Evidence for a PI3-kinase independent pathway in the regulation of Rap1b activation downstream of the P2Y12 receptor in platelets

Abstract

Platelet activation by adenosine diphosphate (ADP) is mediated through two G-protein-coupled receptors, P2Y1 and P2Y12, which signal through Gq and Gi, respectively. P2Y1 stimulation leads to phospholipase C activation and an increase in cytosolic calcium necessary for CalDAG-GEF1 activation. Engagement of P2Y12 inhibits adenylate cyclase, which reduces cAMP, and activation of PI3-kinase, which inhibits RASA3 resulting in sustained activated Rap1b. In this study we activated human platelets with 2-MeSADP in the presence of LY294002, a PI3-kinase inhibitor, AR-C69931MX, a P2Y12 antagonist or MRS2179, a P2Y1 antagonist. We measured the phosphorylation of Akt on Ser473 as an indicator of PI3-kinase activity. As previously shown, LY294002 and ARC69931MX abolished 2MeSADP-induced Akt phosphorylation. MRS2179 reduced ADP-induced Akt phosphorylation but did not abolish it. Rap1b activation, however, was only reduced, but not ablated, using LY294002 and was completely inhibited by ARC69931MX or MRS2179. Furthermore, 2MeSADP-induced Rap1b activation was abolished in either P2Y1 or P2Y12 null platelets. These data suggest that ADP-induced Rap1b activation requires both P2Y1 and P2Y12. In addition, although stimulation of P2Y12 results in PI3-kinase activation leading to Akt phosphorylation and Rap1b activation, Rap1b activation can occur independently of PI3-kinase downstream of P2Y12. Thus, we propose that the P2Y12 receptor can regulate Rap1b, possibly through RASA3, in a pathway independent of PI3-kinase.

Keywords:

• PI3K
• Rap1B
• RASA3

Acknowledgements

We thank Monica Wright for her expert technical assistance in mouse colony maintenance, breeding, genotyping and in vivo thrombosis experiments.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by grants HL155694, HL137721, HL137207, HL132171, and HL93231 from the National Institutes of Health (to S. P. K). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health; NHLBI [HL132171, HL137207, HL137721, HL155694, HL93231].