Presence Of PGI2 Research Articles

158 The Anti-Platelet Effectiveness of P2Y12Receptor Blockade is Strongly Influenced by the Endothelium-Derived Mediators Nitric Oxide and Prostacyclin

Introduction P2Y12 receptor antagonists that block the pro-aggregatory effects of ADP on platelets are commonly prescribed alongside aspirin, as dual antiplatelet therapy (DAPT) for secondary prevention of atherothrombotic events. It is emerging that these agents exert part of their effects through potentiation of the endogenous, endothelial-derived platelet inhibitors, nitric oxide (NO) and prostacyclin (PGI2), which constantly bathe circulating platelets reducing their reactivity through elevating levels of cyclic nucleotides. Thus, if the in vivosynergy between NO and PGI2 amplifies the anti-thrombotic effects of P2Y12 inhibitors, within the circulation variances in the levels of endothelial-derived mediators will be an important determinant of the efficacy of DAPT, suggesting that for the individual patient, endothelial function influences the therapeutic potential of these powerful agents. Here, we characterised the interplay of PGI2, NO and P2Y12 blockade on platelet function. Methods 8 healthy male volunteers received aspirin (75 mg) and prasugrel (10 mg) for 7 days. Platelet responses to TRAP-6 (25 iM) or collagen (4 ig/ml) in the presence of PGI2 (1 nM), NO (100 nM), PGI2+NO or vehicle were assessed by LTA and lumi-aggreggometry. Experiments were replicated in vitro (n = 4) utilising aspirin (30 iM) and prasugrel active metabolite (PAM; 6, 3 or 1.5 iM) representing maximal and partial P2Y12 blockade. Results Ex vivo, prior to DAPT, PGI2, NO, or PGI2+NO had little effect upon platelet aggregation. TRAP-6 (25 iM): vehicle, 74 ± 3%; PGI2+NO, 66 ± 3%. Following DAPT, PGI2 or NO alone caused minor inhibition: vehicle, 57 ± 4%; PGI2, 47 ± 6%; NO 49 ± 6%. Conversely, the combination of PGI2+NO produced strong inhibitory effects, 19 ± 6% (p In vitro, with maximal P2Y12 inhibition (PAM, 6 iM) aggregation to TRAP-6 (25 iM) was reduced: vehicle, 56 ± 5%; PGI2, 30 ± 12%; NO, 46 ± 7%; PGI2+NO, 15 ± 8% (p Conclusion We demonstrate that PGI2 and NO synergise with P2Y12 blockade to produce the greatest platelet inhibition and that platelet reactivity, despite DAPT is powerfully influenced by the presence of NO and PGI2. This highlights that in vivo platelet reactivity will be a function of the level of P2Y12 receptor blockade and levels of extrinsic inhibitory endothelial mediators. This may be relevant to the efficacy of anti-platelet therapies in patients with endothelial dysfunction and introduces the concept of assessing endothelial function in patients undergoingex vivo platelet function testing to help bridge the disconnect between results of platelet testing and thrombotic outcomes.

213 Synergy between Endothelial Mediators and P2Y12 Receptor Blockade as a Potential Determinant in Tailoring Anti-Platelet Therapy

