The ability of Staphylococcus aureus to adhere to adsorbed fibrinogen and fibrin is believed to be an important step in the initiation of bioinaterial 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-terminus of the fibrinogen y chain. S. aureus Newman cells expressing ClfA adhered to microtitre wells coated with recombinant fibrinogen purified from BHK cells, but did not adhere to wells coated with a purified recombinant fibrinogen variant where the 4 C-terminal residues of the y chain were replaced by 20 unrelated residues. In addition, a synthetic peptide corresponding to the 17 C-terminal amino acids of the fibrinogen y chain effectively inhibited adherence of ClfA-expressing cells to fibrinogen. In western ligand blots, a recombinant truncated ClfA protein called Clf33 (residues 221 -550) recognized intact recombinant fibrinogen y chains, but failed to recognize recombinant fibrinogen y chains where the 4 C-terminal amino acids were altered by deletion or substitution. Previous studies have shown that the C-terminal domain of fibrinogen y chains contains a binding site for the integrin . We now show that Clf33 inhibits ADPinduced, fibrinogen-dependent platelet aggregation in a concentration-dependent manner and inhibits adhesion of platelets to immobilized fibrinogen under fluid shear stress, indicating that the binding sites for the platelet integrin and the staphylococcal adhesin overlap. The interaction between Clf33 and fibrinogen was further characterized using the BIAcore biosensor. When soluble Clf33 was allowed to bind to immobilized fibrinogen, a Kd of 0.51 t-0.19 pM was experimentally determined using equilibrium binding data. It was also shown that the synthetic C-terminal y-chain peptide effectively inhibited this interaction.

The clumping factor (ClfA) is a cell surface-associated protein of Staphylococcus aureus that promotes binding of fibrinogen or fibrin to the bacterial cell. Previous studies have shown that ClfA and the platelet integrin ␣ IIb ␤ 3 recognize the same domain at the extreme C terminus of the fibrinogen ␥-chain. ␣ IIb ␤ 3 interaction with this domain is known to occur in close proximity to a Ca 2؉ -binding EF-hand structure in the ␣-subunit. Analysis of the primary structure of ClfA indicated the presence of a potential Ca Staphylococcus aureus causes a wide range of opportunistic infections that range from superficial skin infections to lifethreatening diseases including endocarditis, pneumonia, and septicemia. Adherence of bacteria to host matrix components that is mediated by bacterial surface adhesins is the initial critical event in the pathogenesis of most infections. The extracellular matrix (ECM) 1 contains numerous glycoproteins and proteoglycans assembled into insoluble matrices that serve as substrata for the adhesion and migration of tissue cells. These processes involve integrins, a family of heterodimeric (␣␤) cellsurface receptors that recognize specific ECM proteins. It has become increasingly evident that bacteria, including S. aureus, also utilize the ECM as substrata for their adhesion by way of a family of adhesins called MSCRAMM (microbial surface components recognizing adhesive matrix molecules) (1) that specifically recognize host matrix components. One important component of the ECM, also occurring in soluble form in blood plasma, is fibrinogen, a 340-kDa hexamer composed of 2␣-, 2␤-, and 2␥-chains linked by disulfide bonds. This protein is recognized by several integrins including the platelet integrin ␣ IIb ␤ 3 . Activation of platelets and integrin ␣ IIb ␤ 3 results in fibrinogen-dependent aggregation in vitro and the formation of platelet-fibrin thrombi in vivo.

This study demonstrates the development and use of a promising new method that uses the short-lived radioisotope Ta-178 and MPC for noninvasive quantification of murine ventricular function, that produces accurate and highly reproducible results, and that can be applied in multiple serial studies.