Previously we defined binding sites for high molecular weight kininogen (HK) and thrombin in the Apple 1 (Al) domain of factor XI (FM). 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 Al domain (rAl, Glu(1)-Ser(90)) inhibited the saturable, specific and reversible binding of prothrombin to FXI, whereas neither the rA2 domain (Ser(90)-Ala(181)), rA3 domain (Ala(181)-Val(271)), nor rA4 domain (Phe(272)-Glu(361)) inhibited prothrombin binding to FXI. Kinetic binding studies using surface plasmon resonance showed binding of FXI (K-d similar to 71 nn) and the rAl domain (K-d similar to 239 nM) but not rA2, rA3, or rA4 to immobilized prothrombin. Reciprocal binding studies revealed that synthetic peptides (encompassing residues Ala(45)-Ser(86)) containing both HK- and thrombin-binding sites, inhibit I-125-rAl (Glu(1)-Ser(90)) binding to prothrombin, I-125-prothrombin binding to FXI, and 125I-prothrombin fragment 2 (Ser(156)-Arg(271)) binding to FXI. However, homologous prekallikrein-derived peptides (encompassing Pro(45)-Gly(86)) did not inhibit FXI rAl binding to prothrombin. The peptides Ala(45)-Arg(54), Phe(56)-Val(71), and Asp(72)-Ser(86), derived from sequences of the Al domain of FXI, acted synergistically to inhibit I-125-rAl binding to prothrombin. Mutant rAl peptides (V64A and I77A), which did not inhibit FXI binding to III, retained full capacity to inhibit rAl domain binding to prothrombin, and mutant rAl peptides Ala(45)-Ala(54) (D51A) and Val(59)-Arg(70) (E66A), which did not inhibit FXI binding to thrombin, retained full capacity to inhibit rAl domain binding to prothrombin. Thus, these experiments demonstrate that a prothrombin binding site exists in the Al domain of FXI spanning residues Ala(45)-Ser(86) 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.
