Platelet protease-activated receptor (PAR)4, but not PAR1, associated with neutral sphingomyelinase responsible for thrombin-stimulated ceramide-NF-κB signaling in human platelets.
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.
Graduate Institute of Medical Sciences, Taipei Medical University, Taipei, Taiwan.
Wei-Fan
WF
Chen
Jie-Jen
JJ
Lee
Chao-Chien
CC
Chang
Kuan-Hong
KH
Lin
Shwu-Huey
SH
Wang
Joen-Rong
JR
Sheu
D001792:Blood Platelets / Q000187:drug effects* / Q000378:metabolism*; D002518:Ceramides / Q000378:metabolism*; D006801:Humans; D016328:NF-kappa B / Q000378:metabolism*; D015539:Platelet Activation / Q000187:drug effects; D010974:Platelet Aggregation; D011485:Protein Binding; D018179:Receptors, Thrombin / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism*; D013110:Sphingosine / Q000031:analogs & derivatives / Q000494:pharmacology; D053719:Tandem Mass Spectrometry; D013917:Thrombin / Q000494:pharmacology*; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D002518:Ceramides; C064769:N-acetylsphingosine; D016328:NF-kappa B; D018179:Receptors, Thrombin; D048051:p38 Mitogen-Activated Protein Kinases; D013108:Sphingomyelin Phosphodiesterase; D013917:Thrombin; C112908:protease-activated receptor 4; D013110:Sphingosine