CHARACTERIZATION OF THE INTERACTION BETWEEN THE STAPHYLOCOCCUS-AUREUSCLUMPING FACTOR (CLFA) AND FIBRINOGEN

The ability of Staphylococcus aureus to adhere to adsorbed fibrinogen and fibrin is believed to be an important step in the initiation of bi omaterial and wound-associated infections. In this study, we show that the binding site in fibrinogen for the recently identified S. aureus fibrinogen-binding protein clumping factor (ClfA) is within the C-term inus of the fibrinogen gamma chain. S. aureus Newman cells expressing ClfA adhered to microtitre wells coated with recombinant fibrinogen pu rified from BHK cells, but did not adhere to wells coated with a purif ied recombinant fibrinogen variant where the 4 C-terminal residues of the gamma chain were replaced by 20 unrelated residues. In addition, a synthetic peptide corresponding to the 17 C-terminal amino acids of t he fibrinogen gamma chain effectively inhibited adherence of ClfA-expr essing cells to fibrinogen. In western ligand blots, a recombinant tru ncated ClfA protein called Clf33 (residues 221-550) recognized intact recombinant fibrinogen gamma chains, but failed to recognize recombina nt fibrinogen gamma chains when the 4 C-terminal amino acids were alte red by deletion or substitution. Previous studies have shown that the C-terminal domain of fibrinogen gamma chains contains a binding site f or the integrin alpha(IIb)beta(3) (glycoprotein gpIIb/IIIa) receptor o n platelets [Kloczewiak, M., Timmons, S., Bednarek, M. A., Sakon, M. & Mawiger. J. (1989) Biochemistry 28, 2915-1919; Farrell, D. H., Thiaga rajan, P., Chung, D. W. & Davie, E. W. (1992) Proc. Natl Acad Sci. USA 89, 10729-10732; Hettasch, J. M., Bolyard, M. G. & Lord, S. T. (1992) Thromb. Haemostasis 68, 701-706]. We now show that Clf33 inhibits ADP -induced, fibrinogen-dependent platelet aggregation in a concentration -dependent manner and inhibits adhesion of platelets to immobilized fi brinogen under fluid shear stress, indicating that the binding sites f or the platelet integrin and the staphylococcal adhesin overlap. The i nteraction between Clf33 and fibrinogen was further characterized usin g the BIAcore biosensor. When soluble Clf33 was allowed to bind to imm obilized fibrinogen, a K-d of 0.51+/-0.19 mu M was experimentally dete rmined using equilibrium binding data. It was also shown that the synt hetic C-terminal gamma-chain peptide effectively inhibited this intera ction.