title: 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 examined binding of factor XI (FXI) and FXIa to heparin. FXIa binds to heparin (Kd approximately 0.7 x 10(-9) M) >150-fold more tightly than FXI (Kd approximately 1.1 x 10(-7) M). To localize the heparin-binding site on FXI, rationally designed conformationally constrained synthetic peptides were used to compete with 125I-FXI binding to heparin. A peptide derived from the Apple 3 (A3) domain of FXI (Asn235-Arg266) inhibited FXI binding to heparin (Kd approximately 3.4 x 10(-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 (Ki approximately 1.4 x 10(-7) M) indicating that all the information necessary for FXI binding to heparin is contained entirely within the A3 domain. The A3 domain also contains a platelet-binding site (Asn235-Arg266), consisting of three surface-exposed loop structures, Pro229-Gln233, Thr741-Leu246, and Thr249-Phe260 (Baglia, F. A., Jameson, B. A., and Walsh, P. N. (1995) J. Biol. Chem. 270, 6734-6740). Only 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 Thr249-Phe260 which contains most of the binding energy for FXI interaction with platelets also mediates the binding of FXI to heparin.
authors.affiliation: Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
authors.forename: D H
authors.initials: DH
authors.lastname: Ho
authors.forename: K
authors.initials: K
authors.lastname: Badellino
authors.forename: F A
authors.initials: FA
authors.lastname: Baglia
authors.forename: P N
authors.initials: PN
authors.lastname: Walsh
mesh_terms: D000595:Amino Acid Sequence; D016564:Amyloid beta-Protein Precursor; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism; D016384:Consensus Sequence; D005172:Factor XI / Q000378:metabolism*; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D058255:Protease Nexins; D017510:Protein Folding; D011956:Receptors, Cell Surface; D013329:Structure-Activity Relationship
chemical_list: C545487:APP protein, human; D016564:Amyloid beta-Protein Precursor; D002352:Carrier Proteins; D058255:Protease Nexins; D011956:Receptors, Cell Surface; D006493:Heparin; D005172:Factor XI