Background: 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.

Aims: We evaluated the role of PI3 kinase in the activation of Rap1B downstream of ADP receptors

Methods: 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 Rap1B activation by a pulldown assay with a kit from cell signaling. We measured the phosphorylation of Akt on Ser473 as an indicator of PI3-kinase activity.

Results: LY294002 and ARC69931MX abolished 2MeSADP-induced Akt phosphorylation. MRS2179 reduced 2MeSADP-induced Akt phosphorylation but did not abolish it. Rap1b activity, however, was only reduced, but not ablated, using LY294002 and was completely inhibited by ARC69931MX or MRS2179. Similarly, 2MeSADP-induced Akt phosphorylation was abolished in the P2Y12 knock out mice platelets without much effect on Rap1B. Conversely, Rap1B activation was abolished, while Akt phosphorylation was unaffected, in the P2Y1 knock out mouse platelets.

Conclusion(s): 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 Rap1 through RASA3 in a pathway independent of PI3-kinase.

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Figure 1: 2-MeSADP-induced Rap1b activation and Akt-Ser473- in the presence of a PI3K inhibitor or P2Y1 or P2Y12 antagonists. Isolated human platelets were pre-incubated with either 6 M LY294002 for 5 minutes or 100 nM AR-C69931MX for 60 seconds or 10 M MRS2179 for 60 seconds at 37oC and then activated with 200 nM 2-MeSADP for 30 seconds for Rap1b activation or 3 minutes for Akt-Ser473- phosphorylation. Figure 1A is a representative aggregometer tracing and Figure 1B is a representative Western blot. Figures 1C and 1D represent the means and standard error of at least 3 independent experiments.

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Figure 2: 2-MeSADP-induced Rap1b activation and Akt-Ser473- phosphorylation in P2Y12 or P2Y1 deficient platelets. Platelets were isolated from WT, P2Y12 or P2Y1 knockout mice and activated with 200 nM 2-MeSADP for 30 seconds for Rap1b activation or 3 minutes for Akt-Ser473- phosphorylation. Figure 2A is a representative aggregometer tracing and Figure 2B is a representative Western blot. Figures 2C and 2D represent the means and standard errors of 3 independent experiments.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/evidence-for-a-pi3-kinase-independent-pathway-in-the-regulation-of-rap1b-activation-downstream-of-the-p2y12-receptor-in-platelets/