A binding site for thrombin in the apple 1 domain of factor XI

Previously we defined a binding site for high molecular weight kininogen (HK) in the A1 domain of factor XI (FXI). Since thrombin can activate FXI and HK inhibits the activation of FXI by thrombin, we have identified a thrombin binding site in FXI. Both the recombinant A1 domain (Glu1-Ser90) and a synthetic peptide (Phe56-Ser86) containing the HK binding site inhibited FXI activation by thrombin. Both a monoclonal antibody, 5F7, recognizing the A1 domain, and the rA1 domain were shown to be competitive inhibitors of thrombin-catalyzed FXI activation. The peptides Ala45-Arg54 and Val59-Arg70 acted synergistically to inhibit FXI activation by thrombin. Mutant rA1 domain constructs (Val64 --> Ala and Ile77 --> Ala), which do not inhibit FXI binding to HK, retain full capacity to inhibit FXI activation by thrombin. The peptide Ala45-Arg54 inhibited thrombin-catalyzed FXI activation, whereas it had no effect on FXI binding to HK. In contrast, the peptide Asn72-Leu83 (which inhibited FXI binding to HK) did not inhibit FXI activation by thrombin. Thus, a thrombin binding site exists in the A1 domain of FXI spanning residues Ala45-Arg70 that is contiguous with but separate and distinct from the HK binding site. These sites may regulate which ligand is bound to FXI and through which pathway FXI is activated.