Advantages of Fast-Acting ADP Receptor Blockade in Ischemic Heart Disease
The stimulatory role of ADP in platelet aggregation has received much attention in recent years.1 Not only is ADP a major physiological agonist on its own, it also plays a key role in promoting aggregation by other stimuli of which collagen and epinephrine are excellent examples. Released from damaged tissues and blood cells, including secreting platelets, ADP is a major factor in coronary artery disease favoring platelet accumulation at sites of stenosis and fissured atherosclerotic plaques. Recent advances have defined how ADP activates platelets in a process involving two receptors, P2Y1 and P2Y12, both belonging to the G-protein–coupled seven-transmembrane domain receptor family. Studies were advanced by the discovery of patients in which P2Y12 is congenitally deficient2,3⇓ and by the recent cloning of P2Y12 which had proved a most elusive receptor.2–5⇓⇓⇓ According to current thinking, interaction of platelets with P2Y1 leads to shape change, calcium mobilization, and a rapidly reversible aggregation. Simultaneous binding to P2Y12 permits the formation of large, stable platelet aggregates. It appears that P2Y1 starts the aggregation, and then P2Y12 takes over. This role of P2Y12 had been speculated on before its cloning when, as the mysterious “P2T,” it was already considered to be a primary target for antithrombotic therapy. It has now been confirmed that P2Y12 is the site of action of the long-acting antithrombotic drugs clopidogrel and ticlopidine, widely used to downregulate platelet reactivity in coronary artery disease.6 A third purported platelet receptor for ADP, the ion channel P2X1, is now known to react primarily with ATP.
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In a study presented in this issue of the Journal, Wang et al7 provide evidence that pharmacological inhibition of P2Y12 during fibrinolytic therapy can be of benefit in sustaining blood flow after myocardial infarction. To do this, they have used the latest in a generation of ATP-derived antagonists from Astra-Zeneca termed AR-C69931MX. This product, characterized as an ADP receptor antagonist through in vitro studies on human platelets, is known to block carotid artery occlusion in dogs.8 In the work reviewed here, a canine coronary electrolytic injury thrombosis model was used. On complete occlusion, fibrinolytic therapy was initiated by administering tissue plasminogen activator (t-PA) in the presence or absence of AR-C69931MX given intravenously for 2 hours. All animals also received aspirin and heparin. Thrombus lysis time was defined as restoration of coronary blood flow to at least 30% of the baseline as measured with a Doppler ultrasonic flow probe. Myocardial tissue blood flow was measured by using a fluorescent color microsphere technique. Although AR-C69931MX did not accelerate thrombolysis with t-PA, it prolonged reperfusion time and prevented both reocclusion and cyclic flow variations. Significantly, it was also associated with marked improvements in myocardial tissue blood flow and microvascular perfusion. Infarct size was lowered by approximately 50% in the AR-C69931 group. ADP-induced platelet aggregation was severely reduced during the infusion of AR-C69931. Although bleeding time was prolonged, the increase was modest. Interestingly, the findings were also reproduced at half-dosage t-PA levels suggesting an additional potential as a thrombolytic-sparing adjunct to reduce the risk of hemorrhagic complications. These highly significant results confirm once more the pathological role of ADP in arterial thrombosis. Most interesting is the improvement in myocardial tissue blood flow. It suggests that CD39, a natural ADP and ATP hydrolyzing enzyme of endothelial cells,9 is unable to cope and that AR-C69931 provides necessary support in dissociation of microthrombi and preventing platelet re-accumulation.
Clopidogrel and ticlopidine are pro-drugs of proven benefit that need to be administered several hours before the onset of their reactivity. However, they require metabolism in the liver and plasma levels of the active metabolite can vary between subjects, a small proportion of whom may even be refractory to the drug.10 Toxicity worries mean that doses in humans have to be limited. Side effects can be a worry with ticlopidine, and these include neutropenia and thrombotic thrombocytopenic purpura (TTP). Clopidogrel is somewhat faster acting and has safety advantages over ticlopidine although TTP has again been reported.11 A potential advantage of AR-C69931MX and similar drugs is their fast action, which will make them more suitable to acute situations. However, this does not mean that they are potential replacements for the orally available thienopyridines as long term therapy. Intravenous administration may also mean that optimal plasma levels will be easier to maintain over short periods. The short plasma half-life of the drug and the rapid loss of activity following the stopping of the infusion is an appreciable safety advantage. That AR-C69931 is a potential drug for use in humans was confirmed by Storey et al12 who performed a pilot study and assessed its safety, tolerability, and activity in patients with acute coronary syndromes. The degree of inhibition of ADP-induced platelet aggregation seen by Storey et al12 was >80% and often 100%, values that are much greater than those normally seen for patients receiving clopidogrel. This impressive inhibition of ADP-induced platelet aggregation was confirmed here by Wang et al,7 although the fact that epinephrine failed to reverse the inhibition requires explanation. All in all, platelet reactivity may be pivotal to the pathogenesis of acute coronary syndromes.
