AbstractPlatelets are small circulating blood cells that play essential roles in the maintenance of haemostasis via blood clotting. However, they also play critical roles in the regulation of innate immune responses. Inflammatory receptors, specifically Toll-like receptor (TLR)-4, have been reported to modify platelet reactivity. A plethora of studies have reported controversial functions of TLR4 in the modulation of platelet function using various chemotypes and preparations of its ligand, lipopolysaccharide (LPS). The method of preparation of LPS may explain these discrepancies however this is not fully understood. Hence, to determine the impact of LPS on platelet activation, we used ultrapure preparations of LPS from Escherichia coli (LPS_EC), Salmonella minnesota (LPS_SM), and Rhodobacter sphaeroides (LPS_RS) and examined their actions under diverse experimental conditions in human platelets. LPS_EC did not affect platelet activation markers such as inside-out signalling to integrin α_IIbβ₃ or P-selectin exposure upon agonist-induced activation in platelet-rich plasma or whole blood whereas LPS_SM and LPS_RS inhibited platelet activation under specific conditions at supraphysiological concentrations. Overall, our data demonstrate that platelet activation is not largely influenced by any of the ultrapure LPS chemotypes used in this study on their own except under certain conditions.