Platelet protease-activated receptor (PAR)4, but not PAR1, associated with neutral sphingomyelinase responsible for thrombin-stimulated ceramide-NF- B signaling in human platelets

Abstract

Thrombin activates platelets mainly through protease-activated receptor (PAR)1 and PAR4. However, downstream platelet signaling between PAR1 and PAR4 is not yet well understood. This study investigated the relationship between nSMase/ceramide and the NF-κB signaling pathway in PARs-mediated human platelet activation. The LC-MS/MS, aggregometry, flow cytometry, immunoprecipitation, and mesenteric microvessels of mice were used in this study. Human platelets stimulated by thrombin, 3-OMS (a neutral sphingomyelinase [nSMase] inhibitor) and Bay11-7082 (an NF-κB inhibitor) significantly inhibited platelet activation such as P-selectin expression. Thrombin also activated IκB kinase (IKK)β and IκBα phosphorylation; such phosphorylation was inhibited by 3-OMS and SB203580 (a p38 MAPK inhibitor). Moreover, 3-OMS abolished platelet aggregation, IKKβ, and p38 MAPK phosphorylation stimulated by PAR4-AP (a PAR4 agonist) but not by PAR1-AP (a PAR1 agonist). Immunoprecipitation revealed that nSMase was directly associated with PAR4 but not PAR1 in resting platelets. In human platelets, C24:0-ceramide is the predominant form of ceramides in the LC/MS-MS assay; C24:0-ceramide increases after stimulation by thrombin or PAR4-AP, but not after stimulation by PAR1-AP. We also found that C2-ceramide (a cell-permeable ceramide analog) activated p38 MAPK and IKKβ phosphorylation in platelets and markedly shortened the occlusion time of platelet plug formation in vivo. This study demonstrated that thrombin activated nSMase by binding to PAR4, but not to PAR1, to increase the C24:0-ceramide level, followed by the activation of p38 MAPK-NF-κB signaling. Our results showed a novel physiological significance of PAR4-nSMase/ceramide-p38 MAPK-NF-κB cascade in platelet activation.

Resistin increases platelet P-selectin levels via p38 MAPK signal pathway

“…This observation is consistent with other studies showing a similar role for the p38 signal pathway in P-selectin regulation in platelet. 10 In summary, our study demonstrates that resistin contributes to platelet activation by directly increasing platelet P-selectin levels through the p38 MAPK signal pathway. This further helps to understand the mechanisms for the prothrombotic state in individuals with the metabolic syndrome or those with diabetes.…”

Plasma L5 levels are elevated in ischemic stroke patients and enhance platelet aggregation

“…37 Three MAPKs have been identified in platelets, including extracellular signal-regulated kinase (ERK) p38 MAPK and c-Jun N-terminal kinase 1 (JNK1). 9 We found that L51Ab induced the phosphorylation of JNK1 ( Figure 5D) but not ERK or p38 MAPK (data not shown) in human platelets.…”

Role of NF-κB Pathway on Platelet Activation

“…In fact, in the last sentence of the abstract, it is written "On the basis of these data, NF-κB is also identified as a new target to dampen unwanted platelet activation." The second article of Spinelli et al is just an editorial comment of the article published by Gambaryan et al 2 More important still, there is no mention of 7 other articles, [5][6][7][8][9][10][11] including a pioneer work from our group, 5 describing an opposite effect of NF-κB activation in platelets. In fact, using different strategies (including IkB kinase β knockout mice), these studies show that activation of NF-κB pathway promotes platelet activation.…”