Todd M. Quinton

Platelet purinergic receptors.

Activation of P2Y(1) and P2Y(12) receptors, through secreted ADP that is stimulated by agonists such as thrombin, thromboxane and collagen, is a major mechanism of platelet activation. P2X(1) receptors also participate in platelet shape change and potentiation of calcium mobilization. The cloning of the P2Y(12) receptor and its subsequent knockout in mice promises further understanding of its downstream signaling events.

ADP receptors--targets for developing antithrombotic agents.

Platelet P2 receptors--P2Y1, P2Y12, and P2X1--constitute the means by which adenine nucleotides can activate platelets. Coactivation of the Galphaq-coupled P2Y1 and Galphai2-coupled P2Y12 receptors is necessary for ADP-mediated platelet activation, which forms the basis of using P2 antagonists as antithrombotic drugs. P2Y1 receptor antagonists inhibit platelet activation, while P2Y1 knockout mice show longer bleeding times than normal mice but few other problems; however, its ubiquitous expression in other tissues renders P2Y1 questionable as an antithrombotic target. The P2Y12 receptor is expressed nearly exclusively in platelets and brain, making it an attractive antithrombotic target. Antagonists for the P2Y12 receptor have been developed that either require metabolic activation to covalently inhibit P2Y12 and are irreversible, or simply are competitive in nature and thus reversible. Ticlopidine and clopidogrel are irreversible P2Y12 antagonists and have been repeatedly proven as clinical antithrombotic agents. In addition, a recently reported P2Y12 antagonist, CS-747, shows promise as a future antithrombotic drug. The AR-C series of compounds represent reversible P2Y12 antagonists and have been used extensively to characterize the function of P2Y12 in platelets. Clinical studies show that AR-C69931MX is as effective as clopidogrel; furthermore, the combination of AR-C69931MX (cangrelor) and clopidogrel confers greater antagonism of P2Y12 than either antagonist alone. The P2X1 receptor is a calcium channel that functions to potentiate agonist-induced platelet shape change, and its inhibition or loss has little if any effect on hemostasis. A combination of P2Y1 and P2Y12 antagonists may represent an additional course of antithrombotic treatment.

Lipid rafts are required in Gαi signaling downstream of the P2Y12 receptor during ADP‐mediated platelet activation

Summary.  ADP is important in propagating hemostasis upon its secretion from activated platelets in response to other agonists. Lipid rafts are microdomains within the plasma membrane that are rich in cholesterol and sphingolipids, and have been implicated in the stimulatory mechanisms of platelet agonists. We sought to determine the importance of lipid rafts in ADP‐mediated platelet activation via the G protein‐coupled P2Y1 and P2Y12 receptors using lipid raft disruption by cholesterol depletion with methyl‐β‐cyclodextrin. Stimulation of cholesterol‐depleted platelets with ADP resulted in a reduction in the extent of aggregation but no difference in the extent of shape change or intracellular calcium release. Furthermore, repletion of cholesterol to previously depleted membranes restored ADP‐mediated platelet aggregation. In addition, P2Y12‐mediated inhibition of cAMP formation was significantly decreased upon cholesterol depletion from platelets. Stimulation of cholesterol‐depleted platelets with agonists that depend upon Gαi activation for full activation displayed significant loss of aggregation and secretion, but showed restoration when simultaneously stimulated with the Gαz‐coupled agonist epinephrine. Finally, Gαi preferentially localizes to lipid rafts as determined by sucrose density centrifugation. We conclude that Gαi signaling downstream of P2Y12 activation, but not Gαq or Gαz signaling downstream of P2Y1 or α2A activation, respectively, has a requirement for lipid rafts that is necessary for its function in ADP‐mediated platelet activation.

