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

PLoS One. 2014 Jan 28;9(1):e85833. doi: 10.1371/journal.pone.0085833. eCollection 2014.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Blood Platelets / drug effects
  • Blood Platelets / metabolism*
  • Blotting, Western
  • Calpain / genetics
  • Calpain / metabolism*
  • Cells, Cultured
  • Dipeptides / pharmacology
  • Estrenes / pharmacology
  • Flow Cytometry
  • Gallic Acid / analogs & derivatives
  • Gallic Acid / pharmacology
  • Humans
  • Immunoprecipitation
  • Male
  • Myosins / genetics
  • Myosins / metabolism*
  • Protein Transport / drug effects
  • Protein Transport / genetics
  • Pyrrolidinones / pharmacology
  • Signal Transduction / drug effects
  • Signal Transduction / genetics
  • Thrombin / pharmacology
  • Toll-Like Receptor 4 / genetics
  • Toll-Like Receptor 4 / metabolism*
  • rab GTP-Binding Proteins / genetics
  • rab GTP-Binding Proteins / metabolism*
  • rab7 GTP-Binding Proteins

Substances

  • Dipeptides
  • Estrenes
  • Pyrrolidinones
  • TLR4 protein, human
  • Toll-Like Receptor 4
  • rab7 GTP-Binding Proteins
  • 1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione
  • calpeptin
  • 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate
  • Gallic Acid
  • Thrombin
  • Calpain
  • Myosins
  • rab GTP-Binding Proteins

Grants and funding

This work was supported by the National Science Council (NSC 97-2314-B-038-035-MY2, NSC 101-2314-B-038-041-MY3 and NSC 101-2314-B-016-007-MY2) and partial by the National Defense Medical Center (TSGH-C98-25), Taiwan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.