PPARβ/δ Agonists Modulate Platelet Function via a Mechanism Involving PPAR Receptors and Specific Association/Repression of PKCα–Brief Report
Objectives— Peroxisome proliferator-activated receptor β/δ (PPARβ/δ) is a nuclear receptor found in platelets. PPARβ/δ agonists acutely inhibit platelet function within a few minutes of addition. As platelets are anucleated, the effects of PPARβ/δ agonists on platelets must be nongenomic. Currently, the particular role of PPARβ/δ receptors and their intracellular signaling pathways in platelets are not known.
Methods and Results— We have used mice lacking PPARβ/δ (PPARβ/δ−/−) to show the effects of the PPARβ/δ agonist GW501516 on platelet adhesion and cAMP levels are mediated specifically by PPARβ/δ, however GW501516 had no PPARβ/δ-specific effect on platelet aggregation. Studies in human platelets showed that PKCα, which can mediate platelet activation, was bound and repressed by PPARβ/δ after platelets were treated with GW501516.
Conclusions— These data provide evidence of a novel mechanism by which PPAR receptors influence platelet activity and thereby thrombotic risk.
Peroxisome proliferator-activated receptor β/δ (PPARβ/δ) is a nuclear receptor present in platelets. We have previously shown PPARβ/δ ligands inhibit platelet function via a nongenomic pathway which is independent of nuclear events.1 In addition to synthetic ligands, PPARβ/δ can be also be activated by the antithrombotic hormone prostaglandin (PG) I2.1,2 The specific role of PPARβ/δ receptors in the antiplatelet effects of synthetic agonists is not known. Here, we have used platelets from mice lacking PPARβ/δ (PPARβ/δ−/−) to show this receptor mediates in part the antiplatelet effects of GW501516. We have gone on to show in human or mouse platelets that GW501516 activates PPARβ/δ receptors to bind and repress PKCα, thereby providing a novel yet plausible mechanism for how PPAR receptors produce nongenomic responses in platelets.
All experiments were performed according to ethical guidelines, and a full experimental methods section is available as supplemental material (available online at http://atvb.ahajournals.org).
Immunoprecipitations were performed in human platelet rich plasma (PRP) or human/mouse washed platelets incubated with GW501516. Samples were incubated with antibody against PPARβ/δ and Western blots performed with anti-PKCα antibody. To measure PKC activity a phosphor-(Ser) PKC substrate antibody was used, which measures phosphorylation of PKC target proteins.
Mouse (20 to 25g) PRP was pooled and incubated with GW501516. ADP was added and aggregation measured over 16 minutes using a standard light transmission plate reader at 595 nm. Adhesion was measured using a buffer containing p-nitrophenyl phosphate. cAMP release was measured by ELISA.
To investigate the effect of PPARβ/δ activation on PKCα, human PRP was incubated with GW501516, proteins were immunoprecipitated with PPARβ/δ antibodies, and complexes analyzed by Western blot with primary antibodies to PKCα (Figure 1A). GW501516 significantly increased the association between PPARβ/δ and PKCα (Figure 1B). In addition, this protocol was repeated in human and murine washed platelets and provided similar results (see supplemental materials). In line with these observations, PKCα activity was reduced by GW501516, with maximal inhibition seen after 10 minutes incubation with human PRP (Figure 1C) and murine PRP (see supplemental materials).
Platelet activation assays were performed in platelets from wild-type (WT) and PPARβ/δ−/− mice. cAMP release was stimulated in WT platelets after incubation with GW501516 (1 μmol/L), however this was significantly reduced in PPARβ/δ−/− platelets. The ability of GW501516 (10 μmol/L) to inhibit ADP-induced platelet adhesion was significantly reduced in tissue from PPARβ/δ−/− mice compared to WT animals. By contrast, no difference was seen in the ability of ADP to induce platelet aggregation in tissue from WT versus PPARβ/δ−/− mice (Figure 2).
The mechanism by which PPARβ/δ inhibits platelet activation is unknown. However, as platelets are anucleated, inhibitory effects of PPARβ/δ agonists must clearly be independent of the nucleus. In the search for alternative signaling pathways we identified PKCα as a potential target.3,4 In nucleated cells, PPARγ blocks PKCα downstream signaling.5 We show a concentration-dependent binding of PPARβ/δ to PKCα and a time-dependent inhibition of platelet PKCα activity. This suggests that binding and repression of PKCα by PPARβ/δ could account for the nongenomic inhibitor effects of agonists such as GW501516 on platelets. To establish whether GW501516 effects were mediated specifically by PPARβ/δ, murine PPARβ/δ−/− and WT platelets were incubated with GW501516. GW501516 increased cAMP levels in WT platelets but had little effect in PPARβ/δ−/− platelets. Adenylyl cyclase, which converts ATP to cAMP, is a downstream target of PKCα in nucleated cells.6,7 We found selective inhibitors of PKCα (Gö6976 and Gö6983) caused basal levels of cAMP in mouse platelets to increase significantly (basal cAMP, 106±6 fmol/mL; +Gö6976 (30 nmol/L), 241±9 fmol/mL; +Gö6983 (30 nmol/L), 236±18 fmol/mL). This suggests PKCα activity negatively regulates adenylyl cyclase, which is consistent with our hypothesis. GW501516 also inhibited platelet adhesion partially mediated by PPARβ/δ, as responses were significantly less in platelets from PPARβ/δ−/− compared to WT. PKCα has been shown to positively regulate platelet adhesion.8 This again is consistent with our hypothesis that PPARβ/δ binds PKCα and, via this mechanism, represses its function. In contrast, the inhibitory effects of GW501516 on aggregation were independent of PPARβ/δ receptors. This observation suggests that GW501516 can inhibit platelet functions independently of the target receptor by pathways yet to be established, but clearly independently of gene induction.
It should be noted that interpretation of our data are limited because platelet responses vary greatly with in vitro conditions, and ADP may only activate PKCα weakly. Nevertheless, in summary, we have shown for the first time that PPARβ/δ is a functionally active protein in platelets and that direct binding and repression of PKCα is a plausible mechanism by which PPARβ/δ regulates platelet reactivity.
Sources of Funding
This work was funded by the British Heart Foundation (PG/06/054).
Received April 1, 2009; revision accepted August 12, 2009.
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