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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

Academic Article
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Overview

authors

  • Bergum, P. W.
  • Cruikshank, A.
  • Maki, S. L.
  • Kelly, C. R.
  • Ruf, Wolfram
  • Vlasuk, G. P.

publication date

  • March 2001

journal

  • Journal of Biological Chemistry  Journal

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 (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.

subject areas

  • Animals
  • Anticoagulants
  • Binding Sites
  • CHO Cells
  • Catalytic Domain
  • Cell Membrane
  • Cricetinae
  • Cysteine Endopeptidases
  • Dose-Response Relationship, Drug
  • Electrophoresis, Polyacrylamide Gel
  • Enzyme Precursors
  • Factor VIIa
  • Factor Xa
  • Helminth Proteins
  • Humans
  • Hydrolysis
  • Kinetics
  • Models, Biological
  • Models, Chemical
  • Mutation
  • Neoplasm Proteins
  • Phospholipids
  • Protein Binding
  • Protein Structure, Tertiary
  • Recombinant Proteins
  • Surface Plasmon Resonance
  • Ticks
  • Time Factors
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Identity

International Standard Serial Number (ISSN)

  • 0021-9258

Digital Object Identifier (DOI)

  • 10.1074/jbc.M009116200

PubMed ID

  • 11139576
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Additional Document Info

start page

  • 10063

end page

  • 10071

volume

  • 276

issue

  • 13

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