Download PDF
Thromb Haemost 2018; 118(03): 490-501
DOI: 10.1055/s-0038-1629920
Cellular Haemostasis and Platelets
Schattauer GmbH Stuttgart

Impact of Multidrug Resistance Protein-4 Inhibitors on Modulating Platelet Function and High on-Aspirin Treatment Platelet Reactivity

Laura Alemanno
,
Isabella Massimi
,
Vanessa Klaus
,
Maria Luisa Guarino
,
Teresa Maltese
,
Luigi Frati
,
Dominick J. Angiolillo
,
Fabio M. Pulcinelli
Further Information

Publication History

07 August 2017

06 December 2017

Publication Date:
15 February 2018 (online)

    Permissions and Reprints

Abstract

Platelet multidrug resistance protein 4 (MRP4) plays a modulating role on platelet activation. Platelet function and thrombus formation are impaired in MRP4 knockout mice models, and, among aspirin-treated patients, high on-aspirin residual platelet reactivity (HARPR) positively correlates with MRP4 levels. To better understand the effects of MRP4 on platelet function, the aim of this investigation was to assess the impact of cilostazol-induced inhibition of MRP4-mediated transport and assess aspirin-induced antiplatelet effects and rates of HARPR in human subjects.

Cilostazol-dependent inhibition of MRP4-mediated transport was assessed with the release of the fluorescent adduct bimane-glutathione and aspirin entrapment. Effect of Cilostazol on cAMP inhibition was evaluated by vasodilator-stimulated phosphoprotein (VASP). Platelet function was studied by collagen and TRAP-6-induced platelet aggregation and secretion.

Cilostazol reduced the release of bimane-glutathione and enhanced aspirin entrapment demonstrating an inhibitory effect on MRP4 in platelets. VASP phosphorylation was absent until 10 seconds after addition of cilostazol, and becomes evident after 30 seconds. An inhibitory effect on platelet aggregation and secretion was found in activated platelets, with threshold concentration of agonists, 10 seconds after addition of cilostazol, supporting a role of MRP4 on platelet function that is cAMP independent. Cilostazol effects were also shown in aspirin-treated platelets. A reduction of platelet aggregation and secretion were observed in aspirin-treated patients with HARPR.

This study supports the role of MRP4 on modulating platelet function which occurs through cAMP-independent mechanisms. Moreover, inhibition of MRP4 induced by cilostazol enhances aspirin-induced antiplatelet effects and reduces HARPR.

Keywords

platelet function - multidrug resistance protein-4 - aspirin

Key Points

1. Role of MRP4 on modulating platelet function through cAMP-independent mechanisms.


2. In patients on chronic aspirin therapy, inhibition of MRP4-mediated transport reduces high on-aspirin residual platelet reactivity.


Authors' Contribution

L. Alemanno designed the study, performed experiments, analysed results and wrote the manuscript. I. Massimi, V. Klaus, M.L. Guarino and T. Maltese, L. Frati and D.J. Angiolillo contributed to the study design, interpreted data and revised the intellectual content of the manuscript. F.M. Pulcinelli was responsible for the concept of the study, designed the study, analysed results and wrote the manuscript.


Supplementary Material

 

