Pathologic platelet activation has been implicated in the pathogenesis of ischemic heart disease. Since cardiomyocytes can be protected from ischemia-reoxygenation injury by poly(ADP-ribose) polymerase (PARP) inhibitors mimicking the adenine/ADP part of NAD+, their structural resemblance to ADP may also enable the blockade of platelet aggregation via binding to ADP receptors.
Blood samples drawn from healthy volunteers were pre-incubated with different concentrations of PARP inhibitors: 4-hydroxyquinazoline, 2-mercapto-4(3 H)-quinazolinone, or HO-3089. ADP-, collagen- and epinephrine-induced platelet aggregation was evaluated according to the method described by Born. The effect of PARP inhibitors on thrombocyte aggregation was also examined when platelets were sensitized by heparin and in the presence of incremental concentrations of ADP.
All examined PARP inhibitors reduced the ADP-induced platelet aggregation in a dose-dependent manner (significant inhibition at 20 μM for HO-3089 and at 500 μM for the other agents; P < 0.05), even if platelets were sensitized with heparin. However, their hindrance on platelet aggregation waned as the concentration of ADP rose (no effect at 40 μM ADP). PARP inhibitors had minimal effect on both collagen- and epinephrine-induced platelet aggregation.
Our study first demonstrates the feasibility of a design for PARP inhibitors that does not only protect against ischemia-reperfusion-induced cardiac damage but may also prevent thrombotic events.
Local hemostasis is initiated when platelets are exposed to subendothelial structures. 1 Adhesion and activation of blood platelets is followed by the synthesis and release of thromboxane A2 (TXA2) and adenosine diphosphate (ADP) from α and dense granules. These proaggregatory molecules amplify the hemostatic process by recruiting additional platelets to the site of vascular injury. 2–4 The common impact of collagen, ADP, and TXA2 is to activate specific signaling pathways and to generate a number of second messengers leading to the functional expression of glycoprotein (GP) IIb/IIIa receptors.
Pathologic platelet aggregation may contribute to the morbidity and mortality of ischemic heart disease. 5,6 Therefore, regular application of antiplatelet agents was proved to be useful in the prevention and treatment of occlusive vascular diseases. 7 An ideal platelet inhibitor should specifically block thrombogenic platelet-dependent mechanisms without interfering with the physiological platelet functions. 8 Numerous trials have verified the beneficial effect of the cyclooxygenase inhibitor acetyl salicylic acid (ASA), 9 but 5 to 9% of the patients seem to be aspirin resistant and an additional 23% of them are semi-responders. 10 Thienopyridine derivates (ticlopidine and clopidogrel) are antiaggregatory drugs that selectively and irreversibly inhibit platelet responses to adenosine diphosphate (ADP) by blocking its surface receptor. 9,11,12 The final common pathway for platelet aggregation is the binding of fibrinogen to the glycoprotein (GP) IIb/IIIa receptor. 8,13 Blocking this receptor with a highly effective antagonist (eg, eptifibatide) completely inhibits platelet aggregation induced by any of the agonists or even their combination. 14,15 Heparin, another widely used anticoagulant in the clinical practice, is used to prevent clotting but sensitizes platelets to form TXA2 and even enhances their aggregability. 16
Here, we report that selected poly(ADP-ribose) polymerase (PARP) inhibitors, which have a molecular structure resembling that of the adenine moiety of NAD+ molecule can also antagonize ADP-induced platelet aggregation in addition to their cardioprotective effect observed during ischemiareperfusion. 17,18 PARP inhibitors also decreased the heparin-evoked sensitization of platelets indicating that beyond their myocardial protective effect, these agents may also be useful in the prevention and treatment of thrombotic events.
From the *First Department of Medicine, Division of Cardiology; †Department of Biochemistry and Medical Chemistry; and ‡Institute of Organic and Medicinal Chemistry, University of Pecs, Medical School, Pecs, Hungary.
Received for publication September 29, 2003; accepted December 8, 2003.
