Pathophysiological consequences of receptor mistraffic:Tales from the platelet P2Y12 receptor

Genetic variations in G protein-coupled receptor (GPCR) genes can disrupt receptor function in a wide variety of human genetic diseases, including platelet bleeding disorders. Platelets are critical for haemostasis with inappropriate platelet activation leading to the development of arterial thrombosis, which can result in heart attack and stroke whilst decreased platelet activity is associated with an increased risk of bleeding. GPCRs expressed on the surface of platelets play key roles in regulating platelet activity and therefore function. Receptors include purinergic receptors (P2Y1 and P2Y12), proteinase-activated receptor (PAR1 and PAR4) and thromboxane receptors (TPα), among others. Pharmacological blockade of these receptors forms a powerful therapeutic tool in the treatment and prevention of arterial thrombosis. With the advance of genomic technologies, there has been a substantial increase in the identification of naturally occurring rare and common GPCR variants. These variants include single-nucleotide polymorphisms (SNPs) and insertion or deletions that have the potential to alter GPCR expression or function. A number of defects in platelet GPCRs that disrupt receptor function have now been characterized in patients with mild bleeding disorders. This review will focus on rare, function-disrupting variants of platelet GPCRs with particular emphasis upon mutations in the P2Y12 receptor gene that affect receptor traffic to modulate platelet function. Further this review will outline how the identification and characterization of function-disrupting GPCR mutations provides an essential link in translating our detailed understanding of receptor traffic and function in cell line studies into relevant human biological systems

Carbonic Anhydrase Inhibitors Suppress Platelet Procoagulant Responses and In Vivo Thrombosis

Carbonic anhydrase (CA) inhibitors have a long history of safe clinical use as mild diuretics, in the treatment of glaucoma and for altitude sickness prevention. In this study, we aimed to determine if CA inhibition may be an alternative approach to control thrombosis. We utilized a high-resolution dynamic imaging approach to provide mechanistic evidence that CA inhibitors may be potent anti-procoagulant agents in vitro and effective anti-thrombotics in vivo. Acetazolamide and methazolamide, while sparing platelet secretion, attenuated intracellular chloride ion entry and suppressed the procoagulant response of activated platelets in vitro and thrombosis in vivo. The chemically similar N-methyl acetazolamide, which lacks CA inhibitory activity, did not affect platelet procoagulant response in vitro. Outputs from rotational thromboelastometry did not reflect changes in procoagulant activity and reveal the need for a suitable clinical test for procoagulant activity. Drugs specifically targeting procoagulant remodeling of activated platelets, by blockade of carbonic anhydrases, may provide a new way to control platelet-driven thrombosis without blocking essential platelet secretion responses

P2Y12 receptor modulation of ADP‐evoked intracellular Ca2+ signalling in THP‐1 human monocytic cells

Background and purpose: The Gi‐coupled, ADP‐activated P2Y12 receptor is well characterised as playing a key role in platelet activation via crosstalk with P2Y1 in ADP‐evoked intracellular Ca2+ response. There is limited knowledge on the role of P2Y12 in ADP‐evoked Ca2+ responses in other blood cells. Here we investigate the role of P2Y12 receptor activation in modulation of ADP‐evoked Ca2+ responses in human THP‐1 monocytic cells. Experimental approach: A combination of intracellular Ca2+ measurements, RT‐PCR, immunocytochemistry, leukocyte isolation and siRNA‐mediated gene knockdown were used to identify the role of P2Y12 receptor activation. Key results: ADP‐evoked intracellular Ca2+ responses (EC50 2.7 M) in THP‐1 cells were abolished by inhibition of phospholipase C (U73122) or sarco/endoplasmic reticulum Ca2+‐ATPase (thapsigargin). Loss of ADP‐evoked Ca2+ responses following treatment with MRS2578 (IC50 200 nM) revealed a major role for P2Y6 in mediating ADP‐evoked Ca2+ responses. ADP‐evoked responses were attenuated either with pertussis toxin treatment, or P2Y12 inhibition with two chemically distinct antagonists (ticagrelor, IC50 5.3 M; PSB‐0739, IC50 5.6 M). ADP‐evoked responses were suppressed following siRNA‐mediated P2Y12 gene knockdown. The inhibitory effects of P2Y12 antagonists were fully reversed following adenylate cyclase inhibition (SQ22536). P2Y12 receptor expression was confirmed in freshly isolated human CD14+ monocytes. Conclusion and implications: Taken together, these data suggest that P2Y12 activation positively regulates P2Y6‐mediated intracellular Ca2+ signalling through suppression of adenylate cyclase activity in human monocytic cells

Inverse agonism at the P2Y₁₂ receptor and ENT1 transporter blockade contribute to platelet inhibition by ticagrelor

Ticagrelor is a potent antagonist of the P2Y(12) receptor (P2Y(12)R) and consequently an inhibitor of platelet activity effective in the treatment of atherothrombosis. Here, we sought to further characterize its molecular mechanism of action. Initial studies showed that ticagrelor promoted a greater inhibition of adenosine 5′-diphosphate (ADP)–induced Ca(2+) release in washed platelets vs other P2Y(12)R antagonists. This additional effect of ticagrelor beyond P2Y(12)R antagonism was in part as a consequence of ticagrelor inhibiting the equilibrative nucleoside transporter 1 (ENT1) on platelets, leading to accumulation of extracellular adenosine and activation of G(s)-coupled adenosine A(2A) receptors. This contributed to an increase in basal cyclic adenosine monophosphate (cAMP) and vasodilator-stimulated phosphoprotein phosphorylation (VASP-P). In addition, ticagrelor increased platelet cAMP and VASP-P in the absence of ADP in an adenosine receptor–independent manner. We hypothesized that this increase originated from a direct effect on basal agonist-independent P2Y(12)R signaling, and this was validated in 1321N1 cells stably transfected with human P2Y(12)R. In these cells, ticagrelor blocked the constitutive agonist-independent activity of the P2Y(12)R, limiting basal G(i)-coupled signaling and thereby increasing cAMP levels. These data suggest that ticagrelor has the pharmacological profile of an inverse agonist. Based on our results showing insurmountable inhibition of ADP-induced Ca(2+) release and forskolin-induced cAMP, the mode of antagonism of ticagrelor also appears noncompetitive, at least functionally. In summary, our studies describe 2 novel modes of action of ticagrelor, inhibition of platelet ENT1 and inverse agonism at the P2Y(12)R that contribute to its effective inhibition of platelet activation