Identification of residues Asn⁸⁹, Ile⁹⁰, and Val ¹⁰⁷ of the factor IXa second epidermal growth factor domain that are essential for the assembly of the factor X-activating complex on activated platelets

Activated platelets promote intrinsic factor X-activating complex assembly by presenting high affinity, saturable binding sites for factor IXa mediated by two disulfide-constrained loop structures (loop 1, Cys⁸⁸-Cys ⁹⁹; loop 2, Cys⁹⁵-Cys¹⁰⁹) within the second epidermal growth factor (EGF2) domain. To identify amino acids essential for factor X activation complex assembly, recombinant factor IXa point mutants in loop 1 (N89A, I90A, K91A, and R94A) and loop 2 (D104A, N105A, and V107A) were prepared. All seven mutants were similar to the native factor IXa by SDS-PAGE, active site titration, and content of γ-carboxyglutamic acid residues. Kinetic constants obtained by either titrating factor X or factor VIIIa on SFLLRN-activated platelets or phospholipid vesicles revealed near normal values of K_m(app) and K_d(app)FVIIIa for all mutants, indicating normal substrate and cofactor binding. In a factor Xa generation assay in the presence of activated platelets and cofactor factor VIIIa, compared with native factor IXa (K_d(app)FIXa ∼ 1.1 nM, V_max ∼ nM min^-1), N89A displayed an increase of ∼20-fold in K _d(app)FIXa and a decrease of ∼20-fold in V_max; I90A had an increase of ∼5-fold in K_d(app)FIXa and ∼10-fold decrease in V_max; and V107A had an increase of ∼3-fold in K _d(app)FIXa and ∼4-fold decrease in V_max. We conclude that residues Asn⁸⁹, Ile⁹⁰, and Val¹⁰⁷ within loops 1 and 2 (Cys⁸⁸-Cys¹⁰⁹) of the EGF2 domain of factor IXa are essential for normal interactions with the platelet surface and for the assembly of the factor X-activating complex on activated platelets.