The role of calpain-myosin 9-Rab7b pathway in mediating the expression of Toll-like receptor 4 in platelets: a novel mechanism involved in α-granules trafficking.
Toll-like receptors (TLRs) plays a critical role in innate immunity. In 2004, Aslam R. and Shiraki R. first determined that murine and human platelets express functional TLRs. Additionally, Andonegui G. demonstrated that platelets express TLR4, which contributes to thrombocytopenia. However, the underlying mechanisms of TLR4 expression by platelets have been rarely explored until now. The aim of this study was to identify the mechanism of TLR4 expression underlying thrombin treatment. The human washed platelets were used in this study. According to flowcytometry and western blot analysis, the surface levels of TLR4 were significantly enhanced in thrombin-activated human platelets and decreased by TMB-8, calpeptin, and U73122, but not Y27632 (a Rho-associated protein kinase ROCK inhibitor) indicating that thrombin-mediated TLR4 expression was modulated by PAR/PLC pathway, calcium and calpain. Co-immunoprecipitation (co-IP) assay demonstrated that the interaction between TLR4 and myosin-9 (a substrate of calpain) was regulated by calpain; cleavage of myosin-9 enhanced TLR4 expression in thrombin treated platelets. Transmission electron microscope data indicated that human platelets used α-granules to control TLR4 expression; the co-IP experiment suggested that myosin-9 did not coordinate with Rab7b to negatively regulate TLR4 trafficking in thrombin treated platelets. In summary, phospholipase Cγ-calpain-myosin 9-Rab7b axis was responsible for the mechanism underlying the regulation of TLR4 containing α-granules trafficking in thrombin-stimulated platelets, which was involved in coagulation.
Graduate Institute of Medical Sciences, National Defense Medical Center, Taipei, Taiwan ; Departments of Internal Medicine, Taipei Medical University, Taipei, Taiwan.
Jui-Chi
JC
Tsai
Division of Cardiovascular Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan.
Yi-Wen
YW
Lin
Departments of Internal Medicine, Taipei Medical University, Taipei, Taiwan ; Division of Cardiology and Cardiovascular Research Center, Taipei Medical University Hospital, Taipei, Taiwan.
Chun-Yao
CY
Huang
Division of Cardiovascular Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan.
Chih-Yuan
CY
Lin
Division of Cardiovascular Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan.
Yi-Ting
YT
Tsai
Departments of Internal Medicine, Taipei Medical University, Taipei, Taiwan ; Division of Cardiology and Cardiovascular Research Center, Taipei Medical University Hospital, Taipei, Taiwan.
Chun-Min
CM
Shih
Division of Cardiovascular Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan.
Chung-Yi
CY
Lee
Graduate Institute of Integrated Medicine, China Medical University, Taichung, Taiwan.
Yung-Hsiang
YH
Chen
Graduate Institute of Clinical Medical Sciences, China Medical University, Taichung, Taiwan ; Department of Anesthesiology, China Medical University Hospital, Taichung, Taiwan.
Chi-Yuan
CY
Li
Departments of Internal Medicine, Taipei Medical University, Taipei, Taiwan ; Division of Cardiology and Cardiovascular Research Center, Taipei Medical University Hospital, Taipei, Taiwan.
Nen-Chung
NC
Chang
Departments of Internal Medicine, Taipei Medical University, Taipei, Taiwan ; Division of Cardiology and Cardiovascular Research Center, Taipei Medical University Hospital, Taipei, Taiwan.
Feng-Yen
FY
Lin
Division of Cardiovascular Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan.
Chien-Sung
CS
Tsai
D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D015153:Blotting, Western; D002154:Calpain / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D004151:Dipeptides / Q000494:pharmacology; D004963:Estrenes / Q000494:pharmacology; D005434:Flow Cytometry; D005707:Gallic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D047468:Immunoprecipitation; D008297:Male; D009218:Myosins / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects / Q000235:genetics; D011760:Pyrrolidinones / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D013917:Thrombin / Q000494:pharmacology; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000378:metabolism*; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D000090802:rab7 GTP-Binding Proteins
D004151:Dipeptides; D004963:Estrenes; D011760:Pyrrolidinones; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D000090802:rab7 GTP-Binding Proteins; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; C071482:calpeptin; C006014:8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate; D005707:Gallic Acid; D013917:Thrombin; D002154:Calpain; D009218:Myosins; D020691:rab GTP-Binding Proteins