Reprints: Prof. Kalman Toth, MD, ScD, FESC, First Department of Medicine, Division of Cardiology, University of Pecs, Medical School, Ifjusag ut 13, 7624 Pecs, Hungary (e-mail: firstname.lastname@example.org).
Chemicals and Reagents
Acetyl salicylic acid was a gift from Sanofi-Synthelabo Ltd. (Le Plessis-Robinson, France); eptifibatide was purchased from Schering-Plough Ltd. (New York, USA). 4-hydroxyquinazoline and 2-mercapto-4(3 H)-quinazolinone were purchased from Sigma-Aldrich Chemie Ltd. (Steinheim, Germany). HO-3089 was synthesized at the Institute of Organic and Medicinal Chemistry, University of Pecs, Medical School. (Its synthesis and exact structure will be published elsewhere.) Structures of PARP inhibitors are demonstrated in Figure 1. ADP, collagen, and epinephrine were purchased from Carat Ltd. (Budapest, Hungary). Sodium-heparin was purchased from Biochemie Ltd. (Vienna, Austria). All other reagents were used at the highest purity commercially available.
Oxidative Challenge of Cultured Cardiomyocytes
H9c2 cardiomyocytes, a clonal line derived from embryonic rat heart, were cultured in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal calf serum and 2 mM pyruvate in a humidified atmosphere of 95% air and 5% CO2 at 37°C. Before reaching confluence, the cells were split, plated at low density in culture dishes (approximately 2 ×104 cell/well) and cultured for 24 hours. Cardiomyocytes were then incubated without (negative control) and with 1 mM hydrogen peroxide for 3 hours either untreated (positive control) or treated with 4-hydroxyquinazoline, 2-mercapto-4(3 H)-quinazolinone (both in 5, 10, 20, 50, 100, 500, 1000, and 1500 μM), or HO-3089 (in 0.1, 0.5, 1, 2, 10, 20, 50, 100, and 500 μM). At the end of the incubation period the survival of cells was determined by the MTT assay as the percentage of survival in negative control samples.
Platelet Aggregation Measurements
Venous blood samples were drawn from normal healthy donors into Vacutainer tubes containing trisodium citrate (3.8%). An experimental PARP inhibitor or a well-known antiplatelet drug (ASA or eptifibatide)—all dissolved and diluted in distilled water—was added to the tubes at various final concentrations: 4-hydroxyquinazoline, 2-mercapto-4(3 H)-quinazolinone (both in 100, 500, 1000, and 1500 μM) and HO-3089 (in 10, 20, 50, 100, and 500 μM). ASA (in 0.25, 0.5, 1, 2, 7, 20, and 70 μM) and eptifibatide (in 100, 200, 300, 400, 500, and 1000 ng/ml) served as control agents. The same volume of distilled water without any agent was added to the control tubes. To ensure optimal drug effect, samples were incubated and continuously mixed at 37°C for 20 minutes.
Following the incubation period platelet-rich plasma (PRP) was separated by centrifugation at 150 g for 10 minutes. After carefully removing PRP, the remaining specimen was further centrifuged at 2500 g for 10 minutes to obtain platelet-poor plasma (PPP). Aliquots of 450 μl PRP or PPP were placed in glass cuvettes and 50 μl of ADP (at a final concentration of 5 and 10 μM); collagen (2 μg/ml) or epinephrine (10 μM) was added to the PRP to induce platelet aggregation. Samples were incubated at 37°C and continuously stirred at 1000 rpm during the measurement. The aggregation curve was recorded for 10 minutes with a Carat TX-4 platelet aggregometer (Carat Ltd., Hungary) using the turbidimetric method described by Born. 19 We compared the maximal aggregation indices of the samples to that of control in each concentration. All the measurements were carried out within 2 hours after vein puncture and were repeated 15 times using blood samples from different healthy individuals for every applied concentration of each compound. It is well documented that in case of hemolysis our method is not suitable to assess platelet aggregation. Therefore the desensitization of ADP receptors by ADP released from hemolyzed erythrocytes could be excluded.
