Studies on the Activity Measurement for Blood Proteases using their Monoclonal Antibodies

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TISSUE FACTOR POTENTIATES THE HYDROLYSIS OF SYNTHETIC ESTER SUBSTRATE CATALYZED BY FACTOR VIIA.We established a simple and sensitive assay method for the activity of factor VIIa-tissue factor complex using N^<alpha>-benzyloxycarbonyl-L-arginine P-nitrobenzyl ester (Z-Arg-ONb) as a substrate. The substrate has been synthesized previously (Zur, M., and Nemerson, Y. (1978) J. Biol. Chem. 253, 2203-2209). The new method was that the amount of P-nitrobenzyl alcohol released during the enzyme reaction is measured by reversed-phase HPLC, using a 3C_<18> column in 45% acetonitrile containing 0.1% trifluoroacetic acid with an isocratic elution. Z-Arg-ONb had a broad specificity for plasma serine proteases, and factor IXa could hydrolyze this substrate, indicating that the method is also useful for assay of the factor IXa activity. Using this assay method, we examined effect of tissue factor on the esterase activity of factor VIIa under various conditions, and found that tissue factor. . potenti … More ates greatly the hydrolysis of Z-Arg-ONb by both human and bovine factor VIIa's. These results did not agree with the previous report. Moreover, phospholipids were not required for the factor VIIa-mediated hydrolysis of Z-Arg-ONb even in the presence of tissue factor. We also determined the kinetic parameters (Km and kcat) of factor VIIa toward the substrate in the presence or absence of tissue factor. The Km value of factor VIIa alone was 6 times higher than that of factor VIIa-tissue factor complex (3.2 versus 0.54 mM), whereas the kcat value was 12 times lower than that of factor VIIa-tissue factor complex (14.3 ve rsus 173 s^<-1>). These results indicate that tissue factor affects the catalytic center of factor VIIa and enhances the factor VIIa-mediated hydrolysis of the ester substrate. The potentiating effect of tissue factor disappeared by removal of y-carboxyglutamic acid (Gla) -domain from factor VIIa, whereas the esterase activity in the absence of tissue factor was not affected by this modification, suggesting that the Gla-domain is required as the potent determinants on factor VIIa for the interaction with tissue factor, even if phospholipids are absent in the reaction mixture. Less