Variable effect of P2Y12 inhibition on platelet thrombus volume in flowing blood

Summary. Background and objectives: Patients treated with percutaneous coronary intervention receive aspirin and P2Y12 ADP receptor inhibitors to reduce thrombotic complications. The choice of methodology for monitoring the effects of treatment and assessing its efficacy is still a topic of debate. We evaluated how decreased P2Y12 function influences platelet aggregate (thrombus) size measured ex vivo. Methods and results: We used confocal videomicroscopy to measure in real time the volume of platelet thrombi forming upon blood perfusion over fibrillar collagen type I at a wall shear rate of 1500 s^?1. The average volume was significantly smaller in 31 patients receiving aspirin and clopidogrel (19) or ticlopidine (12) than in 21 controls, but individual values were above the lower limit of the normal distribution, albeit mostly within the lower quartile, in 61.3% of cases. Disaggregation of platelet thrombi at later perfusion times occurred frequently in the patients. Vasodilator‐stimulated phosphoprotein phosphorylation, reflecting P2Y12 inhibition, was also decreased in the patient group, and only 22.6% of individual values were above the lower normal limit. We found no correlation between volume of thrombus formed on collagen fibrils and level of P2Y12 inhibition, suggesting that additional and individually variable factors can influence the inhibitory effect of treatment on platelet function. Conclusions: Measurements of platelet thrombus formation in flowing blood reflects the consequences of antiplatelet therapy in a manner that is not proportional to P2Y12 inhibition. Combining the results of the two assays may improve the assessment of thrombotic risk.     

P2Y12 regulates platelet adhesion/activation,thrombus growth,and thrombus stability in injured arteries

The critical role for ADP in arterial thrombogenesis was established by the clinical success of P2Y12 antagonists, currently used at doses that block 40-50% of the P2Y12 on platelets. This study was designed to determine the role of P2Y12 in platelet thrombosis and how its complete absence affects the thrombotic process. P2Y12-null mice were generated by a gene-targeting strategy. Using an in vivo mesenteric artery injury model and real-time continuous analysis of the thrombotic process, we observed that the time for appearance of first thrombus was delayed and that only small, unstable thrombi formed in P2Y12-/- mice without reaching occlusive size, in the absence of aspirin. Platelet adhesion to vWF was impaired in P2Y12-/- platelets. While adhesion to fibrinogen and collagen appeared normal, the platelets in thrombi from P2Y12-/- mice on collagen were less dense and less activated than their WT counterparts. P2Y12-/- platelet activation was also reduced in response to ADP or a PAR-4-activating peptide. Thus, P2Y12 is involved in several key steps of thrombosis: platelet adhesion/activation, thrombus growth, and stability. The data suggest that more aggressive strategies of P2Y12 antagonism will be antithrombotic without the requirement of aspirin cotherapy and may provide benefits even to the aspirin-nonresponder population.     

Decreased platelet inhibition by P2Y12 receptor blockers in anaemia

Background

Anaemic patients undergoing angioplasty and stenting are at an increased risk of ischaemic events, which may be caused by an inadequate response to antiplatelet therapy with adenosine diphosphate (ADP) P2Y₁₂ inhibitors. In the current study, we investigated the associations between anaemia and on‐treatment platelet reactivity in clopidogrel‐treated (group 1, n = 306) and prasugrel‐/ticagrelor‐treated (group 2, n = 109) patients undergoing elective and acute angioplasty with stent implantation, respectively.

Materials and methods

Monocyte‐platelet aggregate (MPA) formation was determined by flow cytometry in both groups. On‐treatment residual platelet reactivity in response to ADP was assessed by light transmission aggregometry (LTA) in both groups, and by the VerifyNow P2Y₁₂ assay and the Impact‐R in group 1. P‐selectin expression was measured by flow cytometry in group 2.

