Infective endocarditis (IE) is characterized by thrombus formation on a cardiac valve that can embolize to a distant site. Staphylococcus aureus is the most frequent causative organism of IE. Previously we showed that fibrinogen binding to the S. aureus surface protein Clumping Factor A (ClfA) plays an essential role in mediating platelet aggregation. In this study we investigate the role of ClfA in mediating thrombus formation under pathological shear conditions. Bacteria were coated onto flow chamber glass cover slips overnight at room temperature. Platelets in whole blood were stained with a lipophilic dye. Experiments were recorded in real time and visualized using fluorescence and bright-field microscopy. Platelets failed to interact with immobilized S. aureus at a shear rate of <500s−1 but rapidly formed a thrombus at shear rates greater than 800s−1. Deletion of the ClfA gene from the parent S. aureus strain (Newman) resulted in no thrombus formation at any shear rate. Platelets failed to interact with the Gram positive bacterium Lactococcus lactis at all shear rates, suggesting its suitability as a surrogate host for expressing ClfA. L. lactis over-expressing ClfA supported rapid thrombus formation with a similar profile to S. aureus. Fibrinogen binding to ClfA was found to be essential for thrombus formation although fibrinogen-coated surfaces only produced single-platelet adhesion under all shear conditions. Tirofiban and aspirin inhibited thrombus formation. Specific antibody to ClfA was also required as blood depleted of specific anti-ClfA antibody and an antibody against FcγRIIa (IV.3) inhibited thrombus formation. Our results demonstrate for the first time that, platelets interact with immobilized S. aureus under high shear >800s−1 (arterial conditions) but not low shear conditions <500s−1 (venous conditions). We identify S. aureus surface protein ClfA, fibrinogen and specific IgG are essential for thrombus formation. The rapid nature of the thrombus formation may explain why S. aureus is the major cause of IE.

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