Kinetic comparison of bovine blood coagulation factors IXa.alpha. and IXa.beta. toward bovine factor X

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Abstract
The Vmax/Km (.mu.M-1 min-1) for bovine factor X activation by bovine factor IXa.alpha., in the presence of sufficient [Ca2+] to saturate the initial reaction rate, was 0.007. When factor IXa.beta. was substituted for factor IXa.alpha. in this reaction, the Vmax/Km decreased to 0.001, suggesting that factor IXa.alpha. was a more potent catalyst under these conditions. When phospholipid (PL) vesicles (egg phosphatidylcholine/bovine brain phosphatidylserine, 4:1 wt/wt) were added to these same systems, at levels sufficient to saturate their effects, little change in the Vmax/Km occurred when factor IXa.alpha. was the enzyme. However, when factor IXa.beta. was employed, the Vmax/Km dramatically increased to 0.023, demonstrating that factor IXa.beta. responded to PL addition to a much greater extent than did factor IXa.alpha.. Upon addition of thrombin-activated factor VIII (factor VIIIa,t), at a suboptimal level, to the above systems, the Vmax/Km for factor X activation by factor IXa.alpha./Ca2+/PL/factor VIIIa,t was increased to 1.0, whereas this parameter for factor X activation by factor IXa.beta./Ca2+/PL/factor VIIIa,t under the same conditions was found to be 27.3. The factor X which became activated to factor Xa during the course of reaction apparently participated in several feedback reactions: activation of factor X, activation of factor VIII, and conversion of factor IXa.alpha. to factor IXa.beta.. All feedback reactions, which are capable of complicating the kinetic interpretation, were inhibited by performing the studies in a system which contained a rapid factor Xa inhibitor, Glu-Gly-Arg-CH2Cl, thus allowing kinetic constants to be accurately determined. While factor IXa.alpha. is a more efficient enzyme than factor IXa.beta. toward factor X activation in the absence of cofactors, the response of factor IXa.beta. to the reaction cofactors, PL and factor VIIIa,t, apparently is much greater than that of factor IXa.alpha.