Results

In both groups, anaemia was associated with significantly higher MPA formation in response to ADP (both P ≤ .02). Moreover, by LTA maximal aggregation in response to ADP was significantly higher in patients with anaemia in both groups (both P < .05), and anaemic patients in group 1 had a significantly higher on‐treatment platelet reactivity by the VerifyNow P2Y₁₂ assay and the Impact‐R than those without anaemia (both P < .001). In group 2, significantly higher platelet surface expression of P‐selectin was seen in anaemia after stimulation with ADP (P = .02).

Conclusion

Anaemia is associated with decreased platelet inhibition by ADP P2Y₁₂ receptor antagonists after elective and acute percutaneous interventions with stent implantation. However, due to inconsistencies between different platelet function tests additional data are needed to clarify the role of anaemia for platelet inhibition.     

Bleeding risk with AZD6140, a reversible P2Y12 receptor antagonist,vs. clopidogrel in patients undergoing coronary artery bypass grafting in the DISPERSE2 trial

AZD6140, the first reversible oral P2Y₁₂ receptor antagonist, exhibits greater and more consistent inhibition of platelet aggregation than the irreversible thienopyridine clopidogrel. As a result of its reversible effect, AZD6140 may pose less risk for bleeding when antiplatelet treatment cannot be stopped at least 5 days before coronary artery bypass graft (CABG) surgery or other invasive procedures. The Dose conflrmation Study assessing anti‐Platelet Effects of AZD6140 vs. clopidogRel in NSTEMI (DISPERSE2) trial showed overall comparable bleeding rates with antiplatelet treatment with AZD6140 90 mg twice daily or 180 mg twice daily vs. clopidogrel 75 mg once daily in 984 patients with non‐ST‐elevation acute coronary syndromes. A post hoc exploratory analysis of bleeding outcomes in the subset of 84 patients undergoing CABG in DISPERSE2 suggests reduced risk for total bleeding (41% and 58% vs. 62%), all major bleeding (38% and 50% vs. 62%), and life‐threatening bleeding (22% and 38% vs. 54%) with AZD6140 90 mg (n = 32) and 180 mg (n = 26) vs. clopidogrel (n = 26) respectively. Trends suggested that major bleeding rates were reduced with AZD6140 (combined groups) vs. clopidogrel when treatment was stopped ≤ 5 days prior to surgery (39% vs. 63%, p = 0.15) but not when treatment was stopped > 5 days before surgery (50% vs. 60%). This observation is consistent with the reversible binding of AZD6140 to the P2Y₁₂ receptor. Further prospective studies are planned to assess the relationship between this potential clinical benefit of AZD6140 and the reversibility of its antiplatelet effects.     

Potentiation of clopidogrel active metabolite formation by rifampicin leads to greater P2Y12 receptor blockade and inhibition of platelet aggregation after clopidogrel

Summary. Background: The thienopyridine P2Y₁₂ receptor antagonist clopidogrel reduces the risk of arterial thrombosis and individual pharmacodynamic responses to clopidogrel are believed to reflect the levels of active metabolite (AM) generated. Rifampicin increases the inhibitory effect of clopidogrel on platelet aggregation (PA). We studied the response to clopidogrel before and during administration of rifampicin in order to study the relationship between individual AM levels and P2Y₁₂ blockade. Methods: Healthy volunteers received a 600‐mg loading dose of clopidogrel followed by 75 mg daily for 7 days and, after a washout period and treatment with rifampicin [300 mg twice a day (b.i.d.)], received the same regimen of clopidogrel. Clopidogrel AM levels were determined over 4 h after the clopidogrel loading dose and unblocked P2Y₁₂ receptor number was assessed using a ³³P‐2MeSADP binding assay. PA was measured by optical aggregometry with ADP and TRAP. Results: Rifampicin enhanced clopidogrel AM production [area‐under‐the‐curve (AUC): clopidogrel 89 ± 22 ng h mL^?1, clopidogrel + rifampicin 335 ± 86 ng h mL^?1, P < 0.0001], and P2Y₁₂ blockade (unblocked receptors: clopidogrel 48 ± 24, clopidogrel + rifampicin 4 ± 2, P < 0.0001) and reduced PA (5 μmol L^?1 ADP: clopidogrel 20 ± 4, clopidogrel + rifampicin 5 ± 2, P < 0.01). Increasing numbers of unblocked receptors were required for an aggregation response with a decreasing concentration of ADP. PA induced by ADP 2 μmol L^?1 was particularly sensitive to low levels of receptor blockade. Conclusion: Potentiation of clopidogrel AM production by rifampicin leads to greater P2Y₁₂ blockade and consequently greater inhibition of PA. PA responses to low concentrations of ADP are more sensitive to P2Y₁₂ blockade.     

