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

Wei Fan Chen, Jie Jen Lee, Chao Chien Chang, Kuan Hong Lin, Shwu Huey Wang, Joen Rong Sheu

Research output: Contribution to journalArticle

24 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)793-801
Number of pages9
JournalHaematologica
Volume98
Issue number5
DOIs
Publication statusPublished - 2013

Fingerprint

Sphingomyelin Phosphodiesterase
Ceramides
Thrombin
Blood Platelets
p38 Mitogen-Activated Protein Kinases
Platelet Activation
Phosphorylation
Immunoprecipitation
PAR-1 Receptor
P-Selectin
Microvessels
protease-activated receptor 4
Platelet Aggregation
Flow Cytometry
Phosphotransferases

ASJC Scopus subject areas

  • Hematology
  • Medicine(all)

Cite this

Platelet protease-activated receptor (PAR)4, but not PAR1, associated with neutral sphingomyelinase responsible for thrombin-stimulated ceramide-NF-κB signaling in human platelets. / Chen, Wei Fan; Lee, Jie Jen; Chang, Chao Chien; Lin, Kuan Hong; Wang, Shwu Huey; Sheu, Joen Rong.

In: Haematologica, Vol. 98, No. 5, 2013, p. 793-801.

Research output: Contribution to journalArticle

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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.",
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