Lipopolysaccharide potentiates platelet responses via toll-like receptor 4-stimulated Akt-Erk-PLA2 signalling

Abstract

Lipopolysaccharide (LPS) from the cell envelope of Gram-negative bacteria is a principal cause of the symptoms of sepsis. LPS has been reported to modulate the function of platelets although the underlying mechanisms of LPS action in these cells remain unclear. Platelets express the Toll-like receptor 4 (TLR4) which serves as a receptor for LPS, although the potential role of TLR4 and associated cell signalling in controlling platelet responses to LPS has not been extensively explored. In this study, we therefore investigated the actions of LPS prepared from different strains of Escherichia coli on platelet function, the underlying signalling mechanisms, and the potential role of TLR4 in orchestrating these. We report that LPS increased the aggregation of washed platelets stimulated by thromboxane (U46619) or GPVI collagen receptor agonists, effects that were prevented by a TLR4 antagonist. Associated with this, LPS enhanced fibrinogen binding, P-selectin exposure and reactive oxygen species (ROS) release. Increase of ROS was found to be important for the actions of LPS on platelets, since these were inhibited in the presence of superoxide dismutase or catalase. The effects of LPS were associated with phosphorylation of Akt, ERK1/2 and PLA2 in stimulated platelets, and inhibitors of PI3-kinase, Akt and ERK1/2 reduced significantly LPS enhanced platelet function and associated ROS production. Furthermore, inhibition of platelet cyclooxygenase or the thromboxane receptor, revealed an important role for thromboxane A2. We therefore conclude that LPS increases human platelet activation through a TLR4-PI3K-Akt-ERK1/2-PLA2 -dependent pathway that is dependent on ROS and TXA2 formation.

Activated αIIbβ3on platelets mediates flow-dependent NETosis via SLC44A2

“…However, LPSstimulated platelets, which bind of fibrinogen (i.e. α IIb β 3 is activated) and also robustly activate NETosis independent of P-selectin (Clark et al, 2007;Looney et al, 2009;Lopes Pires et al, 2017;McDonald et al, 2012).…”

Platelet TLR4 at the crossroads of thrombosis and the innate immune response

“…Whereas some studies have shown that TLR4 activation by LPS induces platelet activation, most observed that this is not a direct effect but rather potentiates platelet responses elicited by classical agonists including aggregation, ATP release, P‐selectin expression, and the formation of mixed aggregates between platelets and neutrophils . It is worth mentioning that, among these studies, only the experiments by Nocella et al .…”

“…Because SNARE complex formation is involved in platelet secretion, it could be conceivable that the NF‐κB pathway controls platelet secretion mediated by LPS, in particular when acting in combination with subthreshold thrombin concentrations. The observation that platelet stimulation with LPS plus thrombin or collagen triggers PI3K/AKT activation and that AKT phosphorylates IKK‐β renders IKK‐β a relevant potential role as a mediator of LPS‐induced platelet secretion. Moreover, this hypothesis appears to be more comprehensive as other TLR4 ligands such as cellular Fn‐EDA + potentiates thrombin‐induced IKK/NF‐κB activation in WT but not in TLR4‐/‐ platelets …”

“…9 Whereas some studies have shown that TLR4 activation by LPS induces platelet activation, most observed that this is not a direct effect but rather potentiates platelet responses elicited by classical agonists including aggregation, ATP release, P-selectin expression, and the formation of mixed aggregates between platelets and neutrophils. [10][11][12][13] It is worth mentioning that, among these studies, only the experiments by Nocella et al 13 were performed using LPS in the picogram range; all other studies required 10 times more LPS to achieve similar results. The use of such a high LPS concentration raises concerns as it is considered that these LPS levels are not representative of the concentrations observed in the clinical setting (approx.…”

Circulating MicroRNA Levels Indicate Platelet and Leukocyte Activation in Endotoxemia Despite Platelet P2Y12 Inhibition

“…Facilitated by sCD14 derived from plasma [39], endotoxin binds to this receptor and initiates a signaling cascade that involves the adaptor protein MyD88, resulting in activation of the nitric oxide and cyclic guanosine monophosphatedependent protein kinase pathway. This is sufficient to induce secretion of dense and α-granules but does not induce platelet aggregation, as determined by ex vivo experiments [36][37][38]. Instead, endotoxin sensitizes and potentiates the aggregation response to subthreshold concentrations of…”