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.

Cysteinyl leukotrienes (cys-LTs) play a validated role in asthma (1). After 5-lipoxygenase oxidizes arachidonic acid to LTA4 (2), eosinophils, basophils, mast cells, and monocytes conjugate LTA4 to reduced glutathione via leukotriene (LT) C4 synthase (LTC4S) (3), forming LTC4. After export, LTC4 is converted to LTD4 (4), a smooth muscle spasmogen, and then to LTE4 (5), a stable metabolite. Three G protein–coupled receptors, termed the type 1 cys-LT receptor (CysLT1R) (6, 7), type 2 cys-LT receptor (CysLT2R) (8, 9), and GPR99 (10), mediate the effects of cys-LTs. CysLT1R is a high-affinity LTD4 receptor with lower affinity for LTC4 (6, 7). CysLT2R binds LTC4 and LTD4 with equal affinity (8, 9), and GPR99 exhibits a preference for LTE4 (10). CysLT1R-selective antagonists are widely prescribed for asthma (11). Although CysLT2R inhibits dendritic cell priming for Th2 immune responses (12) and GPR99 mediates LTE4-induced skin edema (10), our understanding of the therapeutic applicability of these receptors is limited. Moreover, because many cell types express more than one cys-LT receptor, assignment of receptor-specific functions through in vitro approaches is challenging.

Platelets play an important role in asthma (13) and vascular inflammation (14). Platelets adhere to granulocytes by a CD62P (P-selectin)-P-selectin glycoprotein-1–dependent mechanism. Adherent platelets upregulate leukocyte integrin avidity (15) and permit transcellular metabolism of arachidonic acid (16). Platelets contain LTC4S and convert granulocyte-derived LTA4 to LTC4 through a transcellular pathway, amplifying the production of cys-LTs (13). Human platelets express both CysLT1R and CysLT2R (17). To date, however, no study has definitively addressed whether cys-LTs influence platelet functions or determined which receptors are most essential.

We report that LTC4, but not LTD4 or LTE4, activates mouse platelets entirely through CysLT2R. LTC4 induces expression of platelet CD62P. This response requires CysLT2R, but not CysLT1R or GPR99. LTC4 induces platelets to release inflammatory mediators, and to augment allergen-induced airway inflammation. CysLT2R-dependent platelet activation requires amplification from P2Y12 receptors and ADP. LTC4 may facilitate local activation of platelets in a synapse with leukocytes, in turn amplifying inflammatory responses. This function is distinct from those of its extracellular metabolites. Moreover, CysLT2R can function as an LTC4 receptor with high specificity despite its ability to bind LTD4 in transfected cells (8).

Tbxa2r−/− mice were obtained from Dr. Thomas Coffman (Duke University, Durham, NC) (18). P2ry12−/− mice were from Portola Pharmaceuticals (San Francisco, CA) (19). Cysltr1−/−, Cysltr2−/−, and Gpr99−/− mice were generated in our institution (10, 20, 21). Mice were sensitized i.p. on days 0 and 7 with Alum-precipitated chicken egg OVA (10 μg; Sigma) and challenged by inhalation of 0.1% OVA with or without intranasal cys-LTs as described previously (22). Platelets were depleted by an i.p. injection of an anti-CD41 Ab (clone MWReg30; Biolegend, San Diego, CA) or an isotype control (23).

Blood was obtained by cardiac puncture using a 21G needle into 4% sodium citrate (Sodium Citrate Enzyme Grade; Fisher Scientific, Pittsburgh, PA). Platelet-rich plasma (PRP) was obtained by slow-spin centrifugation of whole blood at 1000 rpm/900 × g for 15 min. PRP was incubated with CaCl2 (Fisher) ([final]= 5 mM) at 37°C for 10 min.

