A binding site for heparin in the apple 3 domain of factor XI

Abstract

Since heparin potentiates activated factor XI (FXia) inhibition by protease nexin-2 by providing a template to which both proteins bind (Zhang, Y., scandura, J. M., van Nostrand, W. E., and Walsh, P. N. (1997) J. Biol. chem. 272, 26139-26144), we examind binding of factor XI 9FXi) and FXIa to heparin. FXIa binds to heparin (Kd ~0.7 x 10-9M) > 150-fold more tightly than FXI (Kd-1.1 x 10-7 M). To localize the heparin-binding site on FXI, rationally designed conformationally constrained synthetic peptides were used to compte with 125I-FXI binding to heparin. A peptide derived from the Apple 3 (A3) domain of FXI (Asn235-Arg266) inhibited FXI binding to heparin (K(d)~3.4x10-6 M), whereas peptides from the A1 domain (Phe56-Ser86), A2 domain (Ala134-Ala176), and A4 domain (Ala317-Gly350) had no such effect. The recombinant A3 domain (rA3, Ala181-Val271) inhibited FXI binding to heparin (K(i)~1.4 X 10-7M) indicating that all the information necessary for FXI binding to heparin is contained entirely within the A3 domain. The a3 domain also contains a paltelet-binding site (Asn235-Arg266), consisting of three surface-exposed loop structures, Pro229-Gln293, Thr741-Leu246, and Thr249-Phe260 (Baglia, F.A., Jameson, B.a., and Walsh, P.N. (1995) J. Biol. Chem. 270, 6734-6740). ony peptide Thr249-Phe260 (which contains a heparin binding consensus sequence, RIKKSKA) inhibits FXI binding to heparin (Ki = 2.1 x 10-7 m), whereas peptides Pro229-Gln233 and Thr241-Leu246 had no effect. Fine mapping of the heparin-binding site using prekallikrein analogue amino acid substitutions of the synthetic peptide Thr249-Phe260 and alanine scanning of the recombinant A3 indicated that the amino acids Lys252 and Lys253 are important for heparin binding. Thus, the sequence Thr253 are important for heparin binding. Thus, the sequence Thr249-Phe260 which contains most of the binding energy for FXI interaction with platelets also mediates the binding of FXI to heparin.