition ◽  
Phenyl Ketone ◽  
Kinetics Of

Ancylostoma caninum Anticoagulant Peptide Blocks Metastasis In Vivo and Inhibits Factor Xa Binding to Melanoma Cells In Vitro

1998 ◽  
Vol 79 (05) ◽  
pp. 1041-1047 ◽  
Author(s):  
Kathleen M. Donnelly ◽  
Michael E. Bromberg ◽  
Aaron Milstone ◽  
Jennifer Madison McNiff ◽  
Gordon Terwilliger ◽  
...  
Keyword(s):  
Active Site ◽  
Thrombin Generation ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Melanoma Cells ◽  
Factor V ◽  
Ancylostoma Caninum ◽  
Metastatic Activity

SummaryWe evaluated the in vivo anti-metastatic activity of recombinant Ancylostoma caninum Anticoagulant Peptide (rAcAP), a potent (Ki = 265 pM) and specific active site inhibitor of human coagulation factor Xa originally isolated from bloodfeeding hookworms. Subcutaneous injection of SCID mice with rAcAP (0.01-0.2 mg/mouse) prior to tail vein injection of LOX human melanoma cells resulted in a dose dependent reduction in pulmonary metastases. In order to elucidate potential mechanisms of rAcAP’s anti-metastatic activity, experiments were carried out to identify specific interactions between factor Xa and LOX. Binding of biotinylated factor Xa to LOX monolayers was both specific and saturable (Kd = 15 nM). Competition experiments using antibodies to previously identified factor Xa binding proteins, including factor V/Va, effector cell protease receptor-1, and tissue factor pathway inhibitor failed to implicate any of these molecules as significant binding sites for Factor Xa. Functional prothrombinase activity was also supported by LOX, with a half maximal rate of thrombin generation detected at a factor Xa concentration of 2.4 nM. Additional competition experiments using an excess of either rAcAP or active site blocked factor Xa (EGR-Xa) revealed that most of the total factor Xa binding to LOX is mediated via interaction with the enzyme’s active site, predicting that the vast majority of cell-associated factor Xa does not participate directly in thrombin generation. In addition to establishing two distinct mechanisms of factor Xa binding to melanoma, these data raise the possibility that rAcAP’s antimetastatic effect in vivo might involve novel non-coagulant pathways, perhaps via inhibition of active-site mediated interactions between factor Xa and tumor cells.

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