Date of Award
PhD (Doctor of Philosophy)
Dr Steven W. Kerrigan
Sepsis, Platelet Activation
Bloodstream infection caused by bacteria can develop into serious life threatening disease such as sepsis and infective endocarditis (IE). Platelet activation is characteristic of these sequelae, manifesting as thrombocytopenia in sepsis and the formation of platelet rich vegetations on cardiac valves in IE. The oral cavity and respiratory tract are a reservoir for bloodstream infections, being colonised by mitis group streptococci including Streptococcus pneumoniae and its close relative Streptococcus oralis. In this thesis, we describe platelet interactions with these bacteria and the molecular mechanisms leading to platelet activation.
Previous studies demonstrate that S. pneumoniae induces serotonin release from platelets and, aidied by fibrin and thrombospondin, forms pneumococcal-platelet complexes. However, research into platelet activation by serotypes associated with invasive pneumococcal disease is lacking. In this thesis, we demonstrate strain specific platelet aggregation induced by S. pneumoniae. Using platelet aggregometry coupled with platelet receptor specific antibodies, pharmacological inhibitors and cultured human embryonic kidney cells transfected with Toll like receptors (TLRs), we demonstrate that S. pneumoniae binds directly to TLR2 stimulating FcyRIIA dependent platelet aggregation. TLR2 stimulation leads to dense granule secretion and P2Yi2 dependent platelet aggregation, independent of thromboxane production. Additionally, S. pneumoniae can activate the small GTPase Rap1 upstream of allb|33. We conclude that S. pneumoniae induces platelet activation via a TLR2/PI3K/Rap1 pathway - with contributions from FcyRIIA. Furthermore this study highlights TLR2 as a potential target to prevent inappropriate platelet activation leading to thrombocytopenia in sepsis.
S. oralis is an important pathogen in IE and can cause sepsis in immunocompromised persons. Previous studies, performed under static conditions, suggest S. oralis does not interact directly with platelets. Using a physiological approach of whole blood perfusion over immobilised bacteria, coupled with real-time epi-fluorescent microscopy, this study demonstrates that S. oralis can support platelet recruitment and adhesion from blood flowing at venous (50-200 s'1) and arterial (800 s'1) shear rates. By removing plasma proteins and using monoclonal antibodies and blood from a Bernard Soulier syndrome patient, we show that immobilised S. oralis can sequester platelets from flowing blood via a direct interaction with the platelet GPIb-IX-V complex, specifically GPIba. In doing so, S. oralis, infecting the cardiac valves, can trigger platelet adhesion: an important step in the development of a septic thrombus or vegetation in IE.
We found that platelet adhesion to S. oralis under physiological shear conditions stimulated microaggregate formation signifying platelet activation. Prior to this study, human platelet activation by S. oralis had not been demonstrated. Using platelet aggregometry, we showed that S. oralis induces strain-dependent platelet aggregation requiring IgG. Consequently, co-immunoprecipitation of the low affinity IgG receptor, FcyRIIA, with Syk, showed tyrosine phosphorylation of FcyRIIA. Ligation of FcyRIIA - by immunecomplexed S. oralis - leads to IgG-FcyRIIA dependent dense granule secretion and ADP dependent platelet aggregation. Total protein pull-down of Rap1-GTP showed P2YI2 dependent activation by IgG-bound S. oralis. We conclude that, following GPIba dependent platelet adhesion to S. oralis, S. oralis immune complexes stimulate an FcyRIIA/P2Y12/Rap1 pathway leading to platelet activation and platelet aggregation. This may facilitate growth of a septic thrombus in IE and contribute to disseminated intravascular coagulation in neutropenic septic patients.
This thesis proposes models of platelet activation for both S. pneumoniae and S. oralis and reveals targets for the development of therapies to prevent pathological platelet activation in bloodstream infection. Significantly, it adds to mounting evidence that signalling through FcyRIIA is a conserved mechanism of human platelet activation by streptococcal species and that FcyRIIA may be a universal actor in the general mechanism of bacterial induced platelet activation
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Tilley, DO. Pathways to human platelet activation : convergence of streptococcus pneumoniae and streptococcus oralis at a universal actor, Fc gamma RIIA. [PhD Thesis]. Dublin: Royal College of Surgeons in Ireland; 2013.