Binding of high-molecular-mass kininogen to the Apple 1 domain of factor XI is mediated in part by Val64 and Ile77

Abstract

We have previously demonstrated the presence of a binding site for high-molecular-mass kininogen (HK), spanning residues Val59-Lys83, in the first Apple (A1) domain in the heavy-chain region of factor XI. We have now prepared conformationally constrained synthetic peptides and recombinant A1 domain (rA1) constructs to identify the specific amino acid residues that constitute the HK-binding site. Expression of the A1 domain (Glu1-Ser90) was achieved in a bacterial expression system following PCR amplification of the A1 domain from factor XI cDNA and ligation into an expression plasmid. The rA1 inhibited factor XI binding to HK [Ki approximately (2-3) x 10(-7) M] in a manner indistinguishable from purified factor XI, indicating that all the information necessary for binding HK is contained within the A1 domain. To identify specific amino acid residues involved in binding HK, conformationally constrained peptides were synthesized containing conservative amino acid substitutions at residues suspected to contain side chains involved in binding, including Val64-->Ala, Glu66-->Ala, Arg73-->Ala and Ile77-->Ala. Because normal results were obtained with all peptides with the exception of Val64-->Ala and Ile77-->Ala, which failed to compete normally with factor XI for binding to HK, we prepared two mutant rA1 domains (Val64-->Ala and Ile77-->Ala) by PCR-based site-directed mutagenesis, both of which exhibited diminished capacity to inhibit factor XI binding to HK. Competition studies with prekallikrein (PK) and a PK-dependent synthetic peptide suggested that PK and factor XI have a common surface in the A1 domain for binding HK of which Val64 is a part. We conclude that the binding of factor XI to HK is mediated at least in part by Val64 and Ile77 in the A1 domain of factor XI.

A Binding Site for Thrombin in the Apple 1 Domain of Factor XI

“…Therefore, the binding sites for HK and thrombin in the A1 domain, although contiguous, are apparently separate and distinct. Another experiment that supports this conclusion is that after reduction and alkylation, the rA1 domain (Glu 1 -Ser 90 ) retains the capacity to inhibit FXI binding to HK (IC 50 ϳ10 Ϫ6 M) (19,20,37), whereas it is unable to inhibit thrombincatalyzed FXI activation (data not shown).…”

“…Therefore, we examined the effects of mutations of these two residues on the capacity of the rA1 domain to inhibit thrombin-catalyzed FXI activation. We found that mutant rA1 domain constructs (Val 64 3 Ala and Ile 77 3 Ala), which have lost the capacity to inhibit FXI binding to HK (37), retain the full capacity of the rA1 domain (Glu ) to inhibit thrombin-catalyzed FXI activation (Fig. 2).…”

“…Previously we have identified specific amino acid residues within the A1 domain involved in binding HK (19,20,37). Utilizing mutational analysis we have determined that the binding of FXI to HK is mediated at least in part by Val 64 and Ile 77 in the A1 domain of FXI (37).…”

“…Utilizing mutational analysis we have determined that the binding of FXI to HK is mediated at least in part by Val 64 and Ile 77 in the A1 domain of FXI (37). Therefore, we examined the effects of mutations of these two residues on the capacity of the rA1 domain to inhibit thrombin-catalyzed FXI activation.…”

Thrombin Activation of Factor XI on Activated Platelets Requires the Interaction of Factor XI and Platelet Glycoprotein Ibα with Thrombin Anion-binding Exosites I and II, Respectively

“…In a current model of FXI activation, the circulating FXI dimer is complexed with either HMWK (17)(18)(19) or prothrombin (18,20), both of which bind to the A1 domain of FXI (21).…”