Induction of novel agonist selectivity for the ADP-activated P2Y1 receptor versus the ADP-activated P2Y12 and P2Y13 receptors by conformational constraint of an ADP analog

ADP is the cognate agonist of the P2Y1, P2Y12, and P2Y13 receptors. With the goal of identifying a high potency agonist that selectively activates the P2Y1 receptor, we examined the pharmacological selectivity of the conformationally constrained non-nucleotide analog (N)-methanocarba-2MeSADP [(1'S,2'R, 3'S,4'R,5'S)-4-[(6-amino-2-methylthio-9H-purin-9-yl)-1-diphosphoryloxymethyl]bicyclo[3.1.0]hexane-2,3-diol] among the three ADP-activated receptors. Each P2Y receptor was expressed transiently in COS-7 cells, and inositol lipid hydrolysis was quantified as a measure of receptor activity. In the case of the Gi-linked P2Y12 and P2Y13 receptors, a chimeric G protein, Galphaq/i, was coexpressed to confer a capacity of these Gi-linked receptors to activate phospholipase C. 2MeSADP (2-methylthio-ADP) was a potent agonist at all three receptors exhibiting EC50 values in the sub to low nanomolar range. In contrast, whereas (N)-methanocarba-2MeSADP was an extremely potent (EC50=1.2 +/- 0.2 nM) agonist at the P2Y1 receptor, this non-nucleotide analog exhibited no agonist activity at the P2Y12 receptor and very low activity at the P2Y13 receptor. (N)-Methanocarba-2MeSADP also failed to block the action of 2MeSADP at the P2Y12 and P2Y13 receptors, indicating that the (N)-methanocarba analog is not an antagonist at these receptors. The P2Y1 receptor selectivity of (N)-methanocarba-2MeSADP was confirmed in human platelets where it induced the shape change promoted by P2Y1 receptor activation without inducing the sustained platelet aggregation that requires simultaneous activation of the P2Y12 receptor. These results provide the first demonstration of a high-affinity agonist that discriminates among the three ADP-activated P2Y receptors, and therefore, introduce a potentially important new pharmacological tool for delineation of the relative biological action of these three signaling proteins.     

Evaluating the risk-benefit profile of the direct-acting P2Y(12) inhibitor ticagrelor in acute coronary syndromes

Ticagrelor is a direct-acting, oral, reversibly binding P2Y(12) receptor antagonist. As a cyclopentyltriazolopyrimidine, ticagrelor represents a new chemical class of agents that do not require metabolic activation and have consistent ability to inhibit platelet aggregation. The phase 3 PLATelet Inhibition and Patient Outcomes (PLATO) trial (NCT00391872) evaluated ticagrelor compared with clopidogrel in 18 624 patients with acute coronary syndromes (ACS), and demonstrated a significant reduction in the risk of death from vascular causes/myocardial infarction (MI)/stroke with ticagrelor (9.8% vs 11.7% with clopidogrel [hazard ratio, 0.84; 95% confidence interval, 0.77-0.92]; P < 0.001) without a significant increase in PLATO-defined major bleeding (11.6% vs 11.2%, respectively; P = 0.43). Myocardial infarction and death from vascular causes were separately significantly reduced, and death from any cause and stent thrombosis reductions achieved nominal statistical significance. Ticagrelor showed benefit over clopidogrel in almost all patient subgroups, including patients who had previously received clopidogrel, patients with both planned invasive or noninvasive treatment, patients with ST-segment elevation MI (STEMI) referred for primary percutaneous coronary intervention, patients with non-STEMI, and patients who underwent bypass surgery. Hence, the PLATO population reflected specifically those patients who would ordinarily receive thienopyridine-based antiplatelet therapy in a clinical setting. Although there are limitations in directly translating trial findings to clinical practice, the findings of PLATO suggest that for every 1000 ACS patients admitted to hospital, using ticagrelor instead of clopidogrel for 12 months would result in 14 fewer deaths or 11 fewer MIs. This review places the PLATO data in context, and assesses the role that ticagrelor may play in treating patients with ACS.     

