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 bet a was investigated. For this purpose, the activation intermediate fact ors IX alpha and IXa alpha were purified after cleavage of the Arg(145 )-Ala(146) and Arg(180)-Val(181) bonds, respectively. These intermedia tes were compared for a number of functional properties with factor IX a 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 (k(cat)/K-m approximate to 120 s(-1) M(-1)) but was less efficient in factor X activation. Factor IX alpha was incapa ble of generating factor Xa but displayed reactivity toward p-nitrophe nol p-guanidino-benzoate and the peptide substrate. The catalytic effi ciency, however, was 4-fold lower compared with factor IXa alpha and f actor IXa beta. Factor IX alpha and factor IXa beta had similar affini ty for the inhibitor benzamidine (K-i approximate to 2.5 mM), and amid olytic activity of both species was inhibited by Glu-Gly-Arg-chloromet hyl ketone and antithrombin III. Unlike factor IXa beta, factor IX alp ha was unable to form SDS stable complexes with antithrombin III. More over, inhibition of factor IXa beta and factor IX alpha by Glu-Gly-Arg -chloromethyl ketone followed distinct pathways, because factor IX alp ha was inhibited in a nonirreversible manner and displayed only minor incorporation of the dansylated inhibitor into its catalytic site. The se data demonstrate that the catalytic site of factor IX alpha differs from that of the fully activated factor IXa beta. Factor IX and its d erivatives were also compared with regard to complex assembly with fac tor VIII in direct binding studies employing the immobilized factor VI II light chain. Factor IX alpha and factor IXa beta displayed a 30-fol d higher affinity for the factor VIII light chain (K-d approximate to 12 nM) than the factor IX zymogen. Factor IXa alpha showed lower affin ity (K-d approximate to 50 nM) than factor IX alpha and factor IXa bet a, which may explain the lower efficiency of factor X activation by fa ctor IXa alpha. Collectively, our data indicate that cleavage of the A rg(180)-Val(181) bond develops full amidolytic activity but results in suboptimal binding to the factor VIII light chain. With regard to cle avage of the Arg(145)-Ala(146) bond, we have demonstrated that this re sults in the transition of the factor IX zymogen into an enzyme that l acks proteolytic activity. Moreover, the same cleavage fully exposes t he binding site for the factor VIII light chain, suggesting that cleav age of the Arg(145)-Ala(146) bond serves a previously unrecognized rol e in the assembly of the factor IX-factor VIII complex.