Large enhancement of functional activity of active site-inhibited factor VIIa due to protein dimerization: insights into mechanism of assembly/disassembly from tissue factor.
Active site-inhibited blood clotting factor VIIa (fVIIai) binds to tissue factor (TF), a cell surface receptor that is exposed upon injury and initiates the blood clotting cascade. FVIIai blocks binding of the corresponding enzyme (fVIIa) or zymogen (fVII) forms of factor VII and inhibits coagulation. Although several studies have suggested that fVIIai may have superior anticoagulation effects in vivo, a challenge for use of fVIIai is cost of production. This study reports the properties of dimeric forms of fVIIai that are cross-linked through their active sites. Dimeric wild-type fVIIai was at least 75-fold more effective than monomeric fVIIai in blocking fVIIa association with TF. The dimer of a mutant fVIIai with higher membrane affinity was 1600-fold more effective. Anticoagulation by any form of fVIIai differed substantially from agents such as heparin and showed a delayed mode of action. Coagulation proceeded normally for the first minutes, and inhibition increased as equilibrium binding was established. It is suggested that association of fVIIa(i) with TF in a collision-dependent reaction gives equal access of inhibitor and enzyme to TF. Assembly was not influenced by the higher affinity and slower dissociation of the dimer. As a result, anticoagulation was delayed until the reaction reached equilibrium. Properties of different dissociation experiments suggested that dissociation of fVIIai from TF occurred by a two-step mechanism. The first step was separation of TF-fVIIa(i) while both proteins remained bound to the membrane, and the second step was dissociation of the fVIIa(i) from the membrane. These results suggest novel actions of fVIIai that distinguish it from most of the anticoagulants that block later steps of the coagulation cascade.
Stone
Matthew D
MD
Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, 6-155 Jackson Hall, 321 Church Street SE, Minneapolis, Minnesota 55455, USA.
Harvey
Stephen B
SB
Martinez
Michael B
MB
Bach
Ronald R
RR
Nelsestuen
Gary L
GL
D001665:Binding Sites; D001667:Binding, Competitive; D019281:Dimerization; D005167:Factor VII / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015942:Factor VIIa / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D046912:Multiprotein Complexes; D009154:Mutation; D011485:Protein Binding; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013925:Thromboplastin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D046912:Multiprotein Complexes; D011498:Protein Precursors; D011994:Recombinant Proteins; C071874:prepro-factor VII; D005167:Factor VII; D013925:Thromboplastin; D015942:Factor VIIa