Fabio M. Pulcinelli

Aspirin resistance: position paper of the Working Group on Aspirin Resistance

A. D . MICHELSON,* M. CATTANEO, J . W . E IKELBOOM,§ P . GURBEL ,– K . KOTT KE-MARCHANT ,** T . J . K UNIC K I , F . M. PULC INELL I , C . C ERLETT I §§ and A . K . RAO,–– ON BEHALF OF THE PLATELET PHYS IOLOGY SUBC OMMI TTEE O F T HE SC I ENT IF I C AND STA NDARDI ZAT I ON COMMITT EE OF THE IN TERN AT IONA L SOC IETY ON T HROMBOS IS A ND HA EMOSTA S IS* ** *Chair, Working Group on Aspirin Resistance, International Society on Thrombosis and Haemostasis; Department of Pediatrics, Center for Platelet Function Studies, University of Massachusetts Medical School, Worcester, MA, USA; Department of Medicine, Surgery, and Dentistry, Unit of Hematology and Thrombosis, University of Milan, Ospedale San Paolo, Milan, Italy; §Department of Haematology, School of Medicine and Pharmacology, University of Western Australia, Royal Perth Hospital, Perth, Australia; –Sinai Center for Thrombosis Research, Sinai Hospital, Baltimore, MD, USA; **Hemostasis and Thrombosis, Department of Clinical Pathology, Cleveland Clinic Foundation, Cleveland, OH, USA; Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA, USA; Department of Experimental Medicine and Pathology, University ‘‘La Sapienza’’, Rome, Italy; §§Center for High Technology Research and Education in Biomedical Sciences, Catholic University, Campobasso, Italy; ***Chairperson, ISTH-SSC Platelet Physiology Sub-Committee; and ––Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA, USA

Platelet Activation by Superoxide Anion and Hydroxyl Radicals Intrinsically Generated by Platelets That Had Undergone Anoxia and Then Reoxygenated

BACKGROUND Platelet activation has been demonstrated in experimental and clinical models of ischemia-reperfusion, but the underlying mechanism is still unclear. We mimicked the ischemia-reperfusion model in vitro by exposing platelets to anoxia-reoxygenation (A-R) and evaluated the role of oxygen free radicals (OFRs), which are usually produced during the reperfusion phase, in inducing platelet activation. METHODS AND RESULTS Human platelets were exposed to 15 and 30 minutes of anoxia and then reoxygenated. Compared with control platelets kept in atmospheric conditions, platelets exposed to A-R showed spontaneous platelet aggregation (SPA), which was maximal after 30 minutes of anoxia. Superoxide dismutase (SOD) (-74%, P < .005), catalase (-67%. P < .005). SOD plus catalase (-82%, P < .005), and the hydroxyl radical (OH0) scavengers mannitol (-66%, P < .005) and deoxyribose (-55%, P < .005) inhibited SPA. Platelets that had undergone A-R released superoxide anion (0-2), as detected by lucigenin chemiluminescence. Also, platelets exposed to A-R and incubated with salicylic acid generated 2.3- and 2,5-dihydroxybenzoates, which derive from salicylic acid reaction with OH0. SPA was significantly inhibited by the cyclooxygenase enzyme inhibitors aspirin and indomethacin: by SQ29548, a thromboxane (Tx) A2 receptor antagonist; by diphenyliodonium an inhibitor of flavoprotein-dependent enzymes: and by arachidonyl trifluoromethyl ketone, a selective inhibitor of cytosolic phospholipase A2. Platelets exposed to A-R markedly generated inositol 1,3,4-trisphosphate and TxA2, which were inhibited by incubation of platelets with SOD plus catalase. CONCLUSIONS This study shows that platelets exposed to A-R intrinsically generated 0-2 and OH0, which in turn activate arachidonic acid metabolism via phospholipases A2 and C, and provides further support for the use of antioxidant agents as inhibitors of platelet function in ischemia-reperfusion models.

