Identification of a factor IX binding site on the third apple domain of activated factor XI.
Activated factor XI (factor XIa) participates in blood coagulation by activating factor IX. Previous work has demonstrated that a binding site for factor IX is present on the noncatalytic heavy chain of factor XIa (Sinha, D., Seaman, F. S., and Walsh, P. N. (1987) Biochemistry 26, 3768-3775). Recombinant factor XI proteins were expressed in which each of the four apple domains of the heavy chain (designated A1 through A4) were individually replaced with the corresponding domain from the homologous but functionally distinct protease prekallikrein (PK). To identify the site of factor IX binding, the chimeric proteins were activated with factor XIIa and tested for their capacity to activate factor IX in plasma coagulation and purified protein assays. The chimera with the substitution in the third apple domain (factor XI/PKA3) had <1% of the coagulant activity of wild type factor XIa in a plasma coagulation assay, whereas the chimeras with substitutions in A1, A2, and A4 demonstrated significant activity (68-140% of wild type activity). The Km for activation of factor IX by factor XIa/PKA3 (12. 7 microM) is more than 30-fold higher than the Km for activation by wild type factor XIa or the other factor XI/PK chimeras (0.11-0.37 microM). Two monoclonal antibodies (2A12 and 11AE) that recognize epitopes on the factor XI A3 domain were potent inhibitors of factor IX activation by factor XIa, whereas antibodies against the A2 (1A6) and A4 (3G4) domains were poor inhibitors. The data indicate that a binding site for factor IX is present on the third apple domain of factor XIa.
Division of Hematology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-6305, USA.
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D000911:Antibodies, Monoclonal / Q000494:pharmacology; D007611:Aprotinin / Q000494:pharmacology; D001665:Binding Sites; D005164:Factor IX / Q000378:metabolism*; D015956:Factor XIIa / Q000378:metabolism; D015945:Factor XIa / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D011288:Prekallikrein / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D000911:Antibodies, Monoclonal; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D005164:Factor IX; D011288:Prekallikrein; D007611:Aprotinin; D015945:Factor XIa; D015956:Factor XIIa