A kinetic evaluation of activated bovine blood coagulation factor IX toward synthetic substrates.

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The Vmm (&7%) of bovine Factor IXas (6.9 f 0.4 pmol of P-benzoyl-L-arginine ethyl ester (BAEE) cleaved min mg of protein active sites), is increased to 7.9, 8.7,8.7,8.7, and 9.8 in the presence of 10 lll~ Ba2+, M&+, Sr2+, and Ca2+, respectively, at 30°C. The K,,, of BAEE for Factor Eas (20 f 2.5 m) is not affected by the presence of the above divalent cations. A previous study (Byrne, R., and Castellino, F. J. (1978) Arch. Biochem Biophys. 190, 687-692) has shown that the same metal ions cause a qualitatively similar change in the V,, of bovine Factor Ea, toward this substrate. Benzamidine hydrochloride was found to be a competitive inhibitor of the BAEE esterase activity for these enzymes with a KI of 3.3 f 0.3 nm toward Factor IXap This value is not altered by the presence of 10 lll~ Ca. The KI of this inhibitor for Factor IXa, was 2.9 f 0.3 nm in the presence of 10 nm Ca2+ and 6.3 f 0.5 mM in the absence of Caz+. fie-steady state kinetics, using p-nitropheny1-p'guanidinobenzoate (NphBzoGdn) as the substrate, has also been determined for Factors IXas and Ea,, also at 30°C. In the absence of Ca2+, the acylation rate constant for Factor Eas is 0.10 f 0.02 s-l, and the corresponding value for Factor IXa,is 0.11 f 0.02 s-'. These values are essentially unchanged when 10 lll~ Ca2+ is added to either assay mixture. The binding (dissociation) constant (&) of NphBzoGdn to Factor Eaa was found to be 4.4 f 1.1 X M. The KS for NphBzoGdn to Factor IXa, was determined to be 5.2 f 1.3 X M. In both cases, the Ks values were not affected by the binding of Ca2+ to the enzymes. The deacylation rate constant, ks, for this substrate with Factor Ea@ is 3.0 f 0.5 X lo-' s-' and that for Factor IXa, is 8.7 f 1.0 X lo-' s in the absence of Ca2+. These constants were found to be 6.0 k 0.7 X lo-' M and 1.0 f 0.1 X lo-' M for these same enzymes, respectively, in the presence of 10 nm Ca2+. Factor IX is a zymogen which participates in the intrinsic blood coagulation cascade and is the protein which is functionally deficient in hemophilia B. The zymogen, as isolated from bovine plasma, possesses a molecular weight of 55,400 (l), in a single polypeptide chain. Prior to participation in the intrinsic pathway, Factor IX undergoes activation, to Factor IXa, as a consequence of proteolytic cleavages in the Factor IX molecule. One activator of Factor IX is the serine protease (2) Factor XIa in the presence of Ca2+ (3,4). The Factor XIa