Specific protein kinase C isoforms regulate platelet purinergic receptor function

Mundell SJ, Jones ML, Barton JF, Hardy AR, and Poole AW. Department of Pharmacology, University of Bristol, Bristol, UK.

ADP, a critical regulator of platelet activation, mediates its actions through two G protein-coupled receptors (GPCRs), the P2Y1 and P2Y12 purinergic. Recently we have demonstrated that both P2Y1 and P2Y12 receptor responses undergo rapid homologous desensitization in human platelets (Hardy et al., 2005). In this study we examined if specific protein kinase C (PKC) isoforms regulated the surface expression, trafficking and activity, of these two GPCRs.

Studies examining receptor phosphorylation, surface expression and signalling following activation of PKC isoforms by PMA (1 μM; 15 min) were undertaken in 1321N1 cells stably expressing either HA-P2Y1 or HA-P2Y12 receptors or in human platelets. Receptor surface loss was assessed in 1321N1 cells by ELISA whilst in parallel experiments changes in P2Y1 and P2Y12 surface receptor levels were measured in fixed platelets using the P2Y radioligand [3H]2MeSADP in combination with the P2Y1 receptor antagonist A3P5P (1 mM) and the P2Y12 receptor antagonist AR-C69931MX (1 μM). P2Y1 G q-coupled and P2Y12 Gi-coupled receptor activity was assessed as previously described (Hardy et al., 2005). All data is presented as mean + SEM.and statistical analysis was carried out using 1-way ANOVA with Bonferroni’s multiple comparison test.

Initial studies in 1321N1 cells, stably expressing either receptor showed that activation of PKC by PMA promoted heterologous, agonist-independent, phosphorylation, desensitisation (PMA pre-treatment reduced subsequent ADP (10 μM)-induced P2Y1 and P2Y12 signalling by 74.2 ± 3.2 % and 66.8 ± 2.9 % respectively (n=3)) and internalization (P2Y1 and P2Y12 surface receptor expression reduced by 38.6 ± 4.2 % and 39.4 ± 5.6 % respectively following PMA pre-treatment (n=5)) of both P2Y1 and P2Y12. Expression of dominant negative mutant catalytically inactive forms of PKC a and PKC d , two PKC isoforms expressed in both 1321N1 cells and platelets, revealed that both isoforms regulated P2Y1 receptor signalling and surface expression whilst only PKC d was capable of regulating P2Y12 receptor function. In human platelets, similar to 1321N1 cells, activation of PKC promoted the desensitisation (PMA pre-treatment reduced subsequent ADP (10 μM)-induced P2Y1 and P2Y12 signalling by 71.8 ± 3.4 % and 90.8 ± 3.6 % respectively (n=3 )) and internalization ( P2Y 1 and P2Y 12 surface receptor expression reduced by 46.3 ± 8.0 % and 73.2 ± 5.0 % respectively following PMA pre-treatment (n=3) ) of both GPCRs. Use of the PKC isoform specific inhibitors GF109203X (2 μM), an inhibitor of conventional and novel PKC isoforms, Go6976 (1 μM), an inhibitor of calcium-dependent PKC isoforms, including PKC a , and rottlerin (10 μM), which selectively inhibits PKCδ , revealed that PKCδ preferentially regulates P2Y12 receptor function in an agonist-independent manner whilst both PKCα and PKCδ regulated P2Y1 receptor activity. Interestingly pre-treatment of platelets with these PKC inhibitors increases the surface expression of both receptors.

In conclusion this study is the first to reveal a previously unappreciated role of specific PKC isoforms in the regulation of platelet P2Y receptor function.

Hardy et al. (2005). Blood 105, 3552-3560