Role of Zymogen and Activated Factor X as Scaffolds for the Inhibition of the Blood Coagulation Factor VIIa-Tissue Factor Complex by Recombinant Nematode Anticoagulant Protein c2

Abstract

Recombinant nematode anticoagulant protein c2 (rNAPc2) is a potent, factor Xa (fXa)-dependent small protein inhibitor of factor VIIa-tissue factor (fVIIa·TF), which binds to a site on fXa that is distinct from the catalytic center (exo-site). In the present study, the role of other fX derivatives in presenting rNAPc2 to fVIIa·TF is investigated. Catalytically active and active site blocked fXa, as well as a plasma-derived and an activation-resistant mutant of zymogen fX bound to rNAPc2 with comparable affinities (KD = 1-10 nM), and similarly supported the inhibition of fVIIa·TF (Ki* = ∼10 pM). The roles of phospholipid membrane composition in the inhibition of fVIIa·TF by rNAPc2 were investigated using TF that was either detergent-solubilized (TF S), or reconstituted into membranes, containing phosphatidylcholine (TFPC) or a mixture of phosphatidylcholine and phosphatidylserine (TFPCPS). In the absence of the fX derivative, inhibition of fVIIa·TF was similar for all three conditions (Ki ∼1 μM), whereas the addition of the fX derivative increased the respective inhibition by 35-, 150-, or 100,000-fold for TFS, TFPC, and TFPCPS. The removal of the γ-carboxyglutamic acid-containing domain from the fX derivative did not affect the binding to rNAPc2, but abolished the effect of factor Xa as a scaffold for the inhibition of fVIIa·TF by rNAPc2. The overall anticoagulant potency of rNAPc2, therefore, results from a coordinated recognition of an exo-site on fX/fXa and of the active site of fVIIa, both of which are properly positioned in the ternary fVIIa·TF·fX(a) complex assembled on an appropriate phospholipid surface.