Aliquots of PRP (50 μl) were stimulated with thrombin (50 U/ml; Sigma Aldrich, St. Louis, MO), LTC4, LTD4, or LTE4 (25–250 nM; Cayman Chemical, Ann Arbor, MI) at 37°C for 30 min. Samples were stained with PE anti-mouse CD41 (clone MWReg30; Biolegend) and FITC rat anti-mouse CD62P (clone RB40.34; BD Pharmingen, San Diego, CA) for analysis of CD62P expression on CD41+ mouse platelets. PE rat IgG1κ and FITC rat IgG1λ were used for isotype controls (BD Pharmingen). Cells were fixed overnight in 1% paraformaldehyde in PBS (Affymetrix, Cleveland, OH). Some aliquots of PRP were stimulated with at 37°C for 30 min for analysis of released thromboxane (Thromboxane B2 EIA Kit; Cayman), RANTES (eBioscience, San Diego, CA) and CXCL4 (Sigma) by ELISA, or for ADP (Abcam). Some samples were treated with the CysLT2R antagonists BayCysLT2 and HAMI3379 (300 nM each; Cayman Chemical). In some experiments, supernatants were analyzed for conversion of LTC4 to LTD4 and LTE4 by high-performance liquid chromatography (3).

LTC4 is synthesized by cells that express both 5-lipoxygenase and LTC4S (24), or generated through granulocyte-derived LTA4 by adherent LTC4S-expressing platelets (25). Because extracellular enzymes efficiently convert LTC4 to LTD4 and LTE4, LTC4 most likely functions in a synapse between the cells of origin and adjacent endothelium or platelets. However, apart from its role as a precursor, no unique functions have been attributed to LTC4. Human platelets express both CysLT1R and CysLT2R (17), as is the case for many hematopoietic cells (24). Given that cell recruitment (26), bronchoconstriction (27), airway inflammation (22), and fibrosis (20) all involve both cys-LTs and platelet activation (13, 22, 28, 29), we sought to determine whether platelets might respond directly to cys-LTs.

We first stimulated platelets from wild-type (WT) mice with various concentrations of LTC4, LTD4, and LTE4. Only LTC4 elicited an increase in surface CD62P expression (Fig. 1) and was active at the lowest dose tested (25 nM). The response to LTC4 at 250 nM was ∼60% of that elicited by thrombin (Fig. 1). PRP did not convert LTC4 to LTD4 or LTE4 (not shown). The induction of CD62P by LTC4, and the lack of any response to LTD4 and LTE4 at physiologic ranges, suggests that LTC4 has specific functions in the formation of platelet–leukocyte complexes, which depend on induction of CD62P and its interaction with (P-selectin)-P-selectin glycoprotein-1 on the leukocyte surface (25).

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 1 mouse/strain.

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 1 mouse/strain.

Close modal

Given that CysLT1R and CysLT2R each bind LTC4 and LTD4 at low nanomolar ranges (6, 9), the response limited to LTC4 was unexpected. To identify the responsible receptors, we stimulated PRP obtained from mice lacking CysLT1R (Cysltr1−/− mice), CysLT2R (Cysltr2−/− mice), and GPR99 (Gpr99−/− mice). Platelets from Cysltr2−/− mice were unresponsive to LTC4 (Fig. 2A), whereas platelets from the Cysltr1−/− (Fig. 2B) and Gpr99−/− strains (Fig. 2C) were fully responsive. Platelets from all three strains responded to thrombin, and none reacted to LTD4 or LTE4 (Fig. 2A–C). Platelets from WT mice expressed both CysLT1R and CysLT2R proteins, as did human platelets (Fig. 2D). Thus, whereas recombinant CysLT2R has equal binding affinities for LTC4 and LTD4 (8, 9), natively expressed CysLT2R on mouse platelets exhibits a preference for activation by LTC4. Moreover, despite the presence of CysLT1R on platelets, CysLT2R is the dominant effector of responses to LTC4 in this cell type. In mast cells (30) and dendritic cells (12), CysLT1R signaling dominates and CysLT2R serves an inhibitory function. Cell-specific variations in receptor stoichiometry, relative abundances, localization, or G protein coupling may account for these functional differences.

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 three to five separate experiments.

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 three to five separate experiments.