Reversibility of platelet P2Y12 inhibition by platelet supplementation: ex vivo and in vitro comparisons of prasugrel,clopidogrel and ticagrelor

Essentials

• Successful outcome of platelet transfusion depends on specific antiplatelet therapy in use.
• We assessed if ticagrelor, clopidogrel or prasugrel impacts on donor platelet activity ex vivo.
• Ticagrelor and/or its active metabolite in plasma or bound to platelets can inhibit donor platelets.
• This might compromise the effectiveness of platelet transfusion therapy.

Summary

Background

Platelet transfusion is the conventional approach to restore platelet function during acute bleeds or surgery, but successful outcome depends on the specific antiplatelet therapy. Notably ticagrelor is associated with inadequate recovery of platelet function after platelet transfusion. We examined whether plasma and/or platelets from ticagrelor‐treated patients influence donor platelet function, in comparison with clopidogrel and prasugrel.

Methods

Platelet transfusion was mimicked ex vivo by mixing naïve donor platelet‐rich plasma (PRP) or gel‐filtered platelets (GFP) in defined proportions with PRP, plasma or GFP from cardiovascular patients receiving standard care including medication with prasugrel, clopidogrel or ticagrelor (n = 20 each). Blood was taken 4 h after the previous dose. HLA2/HLA28 haplotyping let us distinguish net (all platelet) and individual patient/donor platelet reactivity in mixtures of patient/donor platelets, measured by flow cytometry analysis of ADP‐induced fibrinogen binding and CD62P expression.

Results

ADP responsiveness of donor platelets was dramatically reduced by even low (10%) concentrations of PRP or plasma from ticagrelor‐treated patients. Clopidogrel and prasugrel were associated with more modest donor platelet inhibition. GFP from ticagrelor‐treated patients but not patients receiving clopidogrel or prasugrel also suppressed donor GFP function upon mixing, suggesting the transfer of ticagrelor from patient platelets to donor platelets. This transfer did not lead to recovery of ADP responsiveness of patient's platelets.

Conclusion

Collectively, these observations support the concept that ticagrelor and/or its active metabolite in plasma or bound to platelets can inhibit donor platelets, which might compromise the effectiveness of platelet transfusion therapy.

     

Influence of hepatic impairment on the pharmacokinetics and pharmacodynamics of the P2Y12 receptor antagonist selatogrel

Selatogrel is a potent and selective reversible P2Y12 receptor antagonist in development for early treatment of acute myocardial infarction via subcutaneous (s.c.) self‐injection. Selatogrel is almost exclusively eliminated via the hepatobiliary route. Hepatic impairment is associated with reduced drug clearance and primary hemostasis. This single‐center, open‐label study investigated the effect of mild and moderate hepatic impairment on pharmacokinetics (PK) and pharmacodynamics (PD) of a single s.c. dose of selatogrel (16 mg). The study included groups of eight subjects with mild and moderate hepatic impairment, and matched healthy control subjects. Compared to healthy subjects, exposure to selatogrel in subjects with mild and moderate hepatic impairment was 30% and 108% (maximum plasma concentration [C _max]) and 47% and 212% (area under the concentration‐time curve from zero to infinity [AUC_0–∞]) higher, respectively. Hepatic impairment was associated with lower clearance and volume of distribution, whereas plasma protein binding was not affected. Marked inhibition of platelet aggregation (IPA > 80%) was attained within 30 min in all subjects and hepatic impairment prolonged IPA duration. Area under the effect curve was 60% and 160% higher in subjects with mild and moderate hepatic impairment, respectively. PK/PD modeling identified a change in the relationship between exposure and IPA, with a steeper concentration‐effect relationship in healthy subjects compared to subjects with hepatic impairment. The combination of higher exposure and lower half‐maximum inhibitory concentration resulted in longer lasting effect. In conclusion, hepatic impairment alters the PK/PD relationship leading to prolonged effects. Therefore, dose adjustments may be warranted in subjects with moderate hepatic impairment.