We subsequently evaluated the antiaggregatory effect of PARP inhibitors on thrombocytes stimulated with increasing ADP concentrations. Platelet aggregation was almost completely blocked by adding high concentrations of 4-hydroxyquinazoline, 2-mercapto-4(3 H)-quinazolinone, or HO-3089 to the samples (2000, 2000, and 1000 μM, respectively). Following the 20-minute incubation period, platelet aggregation was induced by various final concentrations of ADP (2, 5, 10, 15, 20, or 40 μM). We compared the level of maximum aggregation to the control measurement in each case.
It is well documented that heparin enhances platelet aggregation stimulated by various inductors both in vitro and in vivo. 20 In the next part of our study we examined whether PARP inhibitors can antagonize heparin-induced platelet hyperreactivity. Besides the experimental agent, sodium-heparin was also added to the samples before the incubation at a final concentration of 5 U/ml. The process of incubation and sample preparation was unchanged. Platelet aggregation was induced by several ADP concentrations: 0.5, 1, 2.5, 5, and 10 μM. The level of maximum aggregation was compared with that of control measurement (sample incubated with the same amount of heparin but without any PARP inhibitor).
Data are presented as means ± SEM. Samples were compared with the control values using the Student t test.
Effect of Poly(ADP-Ribose) Polymerase Inhibitors on Survival of Cardiomyocytes during Oxidative Insult
As a result of the incubation of H9c2 cardiomyocytes with 1 mM hydrogen peroxide for 3 hours, merely 44 ± 6% of the cells survived. 4-hydroxyquinazoline and 2-mercapto-4(3 H)-quinazolinone could not significantly improve the survival of the cells in the concentration range of 5 to 50 μM. Nevertheless, 100 μM and above 4-hydroxyquinazoline and 2-mercapto-4(3 H)-quinazolinone exerted marked protection on the cells compared with the untreated samples (P < 0.01) (Fig. 2). In the meantime, all the examined concentrations of HO-3089 (0.1–500 μM) significantly promoted the survival of H9c2 cells (P < 0.01) (Table 1) (Fig. 2).
Validation of Our In Vitro Model
Two clinically used antiplatelet agents were administered at the first stage of our study to validate our in vitro measurements. ASA decreased collagen- and epinephrine-induced platelet aggregation in a concentration-dependent manner. Significant inhibition could be observed from 1 μg/ml up to the entire examined concentration range (Fig. 3A). An in vitro concentration of 2 μg/ml and 7 μg/ml corresponds to the eventual serum concentration at 100-mg and 325-mg daily oral administration of ASA, respectively. 21,22 At the same time, acetyl salicylic acid had merely minimal impact on ADP-induced platelet aggregation.
The inhibitory effect of eptifibatide was proportional to the applied drug concentration and could totally block platelet aggregation stimulated by any of the inductors even in 400 ng/ml, well below its in vivo serum concentration (approximately 1000 ng/ml during continuous intravenous administration). 23 By blocking the GP IIb/IIIa platelet fibrinogen receptors, eptifibatide completely inhibits platelet aggregation independent of the type of activation (Fig. 3B).
Effect of Poly(ADP-Ribose) Polymerase Inhibitors on ADP-, Collagen-, and Epinephrine-Induced Platelet Aggregation
The commercially available PARP inhibitors, 4-hydroxyquinazoline and 2-mercapto-4(3 H)-quinazolinone markedly decreased both the 5 and 10 μM ADP-induced platelet aggregation at a concentration of 500 μM. At the same time, the experimental compound HO-3089 showed marked inhibition even at 20 μM (Figs. 4A and 4B). Each compound exerted an incremental inhibitory effect as the administered concentration increased and could significantly reduce ADP-induced platelet aggregation in higher concentrations (Table 1). While no significant inhibitory effect of 4-hydroxyquinazoline could be observed on either collagen- (2 μg/ml) or epinephrine- (10 μM) induced platelet aggregation, 2-mercapto-4(3 H)-quinazolinone and HO-3089 were able to impede the aggregation but merely at the highest examined concentrations (1500 μM and 500 μM, respectively) (data not shown).