<h3>Introduction</h3> Following ACS or PCI, P2Y12 receptor blockers such as clopidogrel or prasugrel are standardly prescribed alongside aspirin as dual anti-platelet therapy in a “one size fits all” approach, however recurrent thrombotic events occur. Despite the logical hypothesis that thrombotic risk should be associated with level of P2Y12 platelet inhibition, randomised control trials have repeatedly failed to show any clinical benefit of guiding anti-platelet therapy by platelet function tests (PFT). Whilst traditional PFT measure reactivity to ADP, they crucially ignore that P2Y12 receptor antagonists also produce powerful anti-thrombotic effects by potentiating the actions of the inhibitory endothelial mediators prostacyclin (PGI2) and nitric oxide (NO). <h3>Methods</h3> In vitro, IC50 curves produced from light transmission aggregation (LTA) traces for PGI2, NO and prasugrel active metabolite (PAM: 3 μM) were used to generate isobolograms. <i>Ex vivo</i>, healthy male volunteers (n = 8 each) received either prasugrel (10 mg) or aspirin (75 mg) for 7 days. Platelet-rich plasma (PRP) was obtained by centrifugation before and after treatment. Platelet responses to TRAP-6 amide (25 μM) or collagen (4 μg/ml) in the presence of PGI2 (1 nM) and/or the NO donor, DEA/NONOate (100 nM), or vehicle, were assessed by LTA, p-selectin binding by flow cytometry, and downstream cyclic nucleotide and VASP phosphorylation by immunoassay. <h3>Results</h3> Isobolographic analysis of <i>in vitro</i> studies showed P2Y12 blockade powerfully enhanced the synergy between NO and PGI2 (10 fold for TRAP-6). Ex-vivo platelet aggregation responses to TRAP-6 were unaffected by the addition of PGI2+NO prior to therapy. Aspirin monotherapy produced minimal inhibition of TRAP-6, though these became stronger in the presence of PGI2+NO (58%±8 to 28%±9; p &lt; 0.05). Inhibitory effects were observed with prasugrel in the presence of PGI2 and NO individually (control, 63 ± 3%; PGI2, 43 ± 6%; NO, 50 ± 5%), and these became much greater with the combination (PGI2+NO, 7%±3). P-selectin expression was reduced from pre-treatment (vehicle, 32 ± 6%) to (PGI2+NO, 11 ± 4%) post aspirin, and from (vehicle, 25 ± 6%) to (PGI2+NO, 2 ± 4%) following prasugrel. cAMP levels increased with PGI2+NO in the aspirin group (0.97 ± 0.06 vs. 1.50 ± 0.19) compared to (1.41 ± 0.07 vs. 3.4 ± 0.58) in the prasugrel group. <h3>Conclusions</h3> PGI2 and NO synergise with P2Y12 blockade to cause powerful platelet inhibition suggesting that an individual’s endothelial function and production of NO and PGI2 are central determents of the anti-thrombotic protection from P2Y12 receptor blocker therapy. Whilst a therapeutic window of platelet reactivity is an attractive concept our research suggests that endothelial function testing alongside PFT would permit better risk stratification. Our research also suggests therapeutically that enhancing endothelial-derived mediators or optimising downstream cyclic nucleotide signalling could reduce both thrombotic and bleeding risk.

CLEC‐2‐dependent activation of mouse platelets is weakly inhibited by cAMP but not by cGMP

The activation of platelet CLEC-2 by podoplanin on lymphatic endothelial cells (LECs) has a critical role in prevention of mixing of lymphatic and blood vasculatures during embryonic development. Paradoxically, LECs release cAMP and cGMP-elevating agents, prostacyclin (PGI2 ) and nitric oxide (NO), respectively, which are powerful inhibitors of platelet activation. This raises the question of how podoplanin is able to activate CLEC-2 in the presence of the inhibitory cyclic nucleotides. We investigated the influence of cyclic nucleotides on CLEC-2 signaling in platelets. We used rhodocytin, CLEC-2 monoclonal antibody, LECs and recombinant podoplanin as CLEC-2 agonists on mouse platelets. The effects of the cyclic nucleotide-elevating agents PGI2 , forskolin and the NO-donor GSNO were assessed with light transmission aggregometry, flow cytometry, protein phosphorylation and fluorescent imaging of platelets on LECs. We show that platelet aggregation induced by CLEC-2 agonists is resistant to GSNO but inhibited by PGI2 . The effect of PGI2 is mediated through decreased phosphorylation of CLEC-2, Syk and PLCγ2. In contrast, adhesion and spreading of platelets on recombinant podoplanin, CLEC-2 antibody and LECs is not affected by PGI2 and GSNO. Consistent with this, CLEC-2 activation of Rac, which is required for platelet spreading, is not altered in the presence of PGI2 . The present results demonstrate that platelet adhesion and activation on CLEC-2 ligands or LECs is maintained in the presence of PGI2 and NO.

Selective type III phosphodiesterase inhibitor as an antithrombotic agent

In platelets, an increase in cAMP levels potently inhibits aggregation and release reactions. Cilostazol is an antiplatelet agent that increases intracellular cAMP levels by selective Type III phosphodiesterase (PDE) inhibition. The characteristics of cilostazol are presented in this review. Adenylate cyclase potentiator also shows strong inhibitory actions on platelet functions, but a number of reports suggest that the continuous use of an adenylate cyclase potentiator may lead to a reduction of drug efficacy. On the other hand, such an action has not been seen with cilostazol even after continuous administration of cilostazol (100 mg/kg) for two weeks in rats, which may be due to a feature of this drug, namely, inhibitory actions on PDE. Inhibitory actions of cilostazol on PDE are specific: strong inhibition (IC50 = 0.19 microM) against Type III PDE that comprises most of the PDE activities in platelets and weak inhibition against Type IV PDE that comprises most of the PDE activities in endothelial cells (ECs). This fact (i.e., specificity of cilostazol) brought about important results when the drug reacted in the presence of both platelets and blood vessels. A non-specific PDE inhibitor such as IBMX increases cAMP and decreases PGI2 synthesis in ECs, but such a phenomenon was not seen with cilostazol. The inhibitory actions of cilostazol on platelet functions were potently enhanced in the presence of PGI2.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of prostaglandins I2 (prostacyclin) and F2 alpha on function, energy metabolism, and calcium uptake in ischaemic/reperfused hearts.