Effects of Pomegranate Juice and Extract Polyphenols on Platelet Function

Several studies have shown that polyphenols reduce cardiovascular accidents in high-risk patients; in particular, the inhibition of platelet function may be responsible for part of this benefit. This research studied the antiplatelet effect of Wonderful variety pomegranate (Punica granatum) products, which contain primarily hydrolyzed tannins such as ellagitannins. We have investigated in vitro the effects of treatment with either pomegranate juice (PJ) or the polyphenol-rich extract from pomegranate fruit (POMx) on platelet aggregation, calcium mobilization, thromboxane A(2) production, and hydrogen peroxide formation, induced by collagen and arachidonic acid. PJ and POMx reduce all the platelet responses studied. POMx showed a stronger action in reducing platelet activation; moreover, POMx is active at the concentration that it is possible to obtain after polyphenol-rich food intake (2 microM). These results demonstrated that the cardiovascular health benefits of pomegranate may in part be related to the ability of polyphenols to inhibit platelet function. In fact, PJ and pomegranate extract have similar effects at concentrations expected for normal intake.

Persistent production of platelet thromboxane A2 in patients chronically treated with aspirin.

Summary.  Background: Patients treated with aspirin may have a reduced sensitivity to its antiplatelet effect. The mechanism accounting for such a reduced sensitivity might involve an impaired interaction of aspirin with cyclooxygenase‐1 (COX)‐1. Objective: We sought to investigate whether platelets from patients under chronic treatment with aspirin still produce TxA2 and whether there is any relationship between the eventual persistent TxA2 formation and platelet aggregation. Finally, whether platelet‐derived TxA2 can be inhibited by in vitro addition of aspirin. Methods: Collagen‐induced platelet aggregation and thromboxane‐A2 (TxA2) were measured in 196 patients treated with aspirin (100–330 mg day−1) because of previous vascular events or presence of risk factors of atherosclerosis. Results: Collagen‐induced TxA2 production of the entire cohort was 128.7 ± 21.6 pg 10−8 cells, and was significantly correlated with platelet aggregation (Spearmans correlation coefficient = 0.44; P < 0.0001). Patients in the highest quartile of TxA2 showed higher platelet response to collagen (P < 0.0001) when compared with those in the lowest quartile. In a subgroup of 96 patients, platelets were treated in vitro with a TxA2 receptor antagonist (13‐azaprostanoic acid) or aspirin before stimulation with collagen. 13‐APA acid significantly inhibited platelet aggregation. Aspirin reduced (−72.9%) TxA2 production in patients with TxA2 values above the median but it was ineffective in those with TxA2 values below the median. Conclusion: In some patients chronically treated with aspirin platelet production of TxA2 may persist and account for enhanced platelet aggregation. Incomplete inhibition of COX‐1 seems to be implicated in persistent TxA2 production.

Aspirin Extrusion From Human Platelets Through Multidrug Resistance Protein-4-Mediated Transport Evidence of a Reduced Drug Action in Patients After Coronary Artery Bypass Grafting

OBJECTIVES In this study we investigate: 1) the role of multidrug resistance protein-4 (MRP4), an organic anion unidirectional transporter, in modulating aspirin action on human platelet cyclooxygenase (COX)-1; and 2) whether the impairment of aspirin-COX-1 interaction, found in coronary artery bypass grafting (CABG) patients, could be dependent on MRP4-mediated transport. BACKGROUND Platelets of CABG patients present a reduced sensitivity to aspirin despite in vivo and in vitro drug treatment. Aspirin is an organic anion and could be a substrate for MRP4. METHODS Intracellular aspirin concentration and drug COX-1 activity, measured by thrombin-induced thromboxane B2 (TxB2) production, were evaluated in platelets obtained from healthy volunteers (HV) and hematopoietic-progenitor cell cultures reducing or not reducing MRP4-mediated transport. Platelet MRP4 expression was evaluated, in platelets from HV and CABG patients, by dot-blot or by immunogold-electromicrographs or immunofluorescence-microscopy analysis. RESULTS Inhibition of MRP4-mediated transport by dipyridamole or Mk-571 increases aspirin entrapment and its in vitro effect on COX-1 activity (142.7 ± 34.6 pg/10(8) cells vs. 343.7 ± 169.3 pg/10⁸ cells TxB2-production). Platelets derived from megakaryocytes transfected with MRP4 small interfering ribonucleic acid have a higher aspirin entrapment and drug COX-1 activity. Platelets from CABG patients showed a high expression of MRP4 whose in vitro inhibition enhanced aspirin effect on COX-1 (349 ± 141 pg/10⁸ cells vs. 1,670 ± 646 pg/10⁸ cells TxB2-production). CONCLUSIONS Aspirin is a substrate for MRP4 and can be extruded from platelet through its transportation. Aspirin effect on COX-1 is little-related to MRP4-mediated aspirin transport in HV, but in CABG patients with MRP4 over-expression, its pharmacological inhibition enhances aspirin action in an efficient way.

