CHARACTERIZATION OF A HEPARIN-BINDING SITE ON THE HEAVY-CHAIN OF FACTOR-XI

The glycosaminoglycan heparin enhances several reactions involving coa gulation factor XI (FXI) including activation of FXI by factor XIIa, t hrombin, and autoactivation; and inactivation of activated FXI (FXIa) by serine protease inhibitors. We examined the effect of heparin on in hibition of FXIa by the inhibitors C1-inhibitor (C1-INH) and antithrom bin LII (ATIII), Second order rate constants for inhibition in the abs ence of heparin were 1.57 x 10(3) and 0.91 x 10(3) M-1 s(-1) for C1-IN H and ATIII, respectively. Therapeutic heparin concentrations (0.1-1.0 units/ml) enhanced inhibition by ATIII 20-55-fold compared with 0.1-7 .0-fold for C1-INH. For both inhibitors, the effect of heparin over a wide range of concentrations (10(-1) to 10(5) units/ml) produced bell- shaped curves, demonstrating that inhibition occurs by a template mech anism requiring both inhibitor and protease to bind to heparin, This i mplies that FXI/XIa contains structural elements that interact with he parin, Human FXI contains a sequence of amino acids (R-250-I-K-K-S-K) in the apple 3 domain of the heavy chain that binds heparin (Ho, D., B adellino, K., Baglia, F., and Walsh, P. (1998) J. Biol. Chem. 273, 163 82-16390), To determine the importance of this sequence to heparin-med iated reactions, recombinant FXI molecules with alanine substitutions for basic amino acids were expressed in 293 fibroblasts, and tested in heparin-dependent assays. Inhibition of FXIa by ATIII in the presence of heparin was decreased 4-fold by alanine substitution at Lys(253) ( A253), with smaller effects noted for mutants A255 and A252, FXI under goes autoactivation to FXIa in the presence of heparin, The rate of au toactivation was decreased substantially for A253 with modest decrease s for A255 and A252, Substituting all four charged residues in the seq uence resulted in a profound decrease in autoactivation, significantly greater than for any single substitution. Relative affinity for hepar in was tested by determining the concentration of NaCl required to elu te FXIa from heparin-Sepharose. Wild type FXIa eluted from the column at 320 mM NaCl, whereas FXIa with multiple substitutions (A252-254 or A250-255) eluted at 230 mM NaCl, AU proteins with single substitutions in charged amino acids eluted at intermediate NaCl concentrations. Th e data indicate that FXI/XIa must bind to heparin for optimal inhibiti on by ATIII and for autoactivation. Lys(253) is the most important ami no acid involved in binding, and Lys(255) and Lys(252) also have roles in interactions with heparin.