A BINDING-SITE FOR HEPARIN IN THE APPLE-3 DOMAIN OF FACTOR-XI

Since heparin potentiates activated factor XI (FXIa) inhibition by pro tease nexin-2 by providing a template to which both proteins bind (Zha ng, Y., Scandura, J. M., Van Nostrand, W. E., and Walsh, P. N. (1997) J. Biol. Chem. 272, 26139-26144), we examined binding of factor XI (FX I) and FXIa to heparin. FXIa binds to heparin (K-d -0.7 x 10(-9) M) > 150-fold more tightly than FXI (K-d similar to 1.1 x 10(-7) M). To loc alize the heparin-binding site on FXI, rationally designed conformatio nally constrained synthetic peptides were used to compete with I-125-F XI binding to heparin. A peptide derived from the Apple 3 (A3) domain of FXI (Asn(235)-Arg(266)) inhibited FXI binding to heparin (K-d simil ar to 3.4 x 10(-6) M), whereas peptides from the A1 domain (Phe(56)-Se r(86)), A2 domain (Ala(134)-Ala(176)), and A4 domain (Ala(317)-Gly(350 )) had no such effect. The recombinant A3 domain (rA3, Ala(181)-Val(27 1)) inhibited FXI binding to heparin (K-i similar to 1.4 x 10(-7) M) i ndicating that all the information necessary for FXI binding to hepari n is contained entirely within the A3 domain. The A3 domain also conta ins a platelet-binding site (Asn(235)-Arg(266)), consisting of three s urface-exposed loop structures, Pro(229)-Gln(233), Thr(741)-Leu(246), and Thr(249)-Phe(260) (Baglia, F. A., Jameson, B. A., and Walsh, P. N. (1995) J. Biol. Chem. 270, 6734-6740). Only peptide Thr(249-)Phe(260) (which contains a heparin binding consensus sequence, RIKKSKA) inhibi ts FXI binding to heparin (K-i = 2.1 x 10(-7) M), whereas peptides Pro (229)-Gln(233) and Thr(241)-Leu(246) had no effect. Fine mapping of th e heparin-binding site using prekallikrein analogue amino acid substit utions of the synthetic peptide Thr(249)-Phe(260) and alanine scanning of the recombinant A3 indicated that the amino acids Lys(252) and Lys (253) are important for heparin binding. Thus, the sequence Thr(249)-P he(260) which contains most of the binding energy for FXI interaction with platelets also mediates the binding of FXI to heparin.