A binding site for the kringle II domain of prothrombin in the Apple 1 domain of factor XI

Previously we defined binding sites for high molecular weight kininogen (HK ) and thrombin in the Apple 1 (Al) domain of factor XI (FM). Since prothrom bin (and Ca2+) can bind FXI and can substitute for HK (and Zn2+) as a cofac tor for FXI binding to platelets, we have attempted to identify a prothromb in-binding site in FXI. The recombinant Al domain (rAl, Glu(1)-Ser(90)) inh ibited the saturable, specific and reversible binding of prothrombin to FXI , whereas neither the rA2 domain (Ser(90)-Ala(181)), rA3 domain (Ala(181)-V al(271)), nor rA4 domain (Phe(272)-Glu(361)) inhibited prothrombin binding to FXI. Kinetic binding studies using surface plasmon resonance showed bind ing 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 s tudies 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, ho mologous prekallikrein-derived peptides (encompassing Pro(45)-Gly(86)) did not inhibit FXI rAl binding to prothrombin. The peptides Ala(45)-Arg(54), P he(56)-Val(71), and Asp(72)-Ser(86), derived from sequences of the Al domai n 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 in hibit rAl domain binding to prothrombin. Thus, these experiments demonstrat e that a prothrombin binding site exists in the Al domain of FXI spanning r esidues Ala(45)-Ser(86) that is contiguous with but separate and distinct f rom the HK- and thrombin-binding sites and that this interaction occurs thr ough the kringle II domain of prothrombin.