Characterization of the Defect in Activation of Factor

Factor IXCH is cleaved upon incubation with human Factor XIa and calcium; however, cleavage of this protein is not observed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis under nonreducing conditions. Under reducing conditions, the rate of disappearance of the zymogen parallels both the appearance of the heavy chain and the generation of clotting activity. In addition, a protein band that migrates with an apparent molecular weight of 45,000 also increases in parallel with clotting activity. Factor IXCH and normal Factor IX (Factor IXN), after incubation with Factor XIa and calcium, were subjected to amino terminal sequence analysis. Activated Factor IXN is cleaved at an arginine-alanine (Arg-Ala) bond and an arginine-valine (Arg-Val) bond as demonstrated by formation of the three amino terminal sequences corresponding to the amino terminal of the light chain, heavy chain, and activation peptide. However, activated Factor IXCH has only two amino terminal sequences, corresponding to the original amino terminal sequence and the heavy chain (formed by cleavage at the Arg-Val bond). It is concluded that the major defect in Factor IXCH is, the inability of Factor XIa to cleave the Arg-Ala bond at a significant rate. The rate of formation ofclotting activity ofFactor IXCH is -60% ofthe rate of formation of clotting activity of Factor IXN. The specific clotting activity of activated Factor IXCH is between 20 and 33% of activated Factor IXN. Received for publication 19 May 1981 and in revised form 29 July 1981.