The aim was to examine the effect on cardiac function, energy metabolism, and calcium uptake of either prostaglandin I2 (PGI2, prostacyclin) or prostaglandin F2 alpha (both 28.6 nM) on the response of isolated rat hearts to 25 min of total global ischaemia with or without 30 min reperfusion. Rat hearts were perfused by the Langendorff method and function assessed by left ventricular pressure. Energy metabolites were measured using enzymatic techniques and 45Ca2+ uptake determined by radioisotopic analysis. Although there was no effect of either prostaglandin on contractile depression during ischaemia, both compounds accelerated the onset of and increased the magnitude of ischaemic contracture. High energy phosphate content at the end of the ischaemic period was not affected by prostaglandin treatment; however, tissue lactate levels were increased by PGI2 as was tissue calcium content. Under control conditions mean recovery of left ventricular developed pressure ranged from 66% to 83%. In the presence of PGI2 and PGF2 alpha, recovery of developed pressure was reduced to 20% and 38% of preischaemic values, respectively. The reduced recovery in developed pressure was accompanied by an approximately threefold increase in diastolic pressure (p < 0.05). The depression of functional recovery in reperfused hearts treated with prostaglandins was associated with various disturbances of cellular metabolism including depressed ATP and creatine phosphate content and increased tissue lactate and calcium following 30 min of reperfusion. A significant correlation was found between the changes in developed pressure and diastolic pressure during reperfusion and the reduction in ATP and creatine phosphate repletion. The deficit in recovery of ventricular function also correlated significantly with increased lactate and calcium accumulation in the reperfused heart. Low concentrations of PGI2 and PGF2 alpha can depress contractile recovery of the globally ischaemic heart through a mechanism associated with altered cellular energy metabolism and increased calcium accumulation.

Prostacyclin attenuates both the pressor and adrenocortical response to angiotensin II in human pregnancy

1. The effects of angiotensin II (ANG II) infusion without and with simultaneous infusion of prostacyclin (PGI2; 1.4 pmol min-1 kg-1; 5 ng min-1 kg-1) have been studied in 16 women in second-trimester pregnancy. Ten received one infusion of ANG II alone, followed by its infusion together with PGI2; the remainder received two identical infusions of ANG II alone as controls. 2. PGI2 administration was associated with a small fall in diastolic pressure (P less than 0.01) and a proportionally greater rise in heart rate (P less than 0.001). Small rises in basal plasma renin and ANG II concentrations and a fall in aldosterone concentration were not statistically significant. 3. The diastolic pressor response to ANG II was blunted during PGI2 infusion by comparison with controls (P less than 0.025); this diminution in response was greatest in patients who had initially been most sensitive to ANG II (P less than 0.02). 4. The evoked increment in plasma aldosterone during ANG II infusion was considerably reduced (P less than 0.005) in the presence of PGI2. 5. These data further support the hypothesis of a role for PGI2 in relation to the blunted pressor response to ANG II of normal pregnancy. The apparent inhibitory effects of PGI2 on aldosterone secretion may partly explain the previously described dissociation between the renin-angiotensin system and aldosterone in pregnancy.

In Vitro Effects of Ethanol on Rabbit Platelet Aggregation, Secretion of Granule Contents, and Cyclic AMP Levels in the Presence of Prostacyclin

Ethanol, at physiologically tolerable concentrations, inhibits platelet responses to low concentrations of collagen or thrombin, but does not inhibit responses of washed rabbit platelets stimulated with high concentrations of ADP, collagen, or thrombin. However, when platelet responses to high concentrations of collagen or thrombin had been partially inhibited by prostacyclin (PGI2), ethanol had additional inhibitory effects on aggregation and secretion. These effects were also observed with aspirin-treated platelets stimulated with thrombin. Ethanol had no further inhibitory effect on aggregation of platelets stimulated with ADP, or the combination of ADP and epinephrine. Thus, the inhibitory effects of ethanol on platelet responses in the presence of PGI2 were very similar to its inhibitory effects in the absence of PGI2, when platelets were stimulated with lower concentrations of collagen or thrombin. Ethanol did not appear to exert its inhibitory effects by increasing cyclic AMP above basal levels and the additional inhibitory effects of ethanol in the presence of PGI2 did not appear to be brought about by further increases in platelet cyclic AMP levels.

Forskolin alters sensitivity of the cAMP-generating system to stimulatory as well as to inhibitory agonists. A study with intact human platelets and guinea pig myometrium.

