Regulation of the hemostatic and inflammatory responses triggered by ligands of Toll like receptor 2 and 4 is another non-genomic role of nuclear factor-κB in platelets

RIVADENEYRA, L; CARESTIA, A; FONDEVILA, C; NEGROTTO, S; SCHATTNER, M

Amsterdam

Congreso; XXIV Congress of International Society on Thrombosis and Haemostasis; 2013

Background: Platelets express toll like receptors (TLRs), a family of proteins that recognize molecular components of pathogens. Although several studies reported platelet activation mediated by activation of TLR2 and 4, the data are controversial and the molecular mechanisms are not fully elucidated. In nucleated cells, one of the downstream molecules of the TLRs signaling pathway is the transcription factor nuclear factor-κB (NF-κB), a major regulator of inflammatory genes transcription. We have previously shown that activation of NF-κB mediates platelet activation triggered by classical platelet agonists, indicating that this transcription factor exerts non-genomic functions in platelets.Whether NF-κB is involved in the signaling pathway of TLR2 and 4 have not yet been investigated. Aim: To further examine platelet activation mediated by TLR2 and TLR4 stimulation and determine if activation of NF-κB is a downstream signal. Methods: Washed human platelets were stimulated with Pam(3)CSK(4) or LPS (agonists for TLR2 or 4 respectively) in the absence or presence of BAY11-7082 and Ro106-9920, two non-structurally related inhibitors of NF-κB. Aggregation and ATP release were measured using a Lumi-aggregometer and platelet-leukocyte aggregates were enumerated by cytometry. Activation of NF-κB was examined by the degradation of its inhibitor, I-B, and the phosphorylation of the p65 subunit, by Western Blot. The release of IL-1was determined by ELISA. Results are expressed as X±SEM of 3-4 independent experiments. Results: Stimulation of platelets with Pam(3)CSK(4) (0.1-15 ug/ml) triggered platelet aggregation, ATP release, as well as proinflammatory responses such as IL-1secretion (C: 51±13, Pam(3)CSK(4) (1ug/ml): 288±96 pg/ml) and the formation of platelet-leukocyte aggregates (C: 17±2, Pam(3)CSK(4): 38±4 % of PMN associated with platelets). All Pam(3)CSK(4)-mediated responses were completely inhibited by preincubation of the platelets with BAY11-7082 or Ro106-9920. LPS (10 ug/ml) did not have a direct effect but potentiated aggregation and ATP release induced by threshold thrombin concentrations (0.01 U/ml). This synergism was partially blocked by the NF-κb inhibitors (BAY11-7082: 39±3, Ro106-9920: 37±5% of inhibition). While platelet-leukocyte aggregates were no triggered by LPS, it efficiently induced IL-1release that was prevented in platelets pretreated with BAY11-7082 or Ro106-9920 (C: 51±13, LPS: 460±110, LPS+BAY11-7082: 24±16, LPS+Ro106-9920: 32±17 pg/ml). Stimulation of platelets with 1ug/ml of Pam(3)CSK(4) or LPS resulted in the degradation of I-B and the phosphorylation of the p65 subunit of NF-κB (Pam(3)CSK(4): 5±1, LPS: 6±1, fold increase). Both responses elicited by Pam(3)CSK(4) or LPS were completely inhibited by an antibody against TLR2 or 4 respectively, and synergized by threshold concentrations of thrombin (phosphorylation of p65: Thr: 4±1, Thr+Pam(3)CSK(4): 9±1, Thr + LPS: 12± 4). Conclusion: Activation of TLR2 and 4 trigger platelet hemostatic and inflammatory responses that are mediated by the transcription factor NF-κB. These data reinforce the notion of the relationship between the immune response and platelets, reveal another non-genomic function of NF-κB in platelets and highlight this molecule as a potential target to prevent platelet activation in inflammatory and infectious diseases.