A sequential mechanism for exosite-mediated factor IX activation by factor XIa.

Geng Y, Verhamme IM, Messer A, Sun MF, Smith SB, Bajaj SP, Gailani D
J Biol Chem. 2012 287 (45): 38200-9

PMID: 22961984 · PMCID: PMC3488089 · 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 Arg(145) to form fIXα, and then after Arg(180) to form the protease fIXaβ. FIXα is released from fXIa, and must rebind for cleavage after Arg(180) to occur. Catalytic efficiency of cleavage after Arg(180) is 7-fold greater than for cleavage after Arg(145), 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 K(m) for fIX cleavage after Arg(145) and Arg(180) 25- and ≥ 90-fold, respectively, and markedly decreases k(cat) for cleavage after Arg(180). Similar results were obtained with the isolated fXIa catalytic domain, or fXIa in the absence of Ca(2+). Forms of fXIa lacking the A3 domain exhibit 15-fold lower catalytic efficiency for cleavage after Arg(180) than for cleavage after Arg(145), resulting in fIXα accumulation. Replacing the A2 domain does not affect fIX activation. The results demonstrate that fXIa activates fIX by an exosite- and Ca(2+)-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.

MeSH Terms (20)

Arginine Binding, Competitive Binding Sites Biocatalysis Calcium Catalytic Domain Electrophoresis, Polyacrylamide Gel Factor IX Factor IXa Factor XIa HEK293 Cells Humans Kinetics Mutation Oligopeptides Protein Multimerization Proteolysis Pyrrolidonecarboxylic Acid Recombinant Proteins Substrate Specificity

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