1. In both the intact guinea pig myometrium and human platelets, cAMP accumulation was enhanced by prostaglandin I2 (prostacyclin, PGI2) and forskolin with potentiated responses in the simultaneous presence of both effectors. Under basal conditions, forskolin caused rises in platelet cAMP concentration through a single low-affinity interaction (Kapp = 90 microM) while in myometrium, activation involved both a low-affinity (Kapp = 10 microM) and a high-affinity (Kapp = 0.8 microM) component. The contribution of the high-affinity component could be reduced when endogenous PGI2 was decreased. In both tissues, the synergistic effect of forskolin in the presence of PGI2 was mediated by a single high-affinity interaction (Kapp = 0.3 microM). The data were consistent with a low-affinity interaction of the diterpene with the cyclase catalytic unit C generating the C...forskolin state and with a high-affinity interaction of the diterpene with the activated complex (stimulatory regulatory protein) and C generating the potentiated Gs-C...forskolin state. 2. Both norepinephrine in platelets and carbachol in the myometrium (via Gi, the inhibitory regulatory protein) inhibited PGI2-mediated cAMP accumulation (EC50 = 100 nM and 8 nM respectively). The persistently activated cAMP-generating system induced by cholera toxin in the myometrium was also susceptible to inhibition but the EC50 for carbachol was increased to 50 nM and the extent of inhibition was decreased. Forskolin-mediated effect in platelets was inhibited by norepinephrine as was the PGI2 response. By contrast, the synergistic state of the cyclase resisted the inhibitory action of norepinephrine and carbachol in platelets and myometrium respectively. In the myometrium, where the cAMP response due to forskolin alone partially involved some synergistic Gs-C ... forskolin species, carbachol at 50 microM elicited no more than 30% inhibition. Inhibition was partly improved (60% inhibition at 1 microM carbachol) when the contribution of the Gs-C species was decreased by lowering the concentration of local PGI2. Partial inhibition by norepinephrine was similarly observed in platelets under partial synergistic conditions. The data suggest that receptor-mediated inhibition of cAMP generation could be differentially expressed depending on the nature of the active species of the cyclase involved in the stimulatory responses.

Absence of plasma prostacyclin stimulating activity deficiency in hemolytic uremic syndrome

We compared the effect of plasma from 19 children with hemolytic uremic syndrome (HUS) on prostacyclin (PGI2) production by fresh rat aortic rings to the effect of plasma from 17 age- and sex-matched normal children, taking into account the PGI2 baseline aortic production (PGI2 release in presence of buffer, 21 determinations). After 10, 20, 30, 40, and 60 minutes incubation of rat aortic tissue with either plasma or buffer, the presence of PGI2 was studied by measuring by radioimmunoassay (RIA) the concentration of 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha). 6-keto-PGF1 alpha production increased with time in the two groups of plasma samples and in the presence of buffer, but 6-keto-PGF1 alpha production (ng/mg dried tissue) after 30 minutes incubation and mean 6-keto-PGF1 alpha production (slope of regression line, ng/mg/min) were significantly (P less than 0.01) lower in the presence of normal plasma compared with buffer, and significantly (P less than 0.01) higher in the presence of HUS plasma compared with normal plasma. There was no significant difference between buffer and HUS plasma. We conclude that, under our experimental conditions, normal plasma had an inhibitory activity on 6-keto-PGF1 alpha production by rat aorta. This inhibitory activity was absent in HUS plasma.

Interaction of insulin and prostacyclin production in the rat.

The effects of hyperinsulinemia on production of prostacyclin (PGI2), a potent inhibitor of platelet aggregation, was studied in vitro in the rat. Aortic rings following 1-h preincubation at 37 degrees C and 1 h at 4 degrees C were incubated with and without purified porcine insulin in Krebs' glucose at 37 degrees C for 5 min and immediately percent inhibition of platelet aggregation was determined. PGI2 production in ng/mg aorta was estimated from a curve using PGI2 standard. The mean PGI2 production was significantly decreased in those rings incubated with insulin in concentration of 2500, 500, and 250 microunits/ml. Likewise, incubation of rings at 22 degrees C for 30 min resulted in at least ten times less PGI2 with insulin. Since PGI2 appears to exert its antiaggregatory effect through cyclic AMP, theophylline was added to the incubation medium resulting in potentiation of inhibition of aggregation which was decreased to control levels when insulin was also added to the medium. Pancreatic slices yielded no significant change in insulin obtained when incubated with and without 5 ng of PGI2 in 2 ml of 2.8 mM glucose, but in a high glucose medium (28 mM) the PGI2-treated slices yielded significantly less insulin. Since PGI2 may play a role in the formation of atherosclerotic plaques, these results suggest a possible deleterious effect of elevated insulin levels in type II and in insulin-treated type I diabetics with regard to macrovascular disease. Suppression of insulin production in presence of PGI2 in high glucose medium resembles the action of other prostaglandins and suggests it may inhibit insulin secretion after a glucose load.