Plasma levels of thromboxane A2 on admission are associated with no-reflow after primary percutaneous coronary intervention

AIMS Thromboxane A2 (TXA2) is a key mediator of platelet activation and aggregation, and an important mediator of platelet-induced coronary artery constriction. We sought to investigate whether baseline plasma levels of TXA2 are associated with coronary no-reflow after primary percutaneous coronary intervention (PPCI). METHODS AND RESULTS A total of 47 consecutive patients (age, 62.5 +/- 12.7; male sex, 76.6%) admitted to our hospital for a first ST-segment elevation myocardial infarction and undergoing PPCI within 12 h of onset of symptoms were enrolled. Admission TXA2 plasma levels were measured by enzyme-linked immunosorbent assay (ELISA). Angiographic no-reflow was defined as a final TIMI flow of <or=2 or final TIMI flow of 3 with a myocardial blush grade of <2, whereas ST-segment resolution from baseline value of <or=50% was used as ECG index of no-reflow. At multivariable analysis TXA2 plasma levels, endothelin-1 (ET-1) plasma levels, and left anterior descending coronary artery (LAD) as culprit vessel were significant predictors of angiographic no-reflow (P = 0.04), whereas TXA2 and ET-1 plasma levels were the only independent predictors of lack of ST-segment resolution (P = 0.013 and 0.04, respectively). Of note, TXA2 tertiles were independent predictors of both angiographic no-reflow and lack of ST-segment resolution (OR, 3.5; 95% CI, 1.1-11; P = 0.03 and OR, 3; 95% CI, 1.3-7; P = 0.01, respectively). CONCLUSION TXA2 is an independent indicator of no-reflow that occurs after PPCI. This observation may open new therapeutic opportunity in the setting of PPCI.

Vitamin E inhibits collagen-induced platelet activation by blunting hydrogen peroxide.

In this study, we investigated whether vitamin E at concentrations achievable in blood after supplementation inhibits platelet function in humans. Gel-filtered platelets were incubated 30 minutes with scalar concentrations (50 to 250 mmol/L) of vitamin E and then stimulated with collagen. Compared with controls, vitamin E inhibited collagen-induced platelet aggregation and thromboxane A2 formation in a dose-dependent manner. Furthermore, vitamin E inhibited, in a dose-dependent manner, Ca(2+) mobilization and formation of inositol 1,4,5-triphosphate. Because it was previously shown that hydrogen peroxide formation mediates arachidonic acid metabolism and phospholipase C activation in collagen-induced platelet activation, we investigated whether vitamin E was able to blunt hydrogen peroxide. In experiments performed in unstimulated platelets supplemented with hydrogen peroxide and in collagen-stimulated platelets, vitamin E was able to blunt hydrogen peroxide. In 6 healthy subjects given vitamin E for 2 weeks (600 mg/d), we found a significant decrease of collagen-induced H(2)O(2) formation, platelet aggregation, and calcium mobilization. This study demonstrated in vitro and ex vivo that vitamin E inhibits collagen-induced platelet activation by blunting hydrogen peroxide formation.

