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

Recombinant nematode anticoagulant protein c2 (rNAPc2) is a potent, factor Xa (Ma)-dependent small protein inhibitor of factor VIIa-tissue factor (fVI Ia.TF), which binds to a site on fXa that is distinct from the catalytic ce nter (exo-site), In the present study, the role of other M derivatives in p resenting rNAPc2 to fVIIa.TF is investigated. Catalytically active and acti ve site blocked fXa, as well as a plasma-derived and an activation-resistan t mutant of zymogen M bound to rNAPc2 with comparable affinities (K-D = 1-1 0 nM), and similarly supported the inhibition of fVIIa.TF (K-i* = similar t o 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 (TFS), or reconstituted into membranes, containing phosphatidyl choline (TFPC) or a mixture of phosphatidylcholine and phosphatidylserine ( TFPCPS). In the absence of the M derivative, inhibition of fVIIa TF was sim ilar for all three conditions (K-i similar to1 muM), whereas the addition o f the M derivative increased the respective inhibition by 35-, 150-, or 100 ,000-fold for TFS, TFPC, and TFPCPS. The removal of the gamma -carboxygluta mic acid-containing domain from the M derivative did not affect the binding to rNAPc2, but abolished the effect of factor Xa as a scaffold for the inh ibition of fVIIa.TF by rNAPcQ, The overall. anticoagulant potency of rNAPc2 , therefore, results from a coordinated recognition of an exo-site on fX/fX a and of the active site of fVIIa, both of which are properly positioned in the ternary fVIIa.TF.M(a) complex assembled on an appropriate phospholipid surface.