Kinetic comparison of bovine blood coagulation factors IXa alpha and IXa beta toward bovine factor X.
The Vmax/Km (microM -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 w/w) 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. During these studies, it was discovered that the factor X which became activated to factor Xa during the course of reaction 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. The results show that 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, is much greater than that of factor IXa alpha.
D000818:Animals; D001779:Blood Coagulation Factors / Q000378:metabolism*; D002122:Calcium Chloride / Q000494:pharmacology; D002417:Cattle; D005164:Factor IX / Q000378:metabolism*; D015949:Factor IXa; D005169:Factor VIII / Q000378:metabolism; D005170:Factor X / Q000378:metabolism*; D015951:Factor Xa; D007700:Kinetics
D001779:Blood Coagulation Factors; D005169:Factor VIII; D005164:Factor IX; D005170:Factor X; D015949:Factor IXa; D015951:Factor Xa; D002122:Calcium Chloride