The role of high molecular weight kininogen and prothrombin as cofactors in the binding of factor XI A3 domain to the platelet surface

We have reported that prothrombin (1 mu M) is able to replace high molecula r weight kininogen (45 nM) as a cofactor for the specific binding of factor XI to the platelet (Baglia, F. k, and Walsh, P. N. (1998) Biochemistry 37, 2271-2281). We have also determined that prothrombin fragment 2 binds to t he Apple 1 domain of factor XI at or near the site where high molecular wei ght kininogen binds. A region of 31 amino acids derived from high molecular weight kininogen (HK31-mer) can also bind to factor XI. (Tait, J. F., and Fujikawa, K. (1987) J. Biol Chem. 262, 11651-11656). We therefore investiga ted the role of prothrombin fragment 2 and HR31-mer as cofactors in the bin ding of factor XI to activated platelets. Our experiments demonstrated that prothrombin fragment 2 (1 mu M) or the HK31-mer (8 mu M) are able to repla ce high molecular weight kininogen (45 nM) or prothrombin (1 mu M) as cofac tors for the binding of factor XI to the platelet. To localize the platelet binding site on factor XI, we used mutant full-length recombinant factor X I molecules in which the platelet binding site in the Apple 3 domain was al tered by alanine scanning mutagenesis. The recombinant factor XI with alani ne substitutions at positions Ser(248), Arg(250), Lys(255), Leu(287), Phe(2 60), or GLn(263) were defective in their ability to bind to activated plate lets. Thus, the interaction of factor XI with platelets is mediated by the amino acid residues Ser(248), Arg(250), Lys(255), Leu(257), Phe(260), and G ln(263) within the Apple 3 domain.