Study HighlightsWHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC?Selatogrel is a potent and selective P2Y12 receptor antagonist for subcutaneous self‐administration by patients when they suspect onset of an acute myocardial infarction.WHAT QUESTION DID THIS STUDY ADDRESS?This study investigated the influence of mild and moderate hepatic impairment on the pharmacokinetics (PK) and pharmacodynamics (PD) of selatogrel.WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE?Increased hepatic impairment was associated with higher exposure to selatogrel due to lower clearance and volume of distribution. The concentration‐effect (inhibition of platelet aggregation) relationship changed in hepatic impairment leading to higher drug sensitivity.HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE?Hepatic impairment does not only change the PK of P2Y12 receptor antagonists but, due to its effect on primary hemostasis, also their PD.     

Selective and rapid monitoring of dual platelet inhibition by aspirin and P2Y12 antagonists by using multiple electrode aggregometry

Background

Experimental models of pulmonary embolism (PE) that produce pulmonary hypertension (PH) employ many different methods of inducing acute pulmonary occlusion. Many of these models induce PE with intravenous injection of exogenous impervious objects that may not completely reproduce the physiological properties of autologous thromboembolism. Current literature lacks a simple, well-described rat model of autlogous PE. Objective: Test if moderate-severity autologous PE in Sprague-Dawley (SD) and Copenhagen (Cop) rats can produce persistent PH.

Methods

blood was withdrawn from the jugular vein, treated with thrombin-Ca⁺⁺ and re-injected following pretreatment with tranexamic acid. Hemodynamic values, clot weights and biochemical measurements were performed at 1 and 5 days.

Results

Infusion of clot significantly increased the right ventricular peak systolic pressure to 45-55 mm Hg, followed by normalization within 24 hours in SD rats, and within 5 days in COP rats. Clot lysis was 95% (24 hours) and 97% (5 days) in SD rats and was significantly lower in COP rats (70%, 24 hours; 87% 5 days). Plasma D-dimer was elevated in surgical sham animals and was further increased 8 hours after pulmonary embolism. Neither strain showed a significant increase in bronchoalveolar chemotactic activity, myeloperoxidase activity, leukocyte infiltration, or chemokine accumulation, indicating that there was no significant pulmonary inflammation.

Conclusions

Both SD and COP rats exhibited near complete fibrinolysis of autologous clot PE within 5 days. Neither strain developed persistent PH. Experimental models of PE designed to induce sustained PH and a robust inflammatory response appear to require significant, persistent pulmonary vascular occlusion.     

The reversible P2Y12 antagonist cangrelor influences the ability of the active metabolites of clopidogrel and prasugrel to produce irreversible inhibition of platelet function