Different G protein‐coupled signaling pathways are involved in α granule release from human platelets

Summary.  Alpha granule release plays an important role in propagating a hemostatic response upon platelet activation. We evaluated the ability of various agonists to cause α granule release in platelets. Alpha granule release was measured by determining P‐selectin surface expression in aspirin‐treated washed platelets. ADP‐induced P‐selectin expression was inhibited both by MRS 2179 (a P2Y1 selective antagonist) and AR‐C69931MX (a P2Y12 selective antagonist), suggesting a role for both Gαq and Gαi pathways in ADP‐mediated α granule release. Consistent with these observations, the combination of serotonin (a Gαq pathway stimulator) and epinephrine (a Gαz pathway stimulator) also caused α granule release. Furthermore, U46619‐induced P‐selectin expression was unaffected by MRS 2179 but was dramatically inhibited by AR‐C69931, indicating a dominant role for P2Y12 in U46619‐mediated α granule release. Additionally, the Gα12/13‐stimulating peptide YFLLRNP potentiated α granule secretion in combination with either ADP or serotonin/epinephrine costimulation but was unable to induce secretion by itself. Finally, costimulation of the Gαi and Gα12/13 pathways resulted in a significant dose‐dependent increase in α granule release. We conclude that ADP‐induced α granule release in aspirin‐treated platelets occurs through costimulation of Gαq and Gαi signaling pathways. The P2Y12 receptor plays an important role in thromboxane A2‐mediated α granule release, and furthermore activation of Gα12/13 and Gαq signaling pathway can cause α granule release.

Glycoprotein VI agonists have distinct dependences on the lipid raft environment

Summary.  Background: It has been reported that the association of glycoprotein VI (GPVI) with lipid rafts regulates GPVI signaling in platelets. Objective: Secreted adenosine 5′‐diphosphate (ADP) potentiates GPVI‐induced platelet aggregation at particular agonist concentrations. We have investigated whether the decrease in GPVI signaling, previously reported in platelets with disrupted rafts, is a result of the loss of agonist potentiation by ADP. Methods: We disrupted platelet lipid rafts with methyl‐β‐cyclodextrin and measured signaling events downstream of GPVI activation. Results: Lipid raft disruption decreases aggregation induced by low concentrations of convulxin, but this decrease is almost eliminated in the presence of ADP antagonists. Signaling indicators, such as protein phosphorylation and calcium mobilization, were not affected by raft disruption in collagen or convulxin stimulated platelets. Interestingly, however, raft disruption directly reduced GPVI signaling induced by collagen‐related peptide. Conclusions: Lipid rafts do not directly contribute to signaling by the physiologic agonist collagen. The effects of disruption of lipid rafts in in vitro assays can be attributed to inhibition of ADP feedback that potentiates GPVI signaling.

Do heterotrimeric G proteins redistribute upon G protein-coupled receptor stimulation in platelets?

Previous studies have proposed that stimulation of G protein-coupled receptors can cause a redistribution of G proteins to other receptors. The redistribution would cause a greater functional sensitivity of unsensitized ‘secondary’ receptors toward their agonists. Using platelets as a model system, we utilized a proximal signaling event, intracellular calcium mobilization, to determine if agonist stimulation of particular Gq-coupled receptors would result in increased sensitivity for stimulation of other Gq-coupled receptors. Platelets express three Gq-coupled receptors for thrombin, thromboxane A2, and ADP with different potencies. Varying concentrations of a primary agonist (PAR-1 agonist SFLLRN, or the TXA2 agonist U46619) was followed by a constant submaximal concentration of a secondary agonist (U46619, or the P2Y1 agonist ADP). We observed that initial stimulation by SFLLRN was followed by a decrease in the extent of secondary U46619 or ADP-mediated calcium mobilization in comparison to control responses (i.e. without primary stimulation). To extend these studies we examined calcium mobilization in platelets from mice that were either wild-type or homozygous null for the PAR-4 or P2Y1 receptors, hypothesizing that the loss of PAR-4 or P2Y1 receptors would cause redistribution of its Gαq proteins to other receptors, and elicit a greater response when stimulated with other agonists than in platelets from a wild-type mouse. However, our results showed almost identical levels of peak calcium between wild-type or PAR-4 null mice when stimulated with either ADP or U46619. Similar results were obtained for the P2Y1 null mice stimulated with AYPGKF or U46619. We conclude that stimulation of one Gq coupled receptor does not result in redistribution of Gq to other Gq-coupled receptors.