Diverse signalling of the platelet P2Y1 receptor leads to a dichotomy in platelet function

Abstract

Platelet P2Y receptor signalling via RhoGTPases is necessary for platelet-dependent leukocyte recruitment, where no platelet aggregation is observed. We investigated signalling cascades involved in distinct P2Y-dependent platelet activities in vitro, using specific inhibitors for phospholipase C (PLC) (U73122, to inhibit the canonical pathway), and RhoGTPases: Rac1 (NSC23766) and RhoA (ROCK inhibitor GSK429286). Human platelet rich plasma (for platelet aggregation) or isolated washed platelets (for chemotaxis assays) was treated with U73122, GSK429286 or NSC23766 prior to stimulation with adenosine diphosphate (ADP) or the P2Y specific agonist MRS2365. Aggregation, chemotaxis (towards f-MLP), or platelet-induced human neutrophil chemotaxis (PINC) towards macrophage derived chemokine (MDC) was assessed. Molecular docking of ADP and MRS2365 to P2Y was analysed using AutoDock Smina followed by GOLD molecular docking in the Accelrys Discovery Studio software. Inhibition of PLC, but not Rac1 or RhoA, suppressed platelet aggregation induced by ADP and MRS2365. In contrast, platelet chemotaxis and PINC, were significantly attenuated by inhibition of platelet Rac1 or RhoA, but not PLC. MRS2365, compared to ADP had a less pronounced effect on P2Y-induced aggregation, but a similar efficacy to stimulate platelet chemotaxis and PINC, which might be explained by differences in molecular interaction of ADP compared to MRS2365 with the P2Y receptor. Platelet P2Y receptor activation during inflammation signals through alternate pathways involving Rho GTPases in contrast to canonical P2Y receptor induced PLC signalling. This might be explained by selective molecular interactions of ligands within the orthosteric site of the P2Y receptor.

A dichotomy in platelet activation: Evidence of different functional platelet responses to inflammatory versus haemostatic stimuli

“…Differences in the kinetics of platelet Akt, ERK1/2 and p38 phosphorylation in response to thrombin and to the inflammatory TLR2/1 receptor agonist lipopeptide Pam3CSK4 have been previously reported [22]. Moreover, we have previously shown that ADP stimulation triggering inflammatory events (pulmonary leukocyte recruitment) acts through activating the P2Y 1 receptors and small Rho GTPases Rac1 and RhoA [9,10], whilst it acts through the classical PLC pathway when triggering haemostatic responses, possibly as a result of differential molecular docking of endogenous agonists to the receptor [21]. However, those studies demonstrated the different signalling pathways induced by a single agonist leading to different platelet functional responses, and did not investigate the effect of different platelet stimuli on platelet responses.…”

“…18 trauma, where both haemostatic pathways and host defence are required [48]. Indeed, we and others have shown the requirement for some agents, including ADP, as a primer [39] necessary for robust platelet migration towards fMLP [21], and platelet-induced neutrophil chemotaxis towards MDC [9,21]. Further research is therefore warranted to investigate whether co-stimulation of platelets with low dose ADP and other inflammatory stimuli leads to potentiation of chemokine-induced platelet transmigration or other functions associated with inflammation.…”

“…We have previously shown that platelet-induced neutrophil chemotaxis towards MDC is dependent on the simultaneous presence of ADP, and ADP has also been implicated as a co-agonist alongside chemokines in leukocyte activation and adhesion molecule expression [9,21,39]. This may suggest that platelet-leukocyte complex formation may either be involved in priming platelets, allowing the subsequent potentiation of certain functional responses [48], or that these interactions require the concomitant stimulation of ADP receptors alongside chemokine receptors.…”

“…Furthermore, distinct non-canonical (RhoA, Rac-1) signalling events downstream of platelet P2Y 1 receptor activation by ADP have been shown to control platelet motility, and interactions with neutrophils, in contrast to ADP-induced aggregation and the canonical P2Y 1 receptor PLC signalling cascade [21]. This functional selectivity in signalling reveals the importance of different molecular signalling pathways of platelet activation during functional activities pertinent to inflammation compared to haemostasis [21]. Stimulation of platelets with either inflammatory or haemostatic stimuli also appears to induce different patterns of phosphorylation of intracellular signalling kinases, including Akt, ERK1, ERK2 and p38 [22].…”

“…Either haemostatic or inflammatory agonists were added to the lower compartment volumes followed as per manufacturer's instructions, while washed platelets (2x10 7 /ml), isolated from ACD-anticoagulated human venous blood, were placed in the upper compartment. Chambers were then kept in a humidified atmosphere (5% CO 2 , pH 7.4, 37°C) for 90 minutes, as previously reported [21]. Migrated platelets positively stained for CD41 were then counted by flow cytometry using Flow-Count fluorospheres (Beckman Coulter, Miami, FL, USA).…”