A Sequential Mechanism for Exosite-mediated Factor IX Activation by Factor XIa

• Published in: The Journal of biological chemistry 2012-11, Vol.287 (45), p.38200-38209
• Main Authors: Geng, Yipeng, Verhamme, Ingrid M., Messer, Amanda, Sun, Mao-fu, Smith, Stephen B., Bajaj, S.Paul, Gailani, David
• Format: Article
• Language: English
• Subjects: Arginine - metabolism; Binding Sites - genetics; Binding, Competitive; Biocatalysis - drug effects; Calcium - metabolism; Calcium - pharmacology; Catalytic Domain; Coagulation Factors; Electrophoresis, Polyacrylamide Gel; Enzyme Catalysis; Enzyme Kinetics; Enzyme Mechanisms; Enzyme Structure; Enzymology; Exosite; Factor IX; Factor IX - metabolism; Factor IXa - metabolism; Factor XIa; Factor XIa - chemistry; Factor XIa - genetics; Factor XIa - metabolism; HEK293 Cells; Humans; Kinetics; Mutation; Oligopeptides - metabolism; Protein Multimerization; Proteolysis; Pyrrolidonecarboxylic Acid - analogs & derivatives; Pyrrolidonecarboxylic Acid - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Substrate Specificity
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M112.376343

During blood coagulation, the protease factor XIa (fXIa) activates factor IX (fIX). We describe a new mechanism for this process. FIX is cleaved initially after Arg145 to form fIXα, and then after Arg180 to form the protease fIXaβ. FIXα is released from fXIa, and must rebind for cleavage after Arg180 to occur. Catalytic efficiency of cleavage after Arg180 is 7-fold greater than for cleavage after Arg145, limiting fIXα accumulation. FXIa contains four apple domains (A1–A4) and a catalytic domain. Exosite(s) on fXIa are required for fIX binding, however, there is lack of consensus on their location(s), with sites on the A2, A3, and catalytic domains described. Replacing the A3 domain with the prekallikrein A3 domain increases Km for fIX cleavage after Arg145 and Arg180 25- and ≥90-fold, respectively, and markedly decreases kcat for cleavage after Arg180. Similar results were obtained with the isolated fXIa catalytic domain, or fXIa in the absence of Ca2+. Forms of fXIa lacking the A3 domain exhibit 15-fold lower catalytic efficiency for cleavage after Arg180 than for cleavage after Arg145, resulting in fIXα accumulation. Replacing the A2 domain does not affect fIX activation. The results demonstrate that fXIa activates fIX by an exosite- and Ca2+-mediated release-rebind mechanism in which efficiency of the second cleavage is enhanced by conformational changes resulting from the first cleavage. Initial binding of fIX and fIXα requires an exosite on the fXIa A3 domain, but not the A2 or catalytic domain. Background: Factor XIa proteolytically activates factor IX. Results: XIa cleaves IX after Arg145, forming IXα, and then after Arg180, forming IXaβ. Both reactions require substrate binding to the XIa A3 domain. Conclusion: XIa activates IX by an exosite-mediated release-rebind mechanism. Efficiency of the second cleavage is enhanced by changes resulting from the first cleavage. Significance: The data support a new model for IX activation by XIa.