Antiplatelet Activity of Poly(ADP-Ribose) Polymerase Inhibitors at Different ADP Concentrations
In our experiments PARP inhibitors were found to be effective mainly against ADP-induced platelet aggregation. Considering that the same PARP inhibitor concentrations had reduced impact on platelet aggregation when a higher concentration of ADP (10 μM) was applied, the dependence of their antiplatelet properties on ADP concentration was evaluated. Figure 5 demonstrates that, parallel to the increasing ADP concentrations, the hindrance on platelet aggregation by HO-3089 waned and finally disappeared at 40 μM. Similar phenomena were detected by the application of the other two PARP inhibitors (data not shown). The level of maximum aggregation at this ADP concentration—despite the presence of high PARP inhibitor concentrations—was found to be similar to that of control measurements.
Heparin-Induced Platelet Aggregation
In accordance with previous studies, our findings confirmed that human platelets showed hypersensitivity to ADP in the presence of heparin. ADP stimulated platelet aggregation in a dose-dependent manner, but almost complete aggregation was achieved in 1 μM, far below its commonly used concentration. However, heparin-evoked sensitization of platelets was markedly decreased by the administration of PARP inhibitors (Fig. 6).
Platelet aggregation is an indispensable step in the pathogenesis of cardiovascular diseases, which justifies that antiplatelet therapy has become a useful means of preventing and treating thrombotic events. 7 Numerous compounds have been developed that can interfere with platelet aggregation at receptorial (GP IIb/IIIa blockers, thienopyridines) or enzymatic level (ASA), but they do not exert any additional impact on the survival of cardiac cells. In the current in vitro study, we evaluated the effect of various PARP inhibitors on platelet responses in human platelet-rich plasma.
Previous studies demonstrated that poly(ADP-ribose) polymerase inhibitors can exert remarkable protection in experimental models of cardiac and brain ischemia-reperfusion, AZT- and anticancer agent-induced cardiomyopathy, ultraviolet radiation-induced skin lesion, various forms of shock, chronic heart failure, diabetic endothelial dysfunction, and diabetes. 17,18,24–29 Inhibition of PARP enzyme contributes to the preservation of intracellular nicotinamide adenine dinucleotide (NAD+) and adenosine triphosphate (ATP) pools in oxidatively challenged cells and tissues. The most efficacious PARP inhibitors can compete with ADP-ribose (substrate of the PARP enzyme) for binding to the enzyme due to their structural resemblance. 17,30 This obvious similarity raised the possibility that some PARP inhibitors may also bind to and block the ADP receptors found on platelets. We tested our hypothesis by using 3 different PARP inhibitors that promote the survival of H9c2 cardiomyocytes exposed to hydrogen peroxide-induced oxidative insult. In this widely used setting of oxidative stress, 4-hydroxyquinazoline (IC50 for PARP enzyme inhibition: 7 μM), 2-mercapto-4(3 H)-quinazolinone (IC50 = 35 μM), and HO-3089 (IC50 = 46 nM) protected the cardiomyocytes in concentrations greater than or equal to 100, 100, and 0.1 μM, respectively. These data are also in accordance with the results obtained in an isolated heart perfusion system, where 4-hydroxyquinazoline and 2-mercapto-4(3 H)-quinazolinone could preserve myocardial high-energy phosphate levels during ischemia-reperfusion cycle in 100μM. 18 HO-3089 also significantly promoted the recovery of high-energy phosphate intermediates in the same system in a concentration of 10 μM. 31 Consequently, these PARP inhibitors indeed render protection for oxidatively challenged myocardial cells.
Our initial findings with known, widely used antiplatelet agents validated our in vitro method for the investigation of experimental molecules in the same model. As expected, ASA decreased the collagen- and epinephrine-induced platelet aggregation in a concentration-dependent manner, while eptifibatide completely blocked the aggregation—well below its in vivo serum concentration—stimulated by any of the inductors.
