Kinetics of the activation of bovine coagulation factor X by components of the extrinsic pathway. Kinetic behavior of two-chain factor VII in the presence and absence of tissue factor.

A new assay for the activation of bovine coagulation Factor X by two-chain Factor VII (cu-VIIa) is described. The assay measures the release of the radiolabeled activation peptide which is soluble in 5% trichloroacetic acid. The rate of release is shown to be a linear function of a-VIIa concentration in a system saturated with tissue factor. It could therefore be used to derive kinetic parameters for the action of cu-VIIa on Factor X, its physiological substrate. The bat, 32 s-l, shows that ru-VIIa in the presence of tissue factor is comparable with other serine proteases in its reactivity. The K,, 0.34 PM Factor X, lies above the plasma concentration which suggests that the physiological level of Factor X can influence the rate of formation of Factor Xa via the extrinsic pathway. The sensitivity of the assay is great enough to detect hydrolysis of Factor X by cY-VIIa in the absence of tissue factor, which is 8,000-fold slower than with the cofactor present at a Factor X concentration of 100 pg/ml (1.8 pm). These experiments were performed using benzamidine to inhibit the feedback reactions of the product, Factor Xa. However, benzamidine also inhibits cy-VIIa activity to some degree, which we show occurs with a mixed competitive and uncompetitive mechanism. Comparison of the kinetic parameters obtained in 10 mM benzamidine in the presence and absence of tissue factor shows that the co-factor decreases the K,,, IO-fold and increases kc,, 2,900-fold. The mechanistic implications of these findings are discussed.