title: Selective inhibition of the prothrombinase complex: factor Va alters macromolecular recognition of a tick anticoagulant peptide mutant by factor Xa.
abstract: The prothrombinase complex assembles through reversible interactions between the protease, factor Xa, the cofactor, factor Va, and acidic phospholipid membranes in the presence of calcium ions. Changes in macromolecular recognition by factor Xa which may result from its interaction with factor Va in the prothrombinase complex have been probed using a recombinant derivative of tick anticoagulant peptide where Arg3 has been replaced with Ala (R3A-TAP). In contrast to the wild type inhibitor, R3A-TAP was a weak competitive inhibitor of factor Xa (Ki = 794 nM). The inhibition of the prothrombinase complex by R3A-TAP was characterized by slow, tight-binding kinetics with an increased affinity of approximately 4000-fold (Ki* = 0.195 nM) relative to that of solution-phase factor Xa. Stopped-flow measurements using p-aminobenzamidine (PAB) demonstrated that the reaction between solution-phase factor Xa and R3A-TAP could be adequately described by a single reversible step with rate constants that were consistent with equilibrium binding measurements. The rate-limiting bimolecular combination of R3A-TAP and factor Xa was competitive with PAB binding of the protease. In contrast, the reaction of R3A-TAP with prothrombinase measured using PAB yielded biphasic stopped-flow traces, indicating a multistep pathway for the reaction of the inhibitor with the enzyme complex. The kinetic measurements were consistent with the initial formation of a ternary complex between R3A-TAP, prothrombinase, and PAB followed by two unimolecular steps which lead to PAB dissociation from the enzyme. In this case, prior occupation of the active site by PAB had no effect on the bimolecular reaction between R3A-TAP and prothrombinase. Thus, the interaction of factor Xa with factor Va on the membrane surface alters recognition of R3A-TAP by the protease, leading to changes in the thermodynamics as well as in the observed kinetic mechanism for the reaction. Therefore, a single amino acid substitution in TAP reveals large changes in macromolecular recognition by factor Xa as a consequence of its interaction with the cofactor within the prothrombinase complex.
authors.affiliation: Department of Medicine, Emory University, Atlanta, Georgia 30322, USA.
authors.forename: A
authors.initials: A
authors.lastname: Betz
authors.forename: G P
authors.initials: GP
authors.lastname: Vlasuk
authors.forename: P W
authors.initials: PW
authors.lastname: Bergum
authors.forename: S
authors.initials: S
authors.lastname: Krishnaswamy
mesh_terms: D000818:Animals; D060829:Arthropod Proteins; D001550:Benzamidines / Q000494:pharmacology; D001665:Binding Sites / Q000235:genetics; D002417:Cattle; D005165:Factor V / Q000037:antagonists & inhibitors* / Q000378:metabolism; D015943:Factor Va / Q000494:pharmacology*; D005170:Factor X / Q000037:antagonists & inhibitors* / Q000378:metabolism; D015951:Factor Xa / Q000378:metabolism*; D036341:Intercellular Signaling Peptides and Proteins; D007700:Kinetics; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D010455:Peptides / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D010713:Phosphatidylcholines / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013050:Spectrometry, Fluorescence
chemical_list: D060829:Arthropod Proteins; D001550:Benzamidines; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D010713:Phosphatidylcholines; D015842:Serine Proteinase Inhibitors; C022475:prothrombinase complex; C063967:tick anticoagulant peptide; C006500:4-aminobenzamidine; D015943:Factor Va; D005165:Factor V; D005170:Factor X; D015951:Factor Xa