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

Xia Yang, Yu Jia Chang, Shu Wha Lin, Peter N. Walsh

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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 Km(app) and Kd(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 (Kd(app)FIXa ∼ 1.1 nM, Vmax ∼ nM min-1), N89A displayed an increase of ∼20-fold in K d(app)FIXa and a decrease of ∼20-fold in Vmax; I90A had an increase of ∼5-fold in Kd(app)FIXa and ∼10-fold decrease in Vmax; and V107A had an increase of ∼3-fold in K d(app)FIXa and ∼4-fold decrease in Vmax. 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.

Original languageEnglish
Pages (from-to)46400-46405
Number of pages6
JournalJournal of Biological Chemistry
Issue number45
Publication statusPublished - Nov 5 2004
Externally publishedYes


ASJC Scopus subject areas

  • Biochemistry

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