An Analysis of Cleavage of the Factor IX Activation Sites by Factor XIa

Abstract During blood coagulation, the plasma zymogen factor IX (fIX) is converted to the active protease factor Ixaβ (fIXaβ). The severe bleeding disorder associated with deficiency of fIX (hemophilia B) attests to the importance of this protein in hemostasis. Conversion of fIX to fIXaβ requires two proteolytic cleavages after Arg145 and Arg180, releasing an activation peptide. This process is mediated by the proteases factor VIIa (fVIIa) and factor XIa (fXIa). FVIIa in complex with tissue factor initially cleaves fIX after Arg145 forming an intermediate, factor IXα (fIXα), which is then cleaved after Arg180 to form fIXaβ. Western blots of activation time courses demonstrate fIXα accumulation during this process, indicating cleavage at Arg180 is rate limiting. In contrast, little intermediate accumulation occurs during fIX activation by fXIa. Previously, we showed that fXIa also cleaves fIX initially after Arg145, generating fIXα (Smith et al., J. Biol. Chem. 283;6696: 2008). To account for the lack of intermediate accumulation, then, the subsequent cleavage after Arg180 must occur at least as rapidly as the initial cleavage. We examined the relative rates of conversion of fIX, fIXα, and the alternative intermediate factor IXaα (fIXaα - cleaved after Arg180) to fIXaβ by fXIa. FIXα or fIXaα were prepared from tritium-labeled fIX by incubation with fXIa-Pro192 (discussed below) or Russell's Viper Venom protease, respectively, and purified by anion exchange chromatography. Conversion to fIXaβ was determined by measuring release of the tritiated activation peptide. FXIa converted fIX to fIXaβ with a kcat of 29.4 ± 0.4/min, a value reflecting cleavage at both activation sites. Kcat for conversion of fIXα and fIXaα to fIXaβ were 29.9 ± 0.5 and 30.0 ± 1.0/min, respectively. The rate of conversion of fIX to fIXα, estimated by measuring tritiated activation products separated by SDS-PAGE, was 30.0 ± 0.4/min. Recently, we showed that amino acid substitutions in fXIa for the conserved active site residue Gly193 (chymotrypsin numbering) decreased kcat for fIX activation 7–1000 fold, with residues with long branched side-chains having the greatest effect (Schmidt et al. Biochemistry 47;1326: 2008). Gly193 substitutions had a modestly larger detrimental effect (1.2–1.5 fold) on cleavage of fIX after Arg180 compared to Arg145 that was associated with varying degrees of fIXα accumulation. Similar effects were noted with substitutions for the adjacent residue Lys192. FXIa with Pro192 cleaved fIX after Arg180 >10-fold more slowly than after Arg145, generating fIXα with little subsequent conversion to fIXaβ. Cumulatively, these data support the premise that the rates for the two sequential reactions required for normal fIX activation by fXIa are comparable. Therefore, perturbations causing a greater effect on cleavage after Arg180 compared to Arg145, even if relatively small, result in fIXα accumulation. Initial recognition of fIX by fXIa involves substrate binding exosites distinct from the enzyme active site. At least one exosite appears to be located in the fXIa third apple (A3) domain, and may interact with an epitope on the fIX Ca2+-binding Gla-domain. The rate of fIX activation to fIXaβ by fXIa was significantly reduced in the presence of an antibody to the fXIa A3 domain or by mutations in the A3 domain. Similarly, rates of activation were decreased in the absence of Ca2+, in the presence of an antibody to the fIX Gla-domain, or when fIX with a decarboxylated Gla-domain was the substrate. In all cases, significant fIXα accumulation was noted in time courses, indicating that interfering with this particular substrate-exosite interaction has a significantly greater effect on cleavage after Arg180 than after Arg145. These findings raise the possibility that the exosite on fXIa A3 plays a larger role in conversion of fIXα to fIXaβ than in initial fIX conversion to fIXα, and are consistent with the possibility, recently proposed by Sinha et al. (Biochemistry 46;9830: 2007), that a second fIX binding exosite is present on fXIa.