So, how does the P2Y12 receptor antagonist AR-C69931MX prevent reocclusion after t-PA–induced fibrinolysis? Recently published ex vivo studies may show light on this. For example, AR-C69931MX impaired thrombus formation when human blood was perfused ex vivo over a collagen-coated surface.13 Microscopic analysis showed clusters of spread platelets with single nonspread platelets on top of these. Such a result is compatible with our previous findings which showed how ADP-induced platelet aggregates from a patient deficient in P2Y12 were loosely packed with few contact points.14 ADP-induced aggregates from subjects receiving clopidogrel had a similar ultrastructure,14 while smaller and loosely-packed thrombi were seen for clopidogrel when blood was perfused under flow over collagen.15 In vitro studies also show that interaction of ADP with P2Y12 is largely responsible for activation of αIIbβ3 by ADP, a key step in the adhesive protein binding necessary for platelet aggregation.3 P2Y12 inhibition may therefore reduce platelet consumption on a growing thrombus by blocking an essential role of ADP in αIIbβ3 activation and/or its maintenance in an activated state. Interestingly, platelet adhesion to fibrinogen-coated surfaces was also interfered with by AR-C69931MX while, in contrast, platelet adhesion and spreading on collagen occurred normally.13 This might provide an added security against bleeding and point to a possibly safer alternative to the use of the more powerful anti-αIIbβ3 inhibitors for this integrin also plays a role in platelet spreading on collagen. In conclusion, AR-C69931MX at levels that virtually totally block platelet aggregation to ADP (1) will prevent the new incorporation of platelets as platelet-containing clots disperse and (2) will help stop new thrombi from forming.
AR-C69931MX has also been shown to inhibit shear-rate–induced platelet activation through the binding of von Willebrand factor to GPIb, another function of ADP which should not be underestimated.16 However, two other considerations provide pause for thought. The first is that although P2Y12 is essentially confined to cells of the megakaryocyte lineage, it is also found in brain tissue.4,17⇓ Its role in the brain needs to be elucidated particularly if the current treatment is extrapolated to stroke. Secondly, studies on transgenic mice suggest that the absence of the P2Y1 receptor leads to a severe aggregation defect and resistance to thrombosis,18 while another study shows that combinations of antagonists of P2Y12 and P2Y1 were more effective in inhibiting shear-induced platelet aggregation than either inhibitor alone.19 Antagonists of P2Y1 should therefore also be tested in animal models to compare their efficacy in comparison with the AR-C compounds before a decision is made as to the optimal way of inhibiting the prothrombotic effects of ADP in man. Such inhibitors may additionally influence vascular tone.20
In summary, Wang et al7 have highlighted an important potential use of a fast-acting ADP receptor antagonist during fibrinolysis after myocardial infarction. Reducing the odds of re-occlusion and the rapid improvement of myocardial tissue perfusion are important goals, and these authors point the way for pharmacological progress to further ameliorate the success rate in what remains a major problem in cardiovascular disease.
- ↵Cattaneo M, Lecchi A, Randi AM, McGregor JL, Mannucci PM. Identification of a new congenital defect of platelet function characterized by severe impairment of platelet responses to adenosine diphosphate. Blood. 1992; 80: 2787–2796.
- ↵Nurden P, Savi P, Heilmann E, Bihour C, Herbert JM, Maffrand JP, Nurden AT. An inherited bleeding disorder linked to a defective interaction between ADP and its receptor on platelets. J Clin Invest. 1995; 95: 1612–1622.
- ↵Foster CJ, Prosser DM, Agans JM, Zhai Y, Smith MD, Lachowicz JE, Zhang FL, Gustafson E, Monsma FJ, Wiekowski MT, Abbondanzo SJ, Cook DN, Bayne ML, Lira SA, Chintala MS. Molecular identification and characterization of the platelet ADP receptor targeted by thienopyridine antithrombotic drugs. J Clin Invest. 2001; 107: 1591–1598.
- ↵Wang K, Zhou X, Zhou Z, Tarakji K, Carneiro M, Penn MS, Murray D, Klein A, Humphries RG, Turner J, Thomas JD, Topol EJ, Lincoff M. Blockade of platelet P2Y12 receptor by AR-C69931MX sustains coronary artery recanalization and improves the myocardial tissue perfusion in a canine thrombosis model. Arterioscler Thromb Vasc Biol. 2003; 23: 357–362.
- ↵Huang J, Driscoll EM, Gonzales ML, Park AM, Lucchesi BR. Prevention of arterial thrombosis by intravenously administered platelet P2T receptor antagonist AR-C69931MX in a canine model. J Pharm Exp Ther. 2000; 295: 492–499.
- ↵Becker RC. Platelet surface physiology and its importance in pharmacotherapy design and development: The adenosine diphosphate receptor antagonists. J Thromb Thrombolysis. 2000; 10: 35–53.
- ↵Remijn JA, Wu Y-P, Jeninga EH, IJsseldijk MJW, van Willigen G, de Groot PG, Sixma JJ, Nurden AT, Nurden P. Role of ADP receptor P2Y12 in platelet adhesion and thrombus formation in flowing blood. Arterioscler Thromb Vasc Biol. 2002; 22: 686–691.
- ↵Humbert M, Nurden P, Bihour C, Pasquet J-M, Winckler J, Heilmann E, Savi P, Herbert J-M, Kunicki TJ, Nurden AT. Ultrastructural studies of platelet aggregates from human subjects receiving clopidogrel and from a patient with an inherited defect of an ADP-dependent pathway of platelet activation. Arterioscler Thromb Vasc Biol. 1996; 16: 1532–1543.
- ↵Sakariessen KS, Örning L, Stormorken H. Role of ADP and thromboxanes in human thrombus formation in ex vivo models. Platelets. 1997; 8: 385–390.
- ↵Goto S, Tamura N, Eto K, Ikeda Y, Handa S. Functional significance of adenosine 5′-diphosphate receptor (P2Y12) in platelet activation initiated by binding of von Willebrand factor to platelet GPIbα induced by conditions of high shear rate. Circulation. 2002; 105: 2531–2536.
- ↵Turner NA, Moake JL, McIntyre LV. Blockade of adenosine diphosphate receptors P2Y12 and P2Y1 is required to inhibit platelet aggregation in whole blood under flow. Blood. 2001; 98: 3340–3345.
- ↵Burnstock G. Purinergic signaling and vascular cell proliferation and death. Arterioscler Thromb Vasc Biol. 2002; 22: 364–373.