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

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

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
Volume279
Issue number45
DOIs
Publication statusPublished - Nov 5 2004
Externally publishedYes

Fingerprint

Factor IXa
Factor X
Platelets
Application programs
Epidermal Growth Factor
Blood Platelets
Factor VIIIa
Intrinsic Factor
Factor Xa
Essential Amino Acids
Disulfides
Polyacrylamide Gel Electrophoresis
Catalytic Domain
Phospholipids
Titration
Reference Values
Binding Sites
Assays
Chemical activation
Acids

ASJC Scopus subject areas

  • Biochemistry

Cite this

@article{8704cd5ba9c04ad69ddacdc03e976f1f,
title = "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",
abstract = "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.",
author = "Xia Yang and Chang, {Yu Jia} and Lin, {Shu Wha} and Walsh, {Peter N.}",
year = "2004",
month = "11",
day = "5",
doi = "10.1074/jbc.M406552200",
language = "English",
volume = "279",
pages = "46400--46405",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "45",

}

TY - JOUR

T1 - 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

AU - Yang, Xia

AU - Chang, Yu Jia

AU - Lin, Shu Wha

AU - Walsh, Peter N.

PY - 2004/11/5

Y1 - 2004/11/5

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=8744274428&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=8744274428&partnerID=8YFLogxK

U2 - 10.1074/jbc.M406552200

DO - 10.1074/jbc.M406552200

M3 - Article

VL - 279

SP - 46400

EP - 46405

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 45

ER -