Close modal

Endogenous ADP can amplify platelet activation through P2Y1 and P2Y12 receptors (31). P2Y12 receptors are implicated in cellular responses to cys-LTs (particularly LTE4) (22, 32), but do not bind cys-LTs (22), suggesting an indirect functional relationship to cys-LT receptors. LTC4-mediated induction of CD62P was markedly impaired in P2ry12−/− platelets (Fig. 3A). Treatment of WT platelets with apyrase attenuated their responses to LTC4 (Fig. 3B) while depleting extracellular ADP (Fig. 3C). Although the doses of LTE4 used in this study may exceed those required to demonstrate activity at P2Y12, only LTC4 caused platelets to release ADP; this response required CysLT2R (Fig. 3C). P2Y12-targeted thienopyridine drugs, which prevent cardiovascular events (33), may interfere with the LTC4/CysLT2R-dependent pathway of platelet activation in vivo.

FIGURE 3.

Involvement of P2Y12 receptors and extracellular nucleotides in CysLT2R- mediated platelet activation. (A) Platelets from WT or P2ry12−/− mice were 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 are mean ± SD from three separate experiments.

FIGURE 3.

Involvement of P2Y12 receptors and extracellular nucleotides in CysLT2R- mediated platelet activation. (A) Platelets from WT or P2ry12−/− mice were 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 are mean ± SD from three separate experiments.

Close modal

Activated platelets generate thromboxane A2 (TXA2), a potent inflammatory mediator, and secrete chemokines (34). Human platelets released RANTES when stimulated with cys-LTs in a prior study (17). In our study, LTC4 induced mouse platelets to release large quantities of TXA2, as well as CXCL4 and, to a lesser extent, RANTES (Supplemental Fig. 1A–C), in a CysLT2R- and P2Y12 receptor–dependent manner. Two CysLT2R antagonists, BayCysLT2 and HAMI3379 (300 nM each), suppressed TXA2 release by WT platelets (Supplemental Fig. 1D). Studies using platelets from Tbxa2r−/− mice revealed that TXA2 was not necessary for LTC4-induced activation, although there was a trend toward less activation at the lowest LTC4 doses (Supplemental Fig. 2).

Intrapulmonary administration of LTE4 to sensitized mice challenged with low-dose OVA potentiates eosinophil recruitment in a platelet- and P2Y12-dependent manner (35). We treated sensitized mice intranasally with LTC4 (2 nmol) on 3 consecutive days before low-dose (0.1%) OVA challenges. LTC4 markedly potentiated the recruitment of eosinophils to the bronchoalveolar lavage (BAL) fluid. This response depended on CysLT2R, P2Y12 (Fig. 4A), and platelets (Fig. 4B). LTC4 may therefore contribute to platelet activation in asthma, aspirin-exacerbated respiratory disease (13), myocardial infarction (36), and stroke (37). Moreover, this pathway likely resists blockade by the available antagonists, which do not target CysLT2R, but may be sensitive to P2Y12 receptor–active drugs.

FIGURE 4.

LTC4 amplifies allergen-induced pulmonary inflammation in a platelet, CysLT2R, and P2Y12-dependent manner. Mice were sensitized i.p. with OVA/Alum and challenged 3× with 0.1% OVA with or without intranasal LTC4 (2 nmol). (A) BAL fluid total cell counts (top panel) and eosinophil counts (bottom panel) from mice of the indicated genotypes. (B) Effect of platelet depletion (using anti-CD41 versus 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.

FIGURE 4.

LTC4 amplifies allergen-induced pulmonary inflammation in a platelet, CysLT2R, and P2Y12-dependent manner. Mice were sensitized i.p. with OVA/Alum and challenged 3× with 0.1% OVA with or without intranasal LTC4 (2 nmol). (A) BAL fluid total cell counts (top panel) and eosinophil counts (bottom panel) from mice of the indicated genotypes. (B) Effect of platelet depletion (using anti-CD41 versus 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.

Close modal

This work was supported by the National Institutes of Health (Grants AI078908, AI095219, AT002782, AI082369, HL111113, HL117945, and HL36110) and the Vinik family.

The online version of this article contains supplemental material.

Abbreviations used in this article:

BAL

bronchoalveolar lavage

cys-LT

cysteinyl leukotriene

CysLT1R

type 1 cys-LT receptor

LT

leukotriene

LTC4

leukotriene C4

LTC4S

leukotriene C4 synthase

PRP

platelet-rich plasma

TXA2

thromboxane A2

WT

wild-type.

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The authors have no financial conflicts of interest.