The
autolysis loop (residues 143-154 in
chymotrypsinogen numbering) plays a pivotal role in determining the macromolecular substrate and inhibitor specificity of coagulation
proteases. This loop in
factor IXa (FIXa) has 3 basic residues (Arg143, Lys147, and Arg150) whose contribution to the
protease specificity of
factor IXa has not been studied. Here, we substituted these residues individually with Ala in Gla-domainless forms of recombinant
factor IX expressed in mammalian cells. All mutants exhibited normal amidolytic activities toward a FIXa-specific
chromogenic substrate. However, Arg143 and Lys147 mutants showed a approximately 3- to 6-fold impairment in FX activation, whereas the Arg150 mutant
activated factor X normally both in the absence and presence of
factor VIIIa. By contrast, Arg143 and Lys147 mutants reacted normally with
antithrombin (AT) in both the absence and presence of the cofactor,
heparin. However, the reactivity of the Arg150 mutant with AT was impaired 6.6-fold in the absence of
heparin and 33- to 70-fold in the presence of pentasaccharide and full-length heparins. These results suggest that Arg143 and Lys147 of the
autolysis loop are recognition sites for FX independent of
factor VIIIa, and Arg150 is a specific recognition site for AT that can effectively interact with AT only if the
serpin is in the
heparin-activated conformation.