Succinate independently stimulates full platelet activation via cAMP and phosphoinositide 3‐kinase‐β signaling

Abstract

Summary. Background: The citric cycle intermediate succinate has recently been identified as a ligand for the G-proteincoupled receptor (GPCR) SUCNR1. We have previously found that this receptor is one of the most highly expressed GPCRs in human platelets. Objective: The aim of this study was to investigate the role of SUCNR1 in platelet aggregation and to explore the signaling pathways of this receptor in platelets. Methods and Results: Using real-time-PCR, we demonstrated that SUCNR1 is expressed in human platelets at a level corresponding to that of the P2Y 1 receptor. Light transmission aggregation experiments showed dose-dependent aggregation induced by succinate, reaching a maximum response at 0.5 mM. The effect of succinate on platelet aggregation was confirmed with flow cytometry, showing increased surface expression of activated glycoprotein IIb-IIIa and P-selectin. Intracellular SUCNR1 signaling was found to result in decreased cAMP levels, Akt phosphorylation mediated by phosphoinositide 3-kinase-b activation, and receptor desensitization. Furthermore, succinate-induced platelet aggregation was demonstrated to depend on Src, generation of thromboxane A 2 , and ATP release. Platelet SUCNR1 is subject to desensitization through both homologous and heterologous mechanisms. In addition, the P2Y 12 receptor inhibitor ticagrelor completely prevented platelet aggregation induced by succinate. Conclusions: Our experiments show that succinate induces full aggregation of human platelets via SUCNR1. Succinate-induced platelet aggregation depends on thromboxane A 2 generation, ATP release, and P2Y 12 activation.

Insight into SUCNR1 (GPR91) structure and function

“…Several authors repeatedly confirmed G i activation by demonstrating a decrease of cAMP levels upon succinate binding to SUCNR1 (Fig. 3), in both heterologous and native systems (Gilissen et al, 2015;Gnana-Prakasam et al, 2011 ;Hakak et al, 2009;Hogberg et al, 2011 ;Sundström et al, 2013). However, the view of SUCNR1 being coupled to G q has been challenged and it was proposed by several authors that the observed [Ca 2+ ] i mobilization was a consequence of PLC-β activation by the βγ dimer (Fig.…”

“…In contrast, Robben et al, showed that SUCNR1 is rapidly desensitized and re-sensitized but not internalized in polarized MDCK cells (Robben et al, 2009). Hogberg et al also showed desensitization of SUCNR1 in platelets (Hogberg et al, 2011). The coupling of activated receptor to arrestins 2 and 3 is very weak (Gilissen et al, 2015;Southern et al, 2013).…”

“…Interestingly this pathway is known to be activated by G i in cardiac myocytes and its inhibition to convert survival to apoptotic signaling (Zhu et al, 2001). (Rubic et aL, 2008) ERK1/2 phosphorylation ↑ TNF-α, IL-1β and cytokines (Rubic et al, 2008) iDC migration Platelet (Macaulay et al, 2007) ↓ cAMP and Src kinase activation via G i PI3K activation, Akt phosphorylation via βγ Pselectin and glycoprotein (GP)IIb-IIIa activation (Hogberg et al, 2011 ) Crosstalk with other GPCRs (ADP, TXA2) (Spath et al, 2012) ↓ platelet aggregation (Macaulay et al, 2007;Spath et al, 2012) (Potentiation of (Spath et al, 2012)) platelet aggregation (Hogberg et al, 2011) Inhibition of antiplatelets Monocytes T and B (Rubic et al, 2008) ↑ TNFα and IFN--γ (Rubic et al, 2008) Immunity (Rubic et al, 2008) …”

“…The precise mechanism of this effect remains unknown, but has been rediscovered and investigated more recently (Hogberg et al, 2011;Macaulay et al, 2007;Spath, Hansen, Bokemeyer, & Langer, 2012).…”

“…In addition, SUCNR1 has been identified as one of the most highly expressed GPCRs in human platelets, at a level corresponding to the P2Y 1 receptor, indicating a potential important role in platelet physiology (Amisten et al, 2008 ;Hogberg et al, 2011 ;Macaulay et al, 2007 ;Spath et al, 2012).…”