Residues 88-109 of factor IXa are important for assembly of the factor X activating complex

Abstract

Activated platelets and phospholipid vesicles promote assembly of the intrinsic factor X (FX) activating complex by presenting high-affinity binding sites for blood coagulation FIXa, FVIIIa, and FX. Previous reports suggest that the second epidermal growth factor (EGF)-like domain of FIXa mediates assembly of the FX activating complex (Ahmad, S. S., Rawala, R., Cheung, W. F., Stafford, D. W., and Walsh, P. N. (1995) Biochem. J. 310, 427-431; Wong, M. Y., Gurr, J. A., and Walsh, P. N. (1999) Biochemistry 38, 8948-8960). To identify important residues, we prepared several chimeric FIXa proteins using homologous sequences from FVII: FIXaFVIIEGF2 (FIXΔ88-124, ∇FVII91-127), FIXaloop1 (FIXΔ88-99, ∇FVII91-102), FIXaloop2 (FIXΔ95-109, ∇FVII98-112), FIXaloop3 (FIXΔ111-124, ∇ FVII114-127), and point mutants (FIXaR94D and FIXaloop1G94R). In the presence and absence of FVIIIa, a 2- to 10-fold reduced Vmax of FX activation (nM FXa min-1) was observed for FIXaFVIIEGF2, FIXaloop1, FIXaloop2, and FIXaloop1G94R, whereas FIXaloop3 and FIXaR94D were normal. For all of the FIXa proteins, Km(app) values were normal as were EC50 values for interactions with FVIIIa. However, Kd(app) (in nM) for the FX activating complex assembled on phospholipid vesicles was increased for FIXaFVIIEGF2 (43.3 ± 2.70), FIXaloop1(10.9 ± 2.8), FIXaloop2 (70.5 ± 1.60), and FIXaloop1G94R (17.1 ± 2.90) relative to FIXaN (3.9 ± 0.11), FIXaWT (4.6 ± 0.17), FIXaloop3 (4.5 ± 0.20), and FIXaR94D (2.2 ± 0.09) suggesting that reduced Vmax is a result of impaired complex assembly. These data indicate that residues 88-109 (but not Arg94) are important for normal assembly of the FX activating complex on phospholipid vesicles.