Clinical ResearchAntiplatelet TherapyAspirin Extrusion From Human Platelets Through Multidrug Resistance Protein-4–Mediated Transport: Evidence of a Reduced Drug Action in Patients After Coronary Artery Bypass Grafting

OBJECTIVES In this study we investigate: 1) the role of multidrug resistance protein-4 (MRP4), an organic anion unidirectional transporter, in modulating aspirin action on human platelet cyclooxygenase (COX)-1; and 2) whether the impairment of aspirin-COX-1 interaction, found in coronary artery bypass grafting (CABG) patients, could be dependent on MRP4-mediated transport. BACKGROUND Platelets of CABG patients present a reduced sensitivity to aspirin despite in vivo and in vitro drug treatment. Aspirin is an organic anion and could be a substrate for MRP4. METHODS Intracellular aspirin concentration and drug COX-1 activity, measured by thrombin-induced thromboxane B2 (TxB2) production, were evaluated in platelets obtained from healthy volunteers (HV) and hematopoietic-progenitor cell cultures reducing or not reducing MRP4-mediated transport. Platelet MRP4 expression was evaluated, in platelets from HV and CABG patients, by dot-blot or by immunogold-electromicrographs or immunofluorescence-microscopy analysis. RESULTS Inhibition of MRP4-mediated transport by dipyridamole or Mk-571 increases aspirin entrapment and its in vitro effect on COX-1 activity (142.7 ± 34.6 pg/10(8) cells vs. 343.7 ± 169.3 pg/10⁸ cells TxB2-production). Platelets derived from megakaryocytes transfected with MRP4 small interfering ribonucleic acid have a higher aspirin entrapment and drug COX-1 activity. Platelets from CABG patients showed a high expression of MRP4 whose in vitro inhibition enhanced aspirin effect on COX-1 (349 ± 141 pg/10⁸ cells vs. 1,670 ± 646 pg/10⁸ cells TxB2-production). CONCLUSIONS Aspirin is a substrate for MRP4 and can be extruded from platelet through its transportation. Aspirin effect on COX-1 is little-related to MRP4-mediated aspirin transport in HV, but in CABG patients with MRP4 over-expression, its pharmacological inhibition enhances aspirin action in an efficient way.

JNK2 is activated during ER stress and promotes cell survival.

Adaptation to endoplasmic reticulum (ER) stress relies on activation of the unfolded protein response (UPR) and induction of autophagy. Indeed, cells die if ER stress is not countered by the UPR. Here we show in U937 cells that the ER stressors tunicamycin and thapsigargin cause increased expression of c-Jun N-terminal kinase 2 (JNK2), which allows regulation of the UPR, whose silencing or pharmacological inhibition delays BiP (immunoglobulin heavy-chain binding protein) upregulation, and causes earlier and greater expression of CCAAT/enhancer-binding protein-homologous protein (CHOP). Furthermore, we show that pharmacological inhibition or silencing of JNK2 causes accumulation of both p62 and the acidic compartment, caspase 3 activation and apoptosis. Our results reveal that JNK2 prevents accumulation of the acidic compartment in U937 cells undergoing autophagic flux and, by this mechanism, it keeps stressed cells alive. Our findings highlight a potential role for JNK2 in tumor cell survival, senescence and neurodegenerative diseases, in which ER stress, autophagy and lysosome activity are known to interplay.

Evidence for separate effects of U73122 on phospholipase C and calcium channels in human platelets

U73122 ((1-[6-(( 17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)exyl]-1H-p yrrole-2,5-dione)) is generally used as a selective inhibitor of phospholipase C (PLC) and the related rise in cytosolic Ca2+. Recently, by using hepatocytes, it was suggested that its action sites are different for PLC activation and increase in Ca2+ concentration. To verify whether U73122 has different sites for inhibiting PLC activation and calcium responses in human platelets, aggregation, Mn2+ influx, cytosolic Ca2+ increase and PLC activation were studied in response to thrombin and the synthetic agonist of the thromboxane receptor U46619 (9,11-dideoxy-9alpha,11alpha-methanoepoxyprostaglandin F2alpha). With both agonists, U73122 inhibited aggregation, Mn2+ influx and the enhancement of cytosolic calcium at concentrations of 2 microM or lower, while 10 microM was necessary to inhibit PLC activation. Our results suggested that U73122 is much more active in antagonizing Ca2+ channels, both the intracellular ones, which are activated by formation of inositol 1,4,5 P3 and those present on plasma membrane, than in reducing the activation of PLC.