The biochemical basis for the apparent defect of soluble mutant tissue factor in enhancing the proteolytic activities of factor VIIa.
Tissue factor (TF), an integral membrane protein, is the cofactor for the serine protease, coagulation factor VIIa (FVIIa). Previous studies of the isolated extracellular domain of TF (sTF) reported a kcat for factor X (FX) activation by the sTF.VIIa complex only 4% that of wild-type TF.VIIa and furthermore, a complete inability of sTF to support FVII autoactivation. We now report that in the presence of poly(L-lysine), sTF promoted both FX activation and FVII autoactivation, the latter with an apparent second-order rate constant higher than reported previously for wild-type TF in phospholipid vesicles. This led us to reexamine the cofactor ability of sTF, using high concentrations of phospholipid to promote nearly quantitative binding of sTF.VII(a) complexes to the phospholipid surface. Rate constants for the activation of FX or FVII by sTF.VIIa were similar to those of wild-type TF.VIIa, indicating that the apparent deficiency of sTF is largely attributable to kinetic consequences of relatively weak affinity of FVIIa (and sTF.VIIa) for phospholipid surfaces in these surface-dependent reactions, compared with TF being embedded in the membrane. This supports the notion that sTF recapitulates the protein-protein interactions of wild-type TF with high, and possibly full, catalytic activity. It also provides a biochemical explanation for the specificity of our recently described, sTF-based clotting assay for plasma FVIIa.
Fiore
M M
MM
Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City 73104.
Neuenschwander
P F
PF
Morrissey
J H
JH
D015942:Factor VIIa / Q000378:metabolism*; D005170:Factor X / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D010743:Phospholipids / Q000378:metabolism; D011107:Polylysine / Q000378:metabolism; D012995:Solubility; D013925:Thromboplastin / Q000235:genetics / Q000378:metabolism*
D010743:Phospholipids; D011107:Polylysine; D005170:Factor X; D013925:Thromboplastin; D015942:Factor VIIa