The evaluation of complex-dependent alterations in human factor VIIa.
Factor VIIa is a plasma glycoprotein which, when bound to the integral membrane glycoprotein tissue factor, forms an enzymatic complex that is essential for normal hemostasis. We have developed a fluorescent substrate (6-(Mes-D-Leu-Gly-Arg)amino-1-naphthalenediethylsulfamide) which can be used to directly measure the enzymatic activity of factor VIIa in the presence and absence of tissue factor and phospholipid. The sensitivity of this substrate allows for detection of factor VIIa at concentrations below 10(-9) M. The kinetics of substrate hydrolysis by factor VIIa were evaluated and it was observed that the binding of factor VIIa to tissue factor increases the catalytic efficiency (kcat/Km) of factor VIIa substrate hydrolysis greater than 100-fold. The increase in enzymatic efficiency of factor VIIa, when complexed to tissue factor, is mediated primarily by an increase in kcat. These data suggest that tissue factor induces an alteration in the catalytic site of factor VIIa, which allows for more efficient hydrolysis of the small fluorescent substrate. Measurements conducted using various phospholipids and detergents demonstrated that the increase in catalytic efficiency of factor VIIa, when complexed to tissue factor, is independent of the supporting surface. The differential rate of substrate hydrolysis when factor VIIa is complexed to tissue factor was used to estimate the binding of factor VIIa to tissue factor. From these data an apparent dissociation constant for factor VIIa binding to tissue factor was calculated to be between 1.1 and 2.1 nM with a binding stoichiometry of 1.04:1 (factor VIIa:tissue factor). When the reactivity of this small fluorescent substrate toward single-chain factor VII was investigated, both in the presence and absence of tissue factor, no substrate hydrolysis was observed.
Lawson
J H
JH
Department of Biochemistry, University of Vermont, Burlington 05405.
Butenas
S
S
Mann
K G
KG
D002118:Calcium / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D015942:Factor VIIa / Q000378:metabolism*; D005456:Fluorescent Dyes; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D010743:Phospholipids / Q000378:metabolism; D013379:Substrate Specificity; D013925:Thromboplastin / Q000378:metabolism
D005456:Fluorescent Dyes; D010743:Phospholipids; D013925:Thromboplastin; D015942:Factor VIIa; D002118:Calcium