The Factor IXa Second Epidermal Growth Factor (EGF2) Domain Mediates Platelet Binding and Assembly of the Factor X Activating Complex

• Published in: The Journal of biological chemistry 2002-02, Vol.277 (8), p.5734-5741
• Main Authors: Wilkinson, Frank H., Ahmad, Syed S., Walsh, Peter N.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Binding Sites; Epidermal Growth Factor - chemistry; Factor IXa - chemistry; Factor IXa - metabolism; Factor VIIIa - metabolism; Factor Xa - chemistry; Factor Xa - metabolism; Factor Xa - pharmacology; Humans; In Vitro Techniques; Kinetics; Molecular Sequence Data; Peptide Fragments - chemistry; Peptide Fragments - pharmacology; Platelet Adhesiveness - drug effects; Platelet Adhesiveness - physiology; Protein Conformation; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M107753200

Previously we have determined that residues 88–109 (but not Arg94) in the second epidermal growth factor (EGF2)-like domain of factor IXa (FIXa) are important for assembly of the factor X (FX) activating complex on phospholipid vesicles (Wilkinson, F. H., London, F. S., and Walsh, P. N. (2002) J. Biol. Chem. 277, 5725–5733). Here we report that these residues are important for platelet binding affinity, stoichiometry, and assembly of the FX activating complex. We prepared several chimeric FIXa proteins using homologous sequences from factor VII (FVII): FIXaFVIIEGF2 (FIXΔ88–124,∇FVII91–127), FIXaloop1 (FIXΔ88–99,∇FVII91–102), FIXaloop2 (FIXΔ95–109,∇FVII98–112), and FIXaloop3 (FIXΔ111–124,∇FVII114–127) and tested their ability to bind to thrombin-activated platelets. Binding affinities (Kd values in 10−9m) for the proteins were as follows in the presence and absence of FVIIIa, respectively: FIXaN (0.55 ± 0.06, 2.9 ± 0.45), FIXaWT (0.80 ± 0.08, 3.5 ± 0.5), FIXaloop1 (19 ± 4.0, 27 ± 5.0), FIXaloop2 (35 ± 9.0, 65 ± 12.0), and FIXaloop3 (1.1 ± 0.09, 5.0 ± 0.90). TheseKd values are in good agreement withKd(app) values (in 10−9m) determined from the activation of FX (in the presence and absence of FVIIIa, respectively): FIXaN (0.46 ± 0.05, 1.40 ± 0.14), FIXaWT (0.72 ± 0.08, 3.8 ± 0.08), FIXaloop1 (3.2 ± 0.72, 14.0 ± 1.60), FIXaloop2 (18.4 ± 1.60, 26.3 ± 3.40), and FIXaloop3 (0.7 ± 0.05, 3.0 ± 0.15). Moreover, the stoichiometry of binding (sites/platelet) showed an agreement with Vmax of FX activation and was reduced in those proteins that also showed a decreased platelet binding affinity. A peptide corresponding to the FIX EGF2 domain (Leu84-Val128) was an effective inhibitor of FIXa binding to platelets in both the presence (Ki = 0.7 × 10−6m) and the absence (Ki = 1.5 × 10−6m) of FVIIIa and FX. We conclude that residues 88–109 of the FIXa EGF2 domain mediate binding to platelets and assembly of the FX activating complex.