Summary.  Background:  Agents that act as antagonists at P2Y₁₂ ADP receptors on platelets are in use (clopidogrel), and in development for use (cangrelor and prasugrel), in patients with cardiovascular disease. Cangrelor is a direct-acting reversible antagonist being developed for short-term infusion; clopidogrel and prasugrel are oral prodrugs that provide irreversible inhibition via transient formation of active metabolites. At the cessation of cangrelor infusion, patients are likely to receive clopidogrel or prasugrel as a means of maintaining antiplatelet therapy. Objectives:  To apply an experimental in vitro approach to investigate the possibility that cangrelor influences the ability of the active metabolites of clopidogrel and prasugrel to inhibit ADP-mediated platelet function. Methods:  The effects of cangrelor and the active metabolites of clopidogrel (C-AM) and prasugrel (P-AM) on platelet function were assessed by ADP-induced platelet P-selectin expression in whole blood. The method involved rapid removal of the antagonists by dilution, and measurement of residual platelet inhibition. Results:  Cangrelor, C-AM and P-AM markedly inhibited P-selectin expression. The effect of cangrelor, but not of C-AM and P-AM, was reversible following antagonist removal. Preincubation of blood with cangrelor prior to addition of C-AM or P-AM reduced the ability of metabolites to irreversibly antagonize P2Y₁₂. Irreversible inhibition was maintained when blood was preincubated with metabolites prior to cangrelor. Conclusions:  Cangrelor influences the ability of the active metabolites of clopidogrel or prasugrel to inhibit platelet function irreversibly. Careful consideration should be given to the timing of administration of an oral P2Y₁₂ antagonist following cangrelor infusion.     

In the presence of strong P2Y12 receptor blockade,aspirin provides little additional inhibition of platelet aggregation

Summary. Background: Aspirin and antagonists of platelet ADP P2Y₁₂ receptors are often coprescribed for protection against thrombotic events. However, blockade of platelet P2Y₁₂ receptors can inhibit thromboxane A₂ (TXA₂)‐dependent pathways of platelet activation independently of aspirin. Objectives: To assess in vitro whether aspirin adds additional antiaggregatory effects to strong P2Y₁₂ receptor blockade. Methods: With the use of platelet‐rich plasma from healthy volunteers, determinations were made in 96‐well plates of platelet aggregation, TXA₂ production and ADP/ATP release caused by ADP, arachidonic acid, collagen, epinephrine, TRAP‐6 amide and U46619 (six concentrations of each) in the presence of prasugrel active metabolite (PAM; 0.1–10 μmol L^?1), aspirin (30 μmol L^?1), PAM + aspirin or vehicle. Results: PAM concentration‐dependently inhibited aggregation; for example, aggregation in response to all concentrations of ADP and U46619 was inhibited by ≥ 95% by PAM at > 3 μmol L^?1. In further tests of PAM (3 μmol L^?1), aspirin (30 μmol L^?1) and PAM + aspirin, aspirin generally failed to produce more inhibition than PAM or additional inhibition to that caused by PAM. The antiaggregatory effects of PAM were associated with reductions in the platelet release of both TXA₂ and ATP + ADP. Similar effects were found when either citrate or lepirudin were used as anticoagulants, and when traditional light transmission aggregometry was conducted at low stirring speeds. Conclusions: P2Y₁₂ receptors are critical to the generation of irreversible aggregation through the TXA₂‐dependent pathway. As a result, strong P2Y₁₂ receptor blockade alone causes inhibition of platelet aggregation that is little enhanced by aspirin. The clinical relevance of these observations remains to be determined.     

Endothelial injury provokes collateral arterial vasoconstriction: response to a serotonin 2 antagonist, thromboxane antagonist or synthetase inhibition

To assess the influence on collateral arteries of vasoactive factors released from activated platelets we used angiography and blood flow measurement to study the limb blood supply in 29 rabbits, 2 weeks after superficial femoral artery ligation. Minutes after balloon catheter injury to the lower aorta, striking spasm of the collateral arteries was routinely evident on the arteriograms, and limb blood flow fell. Spasm was partly reversed either by blockade of thromboxane synthesis (UK-38,485) or its vascular action (SQ 29,548) or by ketanserin, the serotonin receptor antagonist when used alone. Ketanserin combined with either UK-38,485 or SQ 29,548 reversed the spasm completely. We conclude that a combined action of serotonin and thromboxane induces collateral artery spasm when platelets are activated.     

