Cutting edge: Leukotriene C4 activates mouse platelets in plasma exclusively through the type 2 cysteinyl leukotriene receptor

Abstract

Leukotriene C4 (LTC4) and its extracellular metabolites, LTD4 and LTE4, mediate airway inflammation. They signal through three specific receptors (type 1 cys-LT receptor [CysLT1R], CysLT2R, and GPR99) with overlapping ligand preferences. In this article, we demonstrate that LTC4, but not LTD4 or LTE4, activates mouse platelets exclusively through CysLT2R. Platelets expressed CysLT1R and CysLT2R proteins. LTC4 induced surface expression of CD62P by wild-type mouse platelets in platelet-rich plasma (PRP) and caused their secretion of thromboxane A2 and CXCL4. LTC4 was fully active on PRP from mice lacking either CysLT1R or GPR99, but completely inactive on PRP from CysLT2R-null (Cysltr2(-/-)) mice. LTC4/CysLT2R signaling required an autocrine ADP-mediated response through P2Y12 receptors. LTC4 potentiated airway inflammation in a platelet- and CysLT2R-dependent manner. Thus, CysLT2R on platelets recognizes LTC4 with unexpected selectivity. Nascent LTC4 may activate platelets at a synapse with granulocytes before it is converted to LTD4, promoting mediator generation and the formation of leukocyte-platelet complexes that facilitate inflammation.

Figure 1

Platelet activation by cys-LTs. PRP from WT mice was stimulated with the indicated agonists. CD62P was assessed by flow cytometry. Results are mean ± SD from 5–10 separate experiments using platelets from one mouse/strain.

Figure 2

Cys-LT receptors involved in LTC₄-induced platelet activation. PRP from mice of the indicated genotypes was stimulated with various concentrations of cys-LTs, or with thrombin as a positive control. A. Effect of CysLT₂R deletion. B. Effect of CysLT₁R deletion. C. Effect of GPR99 deletion. D. Western blot of proteins from human and WT mouse platelets showing bands corresponding to the anticipated molecular sizes of CysLT₁R and CysLT₂R. Results in A-C are mean ± SD from 3–5 separate experiments.

Figure 3

Involvement of P2Y₁₂ receptors and extracellular nucleotides in CysLT₂R-mediated platelet activation. A. Platelets from WT or P2ry12−/− micewere stimulated with the indicated concentrations of cys-LTs or thrombin. CD62P induction was assessed by flow cytometry. B. WT platelets were stimulated with cys-LTs or thrombin in the absence or presence of apyrase. PRP from P2ry12−/− mice was included as a control. C. Release of ADP by stimulated platelets and effects of apyrase and genotypes. Results mean ± SD from 3 separate experiments.

Figure 4

LTC₄ amplifies allergen-induced pulmonary inflammation in a platelet, CysLT₂R and P2Y₁₂-dependent manner. Mice were sensitized intraperitoneally with OVA/Alum and challenged 3x with 0.1% OVA with or without intranasal LTC₄ (2 nmol). A. BAL fluid total cell counts (top) and eosinophil counts (bottom) from mice of the indicated genotypes. B. Effect of platelet depletion (using anti-CD41 vs. an isotype control) of WT mice challenged with OVA ± LTC₄ on BAL fluid cell counts and eosinophil counts. Results are mean ± SEM from a single experiment using 5–10 mice/group. Results from a second experiment were similar.