Cleavage at arginine 145 in human blood coagulation factor IX converts the zymogen into a factor VIII binding enzyme.

The transition of the factor IX zymogen into the enzyme factor IXa beta was investigated. For this purpose, the activation intermediate factors IX alpha and IXa alpha were purified after cleavage of the Arg145-Ala146 and Arg180-Val181 bonds, respectively. These intermediates were compared for a number of functional properties with factor IXa beta, which is cleaved at both positions. Factor IXa alpha was equal to factor IXa beta in hydrolyzing the synthetic substrate CH3SO2-Leu-Gly-Arg-p-nitroanilide (kcat/Km approximately 120 s-1 M-1) but was less efficient in factor X activation. Factor IX alpha was incapable of generating factor Xa but displayed reactivity toward p-nitrophenol p-guanidinobenzoate and the peptide substrate. The catalytic efficiency, however, was 4-fold lower compared with factor IXa alpha and factor IXa beta. Factor IX alpha and factor IXa beta had similar affinity for the inhibitor benzamidine (Ki approximately 2.5 mM), and amidolytic activity of both species was inhibited by Glu-Gly-Arg-chloromethyl ketone and antithrombin III. Unlike factor IXa beta, factor IX alpha was unable to form SDS stable complexes with antithrombin III. Moreover, inhibition of factor IXa beta and factor IX alpha by Glu-Gly-Arg-chloromethyl ketone followed distinct pathways, because factor IX alpha was inhibited in a nonirreversible manner and displayed only minor incorporation of the dansylated inhibitor into its catalytic site. These data demonstrate that the catalytic site of factor IX alpha differs from that of the fully activated factor IXa beta. Factor IX and its derivatives were also compared with regard to complex assembly with factor VIII in direct binding studies employing the immobilized factor VIII light chain. Factor IX alpha and factor IXa beta displayed a 30-fold higher affinity for the factor VIII light chain (Kd approximately 12 nM) than the factor IX zymogen. Factor IXa alpha showed lower affinity (Kd approximately 50 nM) than factor IX alpha and factor IXa beta, which may explain the lower efficiency of factor X activation by factor IXa alpha. Collectively, our data indicate that cleavage of the Arg180-Val181 bond develops full amidolytic activity but results in suboptimal binding to the factor VIII light chain. With regard to cleavage of the Arg145-Ala146 bond, we have demonstrated that this results in the transition of the factor IX zymogen into an enzyme that lacks proteolytic activity.(ABSTRACT TRUNCATED AT 250 WORDS)