Functional and Structural Characterization of Factor Xa Dimer in Solution.

Abstract Previous studies of blood coagulation factor Xa (bovine) showed that binding of water soluble phosphatidylserine (C6PS) to factor Xa (FXa) leads to Ca2+ dependent inactive (∼1000-fold inactivation) dimer formation. We show now that human factor Xa activity is also regulated by C6PS-induced dimerization in the presence of 5 mM Ca2+ We also report that the FXa dimer is inactive: despite the fact dimerization does not block the active site; in part because it does block a substrate exosite; the dimer interface involves lysine residues that boarder the active site and exosites, and the structure of FXa in the dimer is altered relative to the monomer. We have measured initial rates of prothrombin activation (using synthetic thrombin substrate S-2238) at varying FXa and substrate concentrations to show that the kcat/Km decreased (kcat decreased significantly and Km increased slightly) with an increase in FXa dimer formation. The observed significant decrease in kcat indicates that dimerization affects the alignment of substrate with the active site, perhaps through altering substrate binding or through altering the structure of the active site. Amidolytic activity of monomeric FXa (using synthetic substrate S-2765) decreased in response to C6PS binding, while that of the dimer increased slightly. This indicates that dimerization did not block the active site but may alter its conformation. CD and mass spectrometry showed that both Ca2+ and C6PS binding alter FXa structure and that dimerization further alters structure. Acetylation of exposed lysine residues and analysis of MS patterns obtained under conditions that favor either monomer or dimer FXa revealed that the dimerization buries lysines residues 222 and 224 (chymotrypsin numbering) that boarder the active site and are in putative exocytes. We used MS data, fluorescence energy transfer data for active site labeled FXa, to model the FXa dimer structure based on a FXa monomer model (from Gla-domainless Xa X-ray structure and Gla-EGFn with Ca2+) but the requirement that known membrane binding sites or paired FXa molecules would be located in plane was failed. Our lack of success supports our other measurements suggesting that the structure of FXa in a dimer is very different from that in a monomer. Supported by grant from the NHBL (HL 072827 to BRL).

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

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615117 ◽

1998 ◽

Vol 79 (05) ◽

pp. 1041-1047 ◽

Cited By ~ 20

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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Characterization of an Abnormal Antithrombin (Milano 2) with Defective Thrombin Binding

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661681 ◽

1986 ◽

Vol 56 (03) ◽

pp. 349-352 ◽

Cited By ~ 2

Author(s):

A Tripodi ◽

A Krachmalnicoff ◽

P M Mannucci

Keyword(s):

Venous Thromboembolism ◽

Active Site ◽

Inhibitory Activity ◽

Antithrombin Iii ◽

Factor Xa ◽

Molecular Defect ◽

Affinity Adsorption ◽

Italian Family ◽

Crossed Immunoelectrophoresis

SummaryFour members of an Italian family (two with histories of venous thromboembolism) had a qualitative defect of antithrombin III reflected by normal antigen concentrations and halfnormal antithrombin activity with or without heparin. Anti-factor Xa activities were consistently borderline low (about 70% of normal). For the propositus’ plasma and serum the patterns of antithrombin III in crossed-immunoelectrophoresis with or without heparin were indistinguishable from those of normal plasma or serum. A normal affinity of antithrombin III for heparin was documented by heparin-sepharose chromatography. Affinity adsorption of the propositus’ plasma to human α-thrombin immobilized on sepharose beads revealed defective binding of the anti thrombin III to thrombin-sepharose. Hence the molecular defect of this variant appears to be at the active site responsible for binding and neutralizing thrombin, thus accounting for the low thrombin inhibitory activity.

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Expression, purification, and characterization of inactive human coagulation factor Xa (Asn322Ala419)

Protein Expression and Purification ◽

10.1016/1046-5928(92)90070-d ◽

1992 ◽

Vol 3 (6) ◽

pp. 518-524 ◽

Cited By ~ 8

Author(s):

Uma Sinha ◽

Tom E. Hancock ◽

Pei-Hua Lin ◽

Stan Hollenbach ◽

David L. Wolfs

Keyword(s):

Factor Xa ◽

Coagulation Factor ◽

Purification And Characterization

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Bacterial expression, purification, and partial characterization of amino acids 94–155 of human tissue factor pathway inhibitor (TFPI) as an inhibitor of blood coagulation factor Xa

Thrombosis Research ◽

10.1016/0049-3848(92)90095-r ◽

1992 ◽

Vol 68 (4-5) ◽

pp. 369-381 ◽

Cited By ~ 5

Author(s):

Kathleen C. Day ◽

Dean J. Welsch

Keyword(s):

Amino Acids ◽

Tissue Factor ◽

Blood Coagulation ◽

Human Tissue ◽

Tissue Factor Pathway Inhibitor ◽

Factor Xa ◽

Coagulation Factor ◽

Bacterial Expression ◽

Tissue Factor Pathway

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Converting blood coagulation factor IXa into factor Xa: dramatic increase in amidolytic activity identifies important active site determinants

The EMBO Journal ◽

10.1093/emboj/16.22.6626 ◽

1997 ◽

Vol 16 (22) ◽

pp. 6626-6635 ◽

Cited By ~ 56

Author(s):

K.-P. Hopfner

Keyword(s):

