Diversity of signaling underlying responses mediated by selective activation of the platelet thromboxane A 2 (TXA 2 ) receptor; pharmacological and kinetic studies in vitro

Abstract

Selective activation of the platelet TXA2 receptor is sufficient to mediate concurrent aggregation, deaggregation and shape change (SC) responses without activation of known Gi-coupled receptors (Platelets 2003; 14: 89). However, Gi-coupled receptor activation strongly influences the hemostasis response in vivo. This study investigated the modulatory effects of two signaling pathways related to Gi-coupled receptor activation, stimulation of phosphoinositide 3-kinases (PI3Ks) and inhibition of adenylyl cyclase (AC), on the aggregation, deaggregation and SC components of the platelet activation response. A novel turbidimetric approach was applied to separate these responses and to characterize their pharmacology and kinetics. The SC response was more sensitive to TXA2 receptor activation (lower EC50 value) but less sensitive to a TXA2 receptor antagonist (higher Kd value) than the net aggregation response. Epinephrine and sulprostone, agonists of the Gi-coupled alpha2A-adrenoceptor and EP3 receptor, respectively, amplified the SC, decelerated deaggregation and enhanced net aggregation responses. SQ22536 and 2',5'-dideoxyadenosine, inhibitors of AC activity, elicited smaller but qualitatively similar effects. The PI3K inhibitor wortmannin did not affect the SC response but accelerated deaggregation and inhibited net aggregation. These data are consistent with a differential modulation of the platelet SC response by each pathway associated with Gi-coupled receptor activation, while both pathways cooperatively enhance the net aggregation response by decelerating deaggregation. We propose that the TXA2 receptor mediated concurrent platelet aggregation and SC responses, that are differentially modulated by different signaling pathways, provide a model for studying the underlying cellular pharmacology of platelet physiology.

The role of prostanoid receptors in mediating the effects of PGE2on human platelet function

“…Promotion of platelet aggregation by sulprostone via an effect at EP3 receptors is already well established [7,10,11,[13][14][15][16][17], as is the ability of EP3 to link with adenylyl cyclase via G i and potentiate platelet aggregation via a decrease in cAMP [10,11,13,17]. We have demonstrated that sulprostone is able to decrease basal VASP phosphorylation and the concentrations needed to bring this about compare very well with the concentrations required to potentiate platelet aggregation.…”

“…There is good evidence that potentiation of platelet function is via effects at EP3 receptors linked to G i and inhibition of adenylyl cyclase [7,10,11,[13][14][15][16][17]. It is currently believed that inhibition of platelet aggregation may be via interaction with IP receptors linked to G s and stimulation of adenylyl cyclase [10,11], although this is mainly based on experiments conducted using mouse platelets [10] and the involvement of other receptors has not been fully explored.…”

Detection of P2Y14 protein in platelets and investigation of the role of P2Y14 in platelet function in comparison with the EP3 receptor

“…We compared the effectsofUDP-glucosewith thoseofthe EP 3 receptor agonist sulprostone, aprostaglandin E 2 (PGE 2 )analogue (18).The stimulated EP 3 receptor potentiates plateletfunction by coupling to inhibitoryGp roteins, whichr esults in inhibitiono fa denylate cyclase (19)(20)(21). Sulprostone has been shown to potentiate primarya ggregation responses caused by several primary plateletagonistsincluding ADP,plateletactivating factor (PAF), 11,9-epoxymethano PGH 2 (U-46619),c ollagen, thrombin receptor activating peptide (TRAP)a nd 5-hydroxytryptamine (18,(22)(23)(24). It wasusedhere as apositivecontrol for the studies on the functionalityofthe P2Y 14 receptor.…”