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 (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 (K(D) = 1-10 nm), and similarly supported the inhibition of fVIIa.TF (K(i)* = approximately 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 (TF(PC)) or a mixture of phosphatidylcholine and phosphatidylserine (TF(PCPS)). In the absence of the fX derivative, inhibition of fVIIa.TF was similar for all three conditions (K(i) approximately 1 microm), whereas the addition of the fX derivative increased the respective inhibition by 35-, 150-, or 100,000-fold for TF(S), TF(PC), and TF(PCPS). The removal of the gamma-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.
Bergum
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PW
Corvas International, Inc., San Diego, California 92121, USA. peter_bergum@corvas.com
Cruikshank
A
A
Maki
S L
SL
Kelly
C R
CR
Ruf
W
W
Vlasuk
G P
GP
D000818:Animals; D000925:Anticoagulants / Q000494:pharmacology; D001665:Binding Sites; D016466:CHO Cells; D020134:Catalytic Domain; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003546:Cysteine Endopeptidases / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors / Q000737:chemistry / Q000378:metabolism*; D015942:Factor VIIa / Q000378:metabolism*; D015951:Factor Xa / Q000737:chemistry / Q000378:metabolism*; D015801:Helminth Proteins / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008954:Models, Biological; D008956:Models, Chemical; D009154:Mutation; D009363:Neoplasm Proteins; D010743:Phospholipids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism*; D020349:Surface Plasmon Resonance; D013987:Ticks; D013997:Time Factors
D000925:Anticoagulants; D004792:Enzyme Precursors; D015801:Helminth Proteins; D009363:Neoplasm Proteins; D010743:Phospholipids; D011994:Recombinant Proteins; C400226:anti-coagulant protein C2, Ancylostoma caninum; D015942:Factor VIIa; D015951:Factor Xa; D003546:Cysteine Endopeptidases; C030910:cancer procoagulant