Blood Coagulation ◽

Active Site ◽

Factor Xa ◽

Coagulation Factor ◽

Amidolytic Activity ◽

Factor Ixa

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The Mechanism of Activation of Human Coagulation Factor X

10.1055/s-0039-1684668 ◽

1979 ◽

Author(s):

K. Mertens ◽

R.M. Bertina

Keyword(s):

Active Site ◽

Human Factor ◽

Polyacrylamide Gel Electrophoresis ◽

Active Form ◽

Factor Xa ◽

Coagulation Factor ◽

Factor X ◽

Process Factor ◽

Molecular Events ◽

Product Factor

During the coagulation process factor X is converted to a serine protease, factor Xa. The present study concerns the molecular events which occur during the activation of human factor X by Russell’s viper venom and by the purified proteins of the extrinsic and intrinsic activator. Conversion of factor X was detected by amidolytic assays and SDS/polyacrylamide-gel electrophoresis.The results show that all activators convert factor X (MW 72,000) to an active form. In the presence of phospholipid the initially formed factor Xa (MW 54,000) complicates the further sequence of reactions by catalysing a) the conversion of factor Xa to a second active form (MW 50,000), b) the conversion of factor X to an inactive product (MW 59,000) by splitting off a peptide containing the active site serine, and c) the further degradation of the 50,000 and 59,000 components to a smaller component (MW 40,000).Comparison of these data with those reported for bovine factor X suggests that the mechanism of activation of human factor X is more complicated. The inactivation of both factor Xa and factor X by product factor Xa might be considered as important regulatory principles.

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Characterization Of The Active Site Of Urokinase With A Series Of Potent Inhibitory Amidines

10.1055/s-0038-1652614 ◽

1981 ◽

Author(s):

J D Geratz ◽

S R Shaver ◽

R R Tidwell

Keyword(s):

Active Site ◽

Factor Xa ◽

Selective Inhibition ◽

The Other ◽

Plasminogen Activation ◽

Steric Factors ◽

The Difference ◽

The One ◽

Striking Contrast

Twenty amidine-substituted indole-like heterocycles were synthesized and examined for their blocking effect against human urokinase and a number of related arginine- or lysine- directed proteases. Kinetic analyses were carried out with the help of peptide anilide substrates and revealed a reversible competitive inhibitory pattern with each compound. The Ki values were therefore interpreted to reflect binding conditions at the active site of the enzymes.A highly potent inhibitor of urokinase was discovered in 5-amidino-l-(4-amidinobenzyl)indole which proved to be 18 times more effective on a molar basis than p-aminobenzamidine and 150 times more effective than benzamidine. The Ki value at 37°C and pH 8.3 was determined as 3.2 × 10-6 M. In striking contrast to the findings with the other proteases studied, urokinase was very sensitive to inhibition by 6-amidinoindoline (Ki 1.8 × 10-5 M), yet was much less susceptible to inhibition by the fully unsaturated analog 6-amidinoindole. Steric factors resulting from the difference in planarity between the two compounds are held responsible for this observation. In plasminogen activation assays the antiurokinase effect of the heterocycles mirrored their potency in the assays employing the synthetic urokinase substrate.The significant differences in the inhibitory activities of amidines against urokinase, on the one hand, and plasmin, thrombin and factor Xa, on the other hand, will be useful for experiments where selective inhibition of plasminogen activation is to be achieved. The compounds will also be of help in characterizing other tissue activators with respect to urokinase.

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Investigation of putative active-site lysine residues in hydroxymethylbilane synthase. Preparation and characterization of mutants in which (a) Lys-55, (b) Lys-59 and (c) both Lys-55 and Lys-59 have been replaced by glutamine

Biochemical Journal ◽

10.1042/bj2710487 ◽

1990 ◽

Vol 271 (2) ◽

pp. 487-491 ◽

Cited By ~ 10

Author(s):

A Hädener ◽

P R Alefounder ◽

G J Hart ◽

C Abell ◽

A R Battersby

Keyword(s):

Escherichia Coli ◽

Active Site ◽

Kinetic Studies ◽

Enzyme Kinetic ◽

Wild Type ◽

Wild Type Enzyme ◽

Over Expression ◽

Putative Active Site ◽

Lysine Residues

A new construct carrying the hemC gene was transformed into Escherichia coli, resulting in approx. 1000-fold over-expression of hydroxymethylbilane synthase (HMBS). This construct was used to generate HMBS in which (a) Lys-55, (b) Lys-59 and (c) both Lys-55 and Lys-59 were replaced by glutamine (K55Q, K59Q and K55Q-K59Q respectively). All three modified enzymes are chromatographically separable from wild-type enzyme. Kinetic studies showed that the substitution K55Q has little effect whereas K59Q causes a 25-fold decrease in Kapp. cat./Kapp. m. Treatment of K55Q, K59Q and K55Q-K59Q separately with pyridoxal 5′-phosphate and NaBH4 resulted in incomplete and non-specific reaction with the remaining lysine residues. Pyridoxal modification of Lys-59 in the K55Q mutant caused greater enzymic inactivation than similar modification of Lys-55 in K59Q. The results in sum show that, though Lys-55 and Lys-59 may be at or near the active site, neither is indispensable for the catalytic activity of HMBS.

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