Platelet activation and aggregation provoked by Staphylococcus aureus secreted proteins

U. Binsker1, J. Wesche1, R. Palankar1, T. P. Kohler1, J. Prucha1, B. Bröker1, U. Mamat2, J. Pané-Farré1, F. Schmidt1, A. Greinacher1, S. Hammerschmidt1 (1Greifswald, Germany, 2Borstel, Germany)

Vascular Wall, Platelets and Acquired Problems
Date: 16.02.2017,
Time: 14:00 - 15:15

Objective: Staphylococcus aureus is an opportunistic human pathogen provoking a wide range of severe community-acquired and nosocomial infections amongst others infective endocarditis (IE) and disseminated intravascular coagulopathy (DIC). Both clinical manifestations constitute an uncontrolled activation of both platelets and coagulation cascade resulting in thrombocytopenia. S. aureus - platelet interactions occur either directly or indirectly via recruitment of serum components. Bacterial factors inducing platelet activation or aggregation are mostly secreted proteins possessing ECM binding activity.

Methods: Fifty-six recombinant secreted or surface-localized staphylococcal proteins were screened for their capacity to activate platelets, measured by the activation markers P-selectin and αIIbβ3 conformation, using whole blood, platelet-rich-plasma (PRP), and washed platelets in buffer from a constant set of donors. Micropattern protein array (MiPA) chips were functionalized with His6-tagged staphylococcal proteins to assess the interactions on a single cell level. Real-time calcium mobilization assay, single platelet imaging and P-selectin expression were used to detect platelet activation.

Results: This study confirmed the potential of the SERAM (secretable expanded repertoire adhesive molecules) protein Eap to induce platelet activation and aggregation. In addition, this study further identified the chemotaxis inhibitory protein CHIPS, the formyl peptide receptor-like 1 inhibitory protein FLIPr, all involved in immune evasion, as well as the major autolysin Atl as potent platelet activators. Furthermore, the domains of Atl and the extracellular adherence protein (Eap), responsible for platelet activation could be narrowed down. MiPa chips enabled to follow platelet activation by the candidates at single platelet level. Likewise, platelet aggregation activity of these candidates in whole blood could be determined.

Conclusion: Taken together, this study identified two members of the SERAM family (Eap and Atl) and two additionally secreted proteins of S. aureus as platelet activators and aggregators. These results emphasize the importance and diversity of S. aureus-platelet interactions. Interestingly, Atl and Eap were also shown to interact with the platelet-derived protein thrombospondin-1 (Kohler et al., 2014, Hussain et al., 2008).