Selective Inhibition of the Prothrombinase Complex: Factor Va Alters Macromolecular Recognition of a Tick Anticoagulant Peptide Mutant by Factor Xa †,‡ Andreas Betz, § George P. Vlasuk, | Peter W. Bergum, | and Sriram Krishnaswamy* ,§ DiVision of Hematology/Oncology, Department of Medicine, Emory UniVersity, Atlanta, Georgia 30322, and Molecular Pharmacology, CORVAS International Inc., 3030 Science Park Road, San Diego, California 92121 ReceiVed August 15, 1996; ReVised Manuscript ReceiVed October 17, 1996 X 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 Arg 3 has been replaced with Ala (R3A-TAP). In contrast to the wild type inhibitor, R3A- TAP was a weak competitive inhibitor of factor Xa (K i ) 794 nM). The inhibition of the prothrombinase complex by R3A-TAP was characterized by slow, tight-binding kinetics with an increased affinity of ∼4000-fold (K i * ) 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 to 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. The prothrombinase complex is formed by association of the protease factor Xa with factor Va on appropriate phospholipid surfaces in the presence of calcium ions (Jackson & Nemerson, 1980; Mann et al., 1988, 1990). This enzyme complex converts prothrombin to the clot forming protease thrombin by two successive cleavages (Jackson & Nemerson, 1980; Mann et al., 1988, 1990). Although factor Xa, the catalytic component of prothrombinase, is also capable of activating prothrombin in solution, incorporation into prothrombinase amplifies its catalytic activity by a factor of ∼10 5 (Mann et al., 1988; Nesheim et al., 1979; Rosing et al., 1980). The full catalytic activity of prothrombinase may result from a cumulative effect of the interaction of all accessory components with factor Xa. However, kinetic studies suggest that factor Xa in solution saturated with high concentrations of factor Va approximates the catalytic efficiency of the membrane-assembled prothrombinase (Bosk- ovic et al., 1990). Hence, the enhanced activity of factor Xa within prothrombinase can largely be attributed to the interaction with factor Va, while the ability of factor Va, factor Xa, and prothrombin to bind to phospholipid mem- branes promotes the formation of the protease-cofactor complex and subsequently the delivery of the substrate to membrane-bound enzyme (Mann et al., 1990; Boskovic et al., 1990; Krishnaswamy et al., 1988; Giesen et al., 1991a,b; Walker & Krishnaswamy, 1994). The effect of factor Va on prothrombin activation may result from alterations induced in the protease and/or the substrate as both prothrombin and factor Xa can bind to the cofactor (Jackson & Nemerson, 1980; Mann et al., 1988; Esmon & Jackson, 1974; Luckow et al., 1989). Alterations in factor Xa resulting from its interaction with factor Va have been inferred from changes in fluorescence intensity and anisotropy of a fluorophore covalently tethered to the active site of factor Xa using the active site label dansylglutamyl- glycylarginyl chloromethyl ketone (Nesheim et al., 1981; Higgins et al., 1985; Husten et al., 1987). Since, in contrast to prothrombin, dansylglutamylglycylarginyl chloromethyl † This work was supported by NIH Grants HL-47465 and HL-52883 to S.K. ‡ A preliminary account of this work was presented in poster form at the ninth symposium of the Protein Society, Boston, MA, July 8-12, 1995 (Betz et al., 1995). * Address all correspondence to this author at Division of Hematol- ogy/Oncology, Department of Medicine, Emory University, 1014 Woodruff Memorial Building, 1639 Pierce Dr., Atlanta, GA 30322. Telephone: (404) 727-3806. Fax: (404) 727-3404. E-mail: skris01@unix.cc.emory.edu. § Emory University. | CORVAS International Inc. X Abstract published in AdVance ACS Abstracts, December 15, 1996. 181 Biochemistry 1997, 36, 181-191 S0006-2960(96)02060-0 CCC: $14.00 © 1997 American Chemical Society