SELECTIVE-INHIBITION OF THE PROTHROMBINASE COMPLEX - FACTOR VA ALTERSMACROMOLECULAR RECOGNITION OF A TICK ANTICOAGULANT PEPTIDE MUTANT BY FACTOR XA

The prothrombinase complex assembles through reversible interactions b etween the protease, factor Xa, the cofactor, factor Va, and acidic ph ospholipid membranes in the presence of calcium ions. Changes in macro molecular recognition by factor Xa which may result from its interacti on with factor Va in the prothrombinase complex have been probed using a recombinant derivative of tick anticoagulant peptide where Arg(3) h as been replaced with Ala (R3A-TAP), In contrast to the wild type inhi bitor, R3A-TAP was a weak competitive inhibitor of factor Xa (K-i = 79 4 nM), The inhibition of the prothrombinase complex by R3A-TAP was cha racterized by slow, tight-binding kinetics with an increased affinity of similar to 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 a nd R3A-TAP could be adequately described by a single reversible step w ith rate constants that were consistent with equilibrium binding measu rements. The rate-limiting bimolecular combination of R3A-TAP and fact or Xa was competitive with PAB binding to the protease, In contrast, t he reaction of R3A-TAP with prothrombinase measured using PAB yielded biphasic stopped-flow traces, indicating a multistep pathway for the r eaction of the inhibitor with the enzyme complex. The kinetic measurem ents were consistent with the initial formation of a ternary complex b etween R3A-TAP, prothrombinase, and PAB followed by two unimolecular s teps which lead to PAB dissociation from the enzyme. In this case, pri or occupation of the active site by PAB had no effect on the bimolecul ar 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. Therefo re, a single amino acid substitution in TAP reveals large changes in m acromolecular recognition by factor Xa as a consequence of its interac tion with the cofactor within the prothrombinase complex.