Abstract/Summary

Cardiovascular disease is a group of inflammatory diseases that is responsible for the plurality of deaths in the world. In ischaemic diseases, aberrant platelet activation leads to thrombus formation and occlusion of key arteries. Furthermore, platelets are critically involved in sepsis, where thrombocytopaenia is correlated with worse patient outcomes. Therefore, immune receptors, such as Toll-like receptor 4 (TLR4), have been identified as potential targets for reducing platelet activation by decoupling immune functions from haemostasis. Multiple experimental techniques were used to determine the impact of ultrapure lipopolysaccharide (LPS) chemotypes on platelet and megakaryocyte function as LPS is a specific ligand for TLR4. To determine whether LPS modulates platelet activity, LPS and platelets were co-incubated under different conditions. We determined that ultrapure LPS derived from various species of Gram-negative bacteria cannot significantly modulate platelet activity, as measured by aggregation, fibrinogen binding, and P-selectin exposure, except under specific conditions. This is potentially due to activation dependent cell�surface expression of TLR4. Subsequently, to investigate whether LPS induces inflammatory signalling in megakaryocytes, a novel megakaryocyte reporter cell line, Meg-01R, was developed and used to determine that ultrapure LPS is not sufficient on its own to modulate megakaryocyte function. Characterisation of this cell line suggests that MyD88-dependent signalling pathways are active in Meg-01R cells but TLR4 is not present in sufficient quantities at the cell surface. As LL37 directly binds to LPS and is also a strong platelet agonist, we investigated the impact of LPS on LL37- induced platelet activation. Here, we discovered a TLR4-independent cell-independent formation of LL37-LPS micelles that prevents LL37-induced platelet activation. Based on these results, LPS and TLR4 are not sufficient, in vitro, to decouple the immune function of platelets from haemostasis or induce changes in megakaryocyte function however it may still play an important role in conjunction with other immune receptors.