Identification of residues Asn89, Ile90, and Val107 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-Cys99; 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 gamma-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 approximately 1.1 nm, Vmax approximately 12 nm min(-1)), N89A displayed an increase of approximately 20-fold in Kd(app)FIXa and a decrease of approximately 20-fold in Vmax; I90A had an increase of approximately 5-fold in Kd(app)FIXa and approximately 10-fold decrease in Vmax; and V107A had an increase of approximately 3-fold in Kd(app)FIXa and approximately 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.
Yang
Xia
X
Sol Sherry Thrombosis Research Center, Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
Chang
Yu-Jia
YJ
Lin
Shu-Wha
SW
Walsh
Peter N
PN
D015055:1-Carboxyglutamic Acid / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000737:chemistry*; D001665:Binding Sites; D001777:Blood Coagulation; D001792:Blood Platelets / Q000378:metabolism*; D002460:Cell Line; D004220:Disulfides; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004815:Epidermal Growth Factor / Q000737:chemistry*; D015949:Factor IXa / Q000737:chemistry*; D015944:Factor VIIIa / Q000737:chemistry; D005170:Factor X / Q000737:chemistry*; D006801:Humans; D007532:Isoleucine / Q000737:chemistry*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D015539:Platelet Activation; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D014633:Valine / Q000737:chemistry*
D004220:Disulfides; D011994:Recombinant Proteins; D007532:Isoleucine; D015055:1-Carboxyglutamic Acid; D004815:Epidermal Growth Factor; D001216:Asparagine; D015944:Factor VIIIa; D005170:Factor X; D015949:Factor IXa; D014633:Valine