Abstract
We recently showed that single-chain zymogen factor VII is converted to two-chain factor VIIa in an autocatalytic manner following complex formation with either cell-surface or solution-phase relipidated tissue factor apoprotein (Nakagaki, T., Foster, D. C., Berkner, K. L., and Kisiel, W. (1991) Biochemistry 30, 10819-10824). We have now performed a detailed kinetic analysis of the autoactivation of human plasma factor VII in the presence of relipidated recombinant tissue factor apoprotein and calcium. Incubation of factor VII with equimolar amounts of relipidated tissue factor apoprotein resulted in the formation of factor VIIa amidolytic activity coincident with the conversion of factor VII to factor VIIa. The time course for the generation of factor VIIa amidolytic activity in this system was sigmoidal, characterized by an initial lag phase followed by a rapid linear phase until activation was complete. The duration of the lag phase was decreased by the addition of exogenous recombinant factor VIIa. Relipidated tissue factor apoprotein was essential for factor VII autoactivation. No factor VII activation was observed following complex formation between factor VII and a recombinant soluble tissue factor apoprotein construct consisting of the aminoterminal extracellular domain in the presence or absence of phospholipids. Kinetic analyses revealed that factor VII activation in the presence of relipidated tissue factor apoprotein can be defined by a second-order reaction mechanism in which factor VII is activated by factor VIIa with an apparent second-order rate constant of 7.2 x 10(3) M-1 S-1. Benzamidine inhibited factor VII autoactivation with an apparent Ki of 1.8 mM, which is identical to the apparent Ki for the inhibition of factor VIIa amidolytic activity by this active site competitive inhibitor. Our data are consistent with a factor VII autoactivation mechanism in which trace amounts of factor VIIa rapidly activate tissue factor-bound factor VII by limited proteolysis.
Highlights
IntroductionPrevious studies demonstrated spontaneous activation of plasma or recombinant factor VI1 following complex formation with tissue factor provided by a human bladder carcinoma cell line, 582 [15].This spontaneous activation was, not observed using an inactive mutant recombinant factor VI1preparation (S344A factor VII) in which the active site serine residue was replaced with alanine, providing presumptive evidence for the autoactivation of factor VI1 in complex with tissue factor [11].In addition, catalytic amounts of recombinant factor VIIa, but not factor Xa, readily activated factor VII-tissue factor in the presence of plasma concentrations of antithrombin 111, the major serine protease inhibitor of blood coagulation proteases [11].In the present study, we have performed a detailed kinetic analysis of the activation of plasma factor VI1 in the presence of fluid-phase, relipidated tissue factor apoprotein
From the Blood S.y.stems Research Foundation Laboratory, Department of Pathology, University of New Mexico Schoolof Medicine, Albuq&rque,New Mexico87131
We recently showed that single-chain zymogen factor VI1 is converted to two-chain factor VIIa in an autocatalyticmanner following complex formation with either cell-surface or solution-phase relipidated tissue factor apoprotein
Summary
Previous studies demonstrated spontaneous activation of plasma or recombinant factor VI1 following complex formation with tissue factor provided by a human bladder carcinoma cell line, 582 [15].This spontaneous activation was, not observed using an inactive mutant recombinant factor VI1preparation (S344A factor VII) in which the active site serine residue was replaced with alanine, providing presumptive evidence for the autoactivation of factor VI1 in complex with tissue factor [11].In addition, catalytic amounts of recombinant factor VIIa, but not factor Xa, readily activated factor VII-tissue factor in the presence of plasma concentrations of antithrombin 111, the major serine protease inhibitor of blood coagulation proteases [11].In the present study, we have performed a detailed kinetic analysis of the activation of plasma factor VI1 in the presence of fluid-phase, relipidated tissue factor apoprotein This analysis, together with previous results ( l l ) , provides a mechanism for the initiation of the extrinsic pathway of blood coagulation in which tissue factor-bound factor VI1 is rapidly converted to functional factor VIIa by trace amounts of circulating factor VIIa
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