Abstract
The binding of recombinant nematode anticoagulant protein c2 (NAPc2) to either factor X or Xa is a requisite step in the pathway for the potent inhibition of VIIa tissue factor. We have used NAPc2 as a tight binding probe of human Xa to investigate protein substrate recognition by the human prothrombinase complex. NAPc2 binds with high affinity (K(d) approximately 1 nm) to both X and Xa in a way that does not require or occlude the active site of the enzyme. In contrast, NAPc2 is a tight binding, competitive inhibitor of protein substrate cleavage by human Xa incorporated into prothrombinase with saturating concentrations of membranes and Va. By fluorescence binding studies we show that NAPc2 does not interfere with the assembly of human prothrombinase. These are properties expected of an inhibitor that blocks protein substrate recognition by targeting extended macromolecular recognition sites (exosites) on the enzyme complex. A weaker interaction (K(d) = 260-500 nm) observed between NAPc2 and bovine X was restored to a high affinity one in a recombinant chimeric bovine X derivative containing 25 residues from the COOH terminus of the proteinase domain of human X. This region implicated in binding NAPc2 is spatially adjacent to a site previously identified as a potential exosite. Despite the weaker interaction with bovine Xa, NAPc2 was a tight binding competitive inhibitor of protein substrate cleavage by bovine prothrombinase as well. Extended enzymic surfaces elucidated with exosite-directed probes, such as NAPc2, may define a unique region of factor Xa that is modulated following its assembly into prothrombinase and in turn determines the binding specificity of the enzyme complex for its protein substrate.
Highlights
The proteolytic activation of prothrombin is catalyzed by the prothrombinase complex of coagulation (2–5)
The findings suggest that nematode anticoagulant protein c2 (NAPc2) binds to factor Xa regardless of the presence of other prothrombinase constituents and modestly perturbs active site function
One hallmark of this strategy is that ligands that block the exositemediated bimolecular interaction between enzyme and substrate are expected to act as competitive inhibitors of protein substrate cleavage without occluding the active site of the proteinase within the enzyme complex or interfering with the assembly of prothrombinase (10)
Summary
Materials—Hen egg L-␣-phosphatidylcholine (PC) and bovine brain L-␣-phosphatidyl-L-serine (PS) were from Avanti Polar Lipids (Alabaster, AL). For the recombinant bovine derivatives, stable clones selected with G418 were identified by a functional assay in which conditioned medium was treated with the purified factor X activator from Russell’s viper venom followed by measurements of the initial rate of SpXa hydrolysis. Equilibrium dissociation constants for the interaction between NAPc2 and factor Xa were determined by a local analysis of initial velocity measurements in reaction mixtures containing 0.5 nM Xa, 45 M SpXa, or 1 nM Xa plus 50 M PCPS, 100 M SpXa, and increasing concentrations of NAPc2. Analysis according to Equations 2 and 3 yielded fitted values for Vmax, Km, n, and Ki. Fluorescence titrations obtained at different fixed concentrations of OG488-hXa, a fixed, saturating concentration of PCPS, and increasing concentrations of Va were analyzed according to the model and experimental considerations previously developed in detail for bovine prothrombinase (41). Nonlinear least squares analysis yielded fitted constants for the indicator interaction (Kd, n, ⌬ρr ) as well as the equilibrium dissociation constant for the membrane-mediated interaction between Xai and Va (KdComp) and moles of Va bound per mol of Xai at saturation (nComp)
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