A Binding Site for the Kringle II Domain of Prothrombin in the Apple 1 Domain of Factor XI

• Published in: The Journal of biological chemistry Vol. 275; no. 41; pp. 31954 - 31962
• Main Authors: Baglia, Frank A., Badellino, Karen O., Ho, David H., Dasari, V. Rao, Walsh, Peter N.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 13-10-2000; American Society for Biochemistry and Molecular Biology
• Subjects: Binding Sites; Binding, Competitive; Factor XI - chemistry; Factor XI - genetics; Factor XI - metabolism; Humans; Iodine Radioisotopes; Kinetics; Kininogen, High-Molecular-Weight - metabolism; Kringles; Peptide Fragments - metabolism; Peptide Fragments - pharmacology; Protein Binding - drug effects; Prothrombin - chemistry; Prothrombin - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Surface Plasmon Resonance; Thrombin - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M005465200

Previously we defined binding sites for high molecular weight kininogen (HK) and thrombin in the Apple 1 (A1) domain of factor XI (FXI). Since prothrombin (and Ca2+) can bind FXI and can substitute for HK (and Zn2+) as a cofactor for FXI binding to platelets, we have attempted to identify a prothrombin-binding site in FXI. The recombinant A1 domain (rA1, Glu1–Ser90) inhibited the saturable, specific and reversible binding of prothrombin to FXI, whereas neither the rA2 domain (Ser90–Ala181), rA3 domain (Ala181–Val271), nor rA4 domain (Phe272–Glu361) inhibited prothrombin binding to FXI. Kinetic binding studies using surface plasmon resonance showed binding of FXI (Kd ∼71 nm) and the rA1 domain (Kd ∼239 nm) but not rA2, rA3, or rA4 to immobilized prothrombin. Reciprocal binding studies revealed that synthetic peptides (encompassing residues Ala45–Ser86) containing both HK- and thrombin-binding sites, inhibit 125I-rA1 (Glu1–Ser90) binding to prothrombin,125I-prothrombin binding to FXI, and125I-prothrombin fragment 2 (Ser156–Arg271) binding to FXI. However, homologous prekallikrein-derived peptides (encompassing Pro45–Gly86) did not inhibit FXI rA1 binding to prothrombin. The peptides Ala45–Arg54, Phe56–Val71, and Asp72–Ser86, derived from sequences of the A1 domain of FXI, acted synergistically to inhibit 125I-rA1 binding to prothrombin. Mutant rA1 peptides (V64A and I77A), which did not inhibit FXI binding to HK, retained full capacity to inhibit rA1 domain binding to prothrombin, and mutant rA1 peptides Ala45–Ala54 (D51A) and Val59–Arg70 (E66A), which did not inhibit FXI binding to thrombin, retained full capacity to inhibit rA1 domain binding to prothrombin. Thus, these experiments demonstrate that a prothrombin binding site exists in the A1 domain of FXI spanning residues Ala45–Ser86 that is contiguous with but separate and distinct from the HK- and thrombin-binding sites and that this interaction occurs through the kringle II domain of prothrombin.