  • References

  • 1 Angiolillo DJ, Ueno M, Goto S. Basic principles of platelet biology and clinical implications. Circ J 2010; 74 (04) 597-607
    CrossrefPubMedGoogle Scholar
  • 2 Angiolillo DJ. The evolution of antiplatelet therapy in the treatment of acute coronary syndromes: from aspirin to the present day. Drugs 2012; 72 (16) 2087-2116
    CrossrefPubMedGoogle Scholar
  • 3 Angiolillo DJ, Cho JR. Aspirin treatment and outcomes in patients undergoing percutaneous coronary intervention: is there a role for pharmacodynamic testing?. J Am Coll Cardiol 2014; 64 (09) 872-874
    CrossrefPubMedGoogle Scholar
  • 4 Sofi F, Marcucci R, Gori AM, Abbate R, Gensini GF. Residual platelet reactivity on aspirin therapy and recurrent cardiovascular events--a meta-analysis. Int J Cardiol 2008; 128 (02) 166-171
    CrossrefPubMedGoogle Scholar
  • 5 Krasopoulos G, Brister SJ, Beattie WS, Buchanan MR. Aspirin “resistance” and risk of cardiovascular morbidity: systematic review and meta-analysis. BMJ 2008; 336 (7637): 195-198
    CrossrefPubMedGoogle Scholar
  • 6 Snoep JD, Hovens MM, Eikenboom JC, van der Bom JG, Huisman MV. Association of laboratory-defined aspirin resistance with a higher risk of recurrent cardiovascular events: a systematic review and meta-analysis. Arch Intern Med 2007; 167 (15) 1593-1599
    CrossrefPubMedGoogle Scholar
  • 7 Frelinger III AL, Li Y, Linden MD. , et al. Association of cyclooxygenase-1-dependent and -independent platelet function assays with adverse clinical outcomes in aspirin-treated patients presenting for cardiac catheterization. Circulation 2009; 120 (25) 2586-2596
    CrossrefPubMedGoogle Scholar
  • 8 Gori AM, Grifoni E, Valenti R. , et al. High on-aspirin platelet reactivity predicts cardiac death in acute coronary syndrome patients undergoing PCI. Eur J Intern Med 2016; 30: 49-54
    CrossrefPubMedGoogle Scholar
  • 9 Fiolaki A, Katsanos AH, Kyritsis AP. , et al. High on treatment platelet reactivity to aspirin and clopidogrel in ischemic stroke: a systematic review and meta-analysis. J Neurol Sci 2017; 376: 112-116
    CrossrefPubMedGoogle Scholar
  • 10 Mayer K, Bernlochner I, Braun S. , et al. Aspirin treatment and outcomes after percutaneous coronary intervention: results of the ISAR-ASPI registry. J Am Coll Cardiol 2014; 64 (09) 863-871
    CrossrefPubMedGoogle Scholar
  • 11 Mattiello T, Guerriero R, Lotti LV. , et al. 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. J Am Coll Cardiol 2011; 58 (07) 752-761
    CrossrefPubMedGoogle Scholar
  • 12 Russel FG, Koenderink JB, Masereeuw R. Multidrug resistance protein 4 (MRP4/ABCC4): a versatile efflux transporter for drugs and signalling molecules. Trends Pharmacol Sci 2008; 29 (04) 200-207
    CrossrefPubMedGoogle Scholar
  • 13 Furmanski BD, Hu S, Fujita KI. , et al. Contribution of ABCC4-mediated gastric transport to the absorption and efficacy of dasatinib. Clin Cancer Res 2013; 19 (16) 4359-4370
    CrossrefPubMedGoogle Scholar
  • 14 Massimi I, Guerriero R, Lotti LV. , et al. Aspirin influences megakaryocytic gene expression leading to up-regulation of multidrug resistance protein-4 in human platelets. Br J Clin Pharmacol 2014; 78 (06) 1343-1353
    CrossrefPubMedGoogle Scholar
  • 15 Temperilli F, Di Franco M, Massimi I. , et al. Nonsteroidal anti-inflammatory drugs in-vitro and in-vivo treatment and Multidrug Resistance Protein 4 expression in human platelets. Vascul Pharmacol 2016; 76: 11-17
    CrossrefPubMedGoogle Scholar
  • 16 Bally M, Dendukuri N, Rich B. , et al. Risk of acute myocardial infarction with NSAIDs in real world use: bayesian meta-analysis of individual patient data. BMJ 2017; 357: j1909
    CrossrefPubMedGoogle Scholar
  • 17 Lien LM, Chen ZC, Chung CL. , et al. Multidrug resistance protein 4 (MRP4/ABCC4) regulates thrombus formation in vitro and in vivo. Eur J Pharmacol 2014; 737: 159-167
    CrossrefPubMedGoogle Scholar
  • 18 Decouture B, Dreano E, Belleville-Rolland T. , et al. Impaired platelet activation and cAMP homeostasis in MRP4-deficient mice. Blood 2015; 126 (15) 1823-1830
    CrossrefPubMedGoogle Scholar
  • 19 Cheepala SB, Pitre A, Fukuda Y. , et al. The ABCC4 membrane transporter modulates platelet aggregation. Blood 2015; 126 (20) 2307-2319
    CrossrefPubMedGoogle Scholar
  • 20 Massimi I, Lotti LV, Temperilli F. , et al. Enhanced platelet MRP4 expression and correlation with platelet function in patients under chronic aspirin treatment. Thromb Haemost 2016; 116 (06) 1100-1110
    Article in Thieme ConnectPubMedGoogle Scholar
  • 21 Goto S. Cilostazol: potential mechanism of action for antithrombotic effects accompanied by a low rate of bleeding. Atheroscler Suppl 2005; 6 (04) 3-11
    PubMedGoogle Scholar
  • 22 Tantry US, Bliden KP, Gurbel PA. Overestimation of platelet aspirin resistance detection by thromboelastograph platelