Quantification of ADP and ATP receptor expression in human platelets

Abstract

Summary. The mechanism of ADP-mediated platelet activation has been difficult to unravel due to the large number of receptors for extracellular nucleotides (P2 receptors). mRNA levels in circulating platelets are very low, but have been shown to be translationally active. By optimizing mRNA extraction and using real time (RT)-PCR we were able to establish a protocol for highly sensitive platelet mRNA quantification in human regular blood samples. In platelets from healthy volunteers, only P2X 1 , P2Y 1 and P2Y 12 were found in significant levels, with the following order of expression: P2Y 12 >> P2X 1 > P2Y 1 . Other P2 receptors (P2Y 2 , P2Y 4 , P2Y 6 , P2Y 11 , P2Y 13 , P2X 4 , P2X 7 ) had very low expression. As a control measurement to exclude contamination, P2 receptors in buffy coat were quantified but had a completely different profile. Incubation in vitro revealed a more rapid degradation rate for P2X 1 receptor mRNA than for P2Y 1 and P2Y 12 , indicating that the level of P2X 1 may be relatively higher in newly released platelets and in megacaryocytes. In conclusion, we have developed the first protocol for quantifying mRNA expression in human platelets limiting the P2 receptor drug development targets to P2Y 12 , P2Y 1 and P2X 1 . Furthermore, the method could be used to study platelet expression for any gene in human materials.Keywords: P2X receptor, P2Y receptor, platelets, real time-PCR, Western blot.Platelets play a crucial role in the maintenance of normal hemostasis and are involved in the development of pathological thrombus formation leading to vascular occlusion which is an important mechanism in myocardial infarction and stroke [1,2]. A large number of endogenous mediators can activate platelets, such as thromboxane A2, adenosine diphosphate (ADP), collagen, von Willebrand factor, thrombin, epinephrine and 5-hydroxitryptamine (5-HT). To get full activation of the platelets, these agonists are dependent on two positive feedback loops: the formation of thromboxane A2 by cyclooxygenase in the platelets and the release of ADP from dense platelet granules. Thromboxane A2 and ADP then activates specific receptors on the extracellular side of the platelet membrane.Therapeutic intervention aimed at the first positive feedback loop by inhibiting cyclooxygenase with aspirin is highly efficient in reducing death and cardiovascular events. However, ADP may be even more important as evidenced by the CAPRIE study, in which the ADP receptor inhibition was more beneficial than aspirin in reducing cardiovascular events [3,4].Recently, progress has been made in the understanding of the mechanisms of ADP mediated platelet aggregation, where at least three receptors are considered to be involved; the P2Y 12 , P2Y 1 , and P2X 1 receptors [5]. The importance of the P2Y 12 receptor is proven by the effects of clopidogrel, which after metabolization in the liver, acts as an irreversible antagonist at P2Y 12 receptors. Knockout of the P2Y 1 receptor in mice has demonstrated its importance for thrombus formation, blee...

The active metabolite of Clopidogrel disrupts P2Y12 receptor oligomers and partitions them out of lipid rafts

“…The oligomeric association of P2Y12 receptors at the plasma membrane was shown to depend on disulfide bond formation as demonstrated by the sensitivity of these complexes to DTT reduction after cell solubilization. This finding indicated that the P2Y12 receptor appears to behave in a similar way as ␤2AR in response to thiol-reducing agents, unlike P2Y1 receptors, which oligomers are resistant to reducing reagents (64). In contrast to some other GPCRs (3-13), the oligomerization of P2Y12 does not seem to be modulated by agonist binding, 2MeS-ADP being ineffective in changing the P2Y12 size distribution (data not shown).…”

ADP Acting on P2Y 13 Receptors Is a Negative Feedback Pathway for ATP Release From Human Red Blood Cells

“…1,2 Extracellular ATP in the circulation is rapidly degraded into ADP, AMP, and adenosine by ectonucleotidases. 3 ATP and ADP activate P2 receptors on endothelium, platelets, 4,5 and other blood cells, 6 regulating several physiological responses including vascular tone, 7 platelet aggregation, and the release of endothelial factors. At least 15 nucleotide-activated cell surface receptors (7 of P2X and 8 of P2Y) have been found in man, with remarkably broad and varied physiological responses.…”

Adenylic Dinucleotides Produced by CD38 Are Negative Endogenous Modulators of Platelet Aggregation

“…P2Y 11 mRNA levels in platelets were reported to be much lower than those of P2Y 1 , P2Y 12 , and P2X 1 , although the level of mRNA depends on both its synthesis and degradation rates and may not linearly correlate with its protein expression (34). Also in human granulocytes, P2Y 11 mRNA is present at lower levels compared with mRNA of other purinergic receptors (40).…”

“…6A; control, n ϭ 5, p Ͻ 0.05). The only purinergic receptor known to stimulate adenylic cyclase is P2Y 11 (10,33), which is also expressed in human platelets (34). Preincubation of platelets for 30 min with 1 M NF157, a specific inhibitor of P2Y 11 (35), reduced cAMP overproduction induced by P18, P24, and Ap2A by ϳ70, 75, and 100%, respectively (Fig.…”