ATP modulates noradrenaline release by activation of inhibitory P2Y receptors and facilitatory P2X receptors in the rat vas deferens

The role of ATP on the modulation of noradrenaline release elicited by electrical stimulation (100 pulses/8 Hz) was studied in the prostatic portion of rat vas deferens preincubated with [3H]noradrenaline. In the presence of P1 antagonists, the nucleotides 2-methylthioadenosine-5'-triphosphate (2-MeSATP), 2-methylthioadenosine 5'-diphosphate (2-MeSADP), ADP, and ATP decreased electrically evoked tritium overflow up to 44%, with the following order of potency: 2-MeSATP > 2-MeSADP > ADP > or = ATP. The P2Y antagonists reactive blue 2 (RB2) and 2-methylthioadenosine 5'-monophosphate (2-MeSAMP) increased, whereas the P2X antagonist pyridoxal-5'-phosphate-6-(2'-naphthylazo-6'-nitro-4',8'-disulfonate) (PPNDS) decreased evoked tritium overflow. The inhibitory effect of 2-MeSATP was antagonized by RB2 (10 microM) and by 2-MeSAMP (10 microM) but not by the selective P2Y1 receptor antagonist 2'-deoxy-N6-methyladenosine 3',5'-bisphosphate (MRS 2179; 10 microM). When, besides P1 receptors, inhibitory P2Y receptors were blocked with RB2, alpha,beta-methyleneadenosine 5'-triphosphate (alpha,beta-meATP), beta,gamma-imidoadenosine 5'-triphosphate (beta,gamma-imidoATP), beta,gamma-methyleneadenosine 5'-triphosphate (beta,gamma-meATP), 2-MeSATP, and ATP enhanced tritium overflow up to 140%, with the following order of potency: alpha,beta-meATP > 2-MeSATP = ATP = beta,gamma-meATP > or = beta,gamma-imidoATP. The facilitatory effects of alpha,beta-MeATP and beta,gamma-imidoATP were prevented by PPNDS. Under the same conditions, apyrase attenuated, whereas the ectonucleotidase inhibitor 6-N,N-diethyl-D-beta,gamma-dibromomethylene 5'-triphosphate enhanced tritium overflow, an effect that was prevented by PPNDS. In the prostatic portion of the rat vas deferens, endogenous ATP exerts a dual and opposite modulation of noradrenaline release: an inhibition through activation of P2Y receptors with a pharmacological profile similar to that of the P2Y12 and P2Y13 receptors and a facilitation through activation of P2X receptors with a pharmacological profile similar to that of P2X1 and P2X3, or PX2/P2X3 receptors.     

A3 and P2Y2 receptors control the recruitment of neutrophils to the lungs in a mouse model of sepsis

We have recently shown that A3 adenosine receptors and P2Y2 purinergic receptors play an important role in neutrophil chemotaxis. Chemotaxis of neutrophils to sites of infections is critical for immune defense. However, excessive accumulation of neutrophils in the lungs can cause acute lung tissue damage. Here we assessed the role of A3 and P2Y2 receptors in neutrophil sequestration to the lungs in a mouse model of sepsis. Sepsis was induced by cecal ligation and puncture (CLP) using adult male C57BL/6J mice (wild type [WT]), homozygous A3 receptor knockout (A3KO) mice, and P2Y2 receptor knockout (P2Y2KO) mice. Animals were killed 2, 4, 6, or 8 h after CLP, and peritoneal lavage fluid and blood were collected. Lungs were removed, and neutrophil infiltration was evaluated using elastase as a marker. Leukocyte and bacterial counts in peritoneal lavage fluid and blood samples were determined. Survival after sepsis was determined in a separate group. Leukocyte counts in the peritoneum were lower in A3KO and P2Y2KO mice than in WT mice. Conversely, initial leukocyte counts in the peripheral blood were higher in KO mice than in WT mice. Neutrophil sequestration to the lungs reached a maximum 2 h after CLP and remained significantly higher in WT mice compared with A3KO and P2Y2KO mice (P < 0.001). Survival after 24 h was significantly lower in WT mice (37.5%) than in A3KO or P2Y2KO mice (82.5%; P < 0.05). These data suggest that A3 and P2Y2 receptors are involved in the influx of neutrophils into the lungs after sepsis. Thus, pharmaceutical approaches that target these receptors might be useful to control acute lung tissue injury in sepsis.