In our in vitro model all 3 PARP inhibitors exerted marked antiplatelet activity when the aggregation was induced with ADP. However, on a molar basis HO-3089 had greater platelet inhibitory effect (significant inhibition could be observed even at 20 μM) than 4-hydroxyquinazoline and 2-mercapto-4(3 H)-quinazolinone (significant inhibition at 500 μM and above) (Table 1). Each compound exerted an incremental inhibitory effect as the administered concentration increased and could significantly reduce ADP-induced platelet aggregation. Although the concentration range required for the development of complete antiplatelet effect proved to be significantly higher than that of delivering protection in H9c2 cells by inhibiting the PARP enzyme. Considering the relevance of oxidative stress-induced platelet activation, even partial inhibition of platelet aggregation by lower concentrations of PARP inhibitors may have an additional beneficial effect under ischemic conditions.
The observed antiplatelet effect of the examined PARP inhibitors was specific on ADP receptors since 4-hydroxyquinazoline could not inhibit either collagen- (2 μg/ml) or epinephrine- (10 μM) induced platelet aggregation; 2-mercapto-4(3 H)-quinazolinone and HO-3089 were able to impede it, but only at the highest examined concentrations.
To clarify the hypothetical mechanism of the antiplatelet activity of PARP inhibitors, we used incremental amounts of ADP as an inductor. In accordance with our theory that PARP inhibitors may compete with ADP for their receptors, elevated concentrations of ADP gradually neutralized the antiplatelet properties of the selected PARP inhibitors. Moreover, when applying ADP at 40 μM, the antiaggregatory effect of the examined agents seemed to disappear. The level of maximum aggregation at this ADP concentration was found to be similar to that of control measurements. This phenomenon can speak for an eventual competitive antagonism as the possible underlying mechanism of the aggregation-blocking property of PARP inhibitors, because the increasing amounts of ligand could antagonize the inhibitory action. Of course, the antiplatelet effect of the examined PARP inhibitors occurred at higher concentrations than their PARP enzyme inhibitory activity, because this represents a receptorial mechanism. In addition, it is also clinically relevant that all the examined PARP inhibitors were capable of hindering thrombocyte aggregation even if those were sensitized to ADP by the pre-administration of heparin.
The use of sodium citrate as the anticoagulant throughout our experiments might be of interest. It is documented that a major consequence of lowering external Ca2+ concentrations in vitro is the activation of platelet thromboxane formation subsequent to stimulation by adrenaline or ADP, resulting in a “secondary” aggregation response. Inhibition of this excessive generation of thromboxane A2 subsequent to ADP or epinephrine induction may lead to an over-interpretation of the antiplatelet effects of both thromboxane synthase inhibitors and thromboxane receptor antagonists. 32,33 It has also been documented that sodium citrate may alter the function of the GPIIb/IIIa receptor complex and may enhance the binding of some antagonists to this receptor. 34 According to our present study PARP inhibitors are thought to compete with ADP for binding to its surface receptor on platelets; this is not affected by low extracellular Ca2+ concentrations. 35 In addition—as discussed previously—the examined agents did not have any effect on epinephrine- or collagen-induced platelet aggregation confirming that PARP inhibitors are not likely to influence platelet thromboxane A2 biosynthesis, even when ADP is used to stimulate platelet aggregation. As a consequence, we can conclude that the observed antiplatelet effect of PARP inhibitors may not be significantly influenced by the use of sodium citrate.
This is the first report on the ability of certain poly(ADP-ribose) polymerase inhibitors to interfere with ADP-induced platelet aggregation. As ADP is involved in physiological thrombocyte activation, our findings may be of crucial relevance in the eventual future therapeutic application of PARP inhibitors. Besides preserving the cellular energy stores and protecting the functioning of vascular endothelial cells, the observed beneficial “side-effect” of selected PARP inhibitors may contribute to the survival of ischemic tissues by hindering intravascular thrombus formation. These results depict an exciting novel feature of selected, ADP-, or adenine-mimicking PARP inhibitors that further adds to our understanding on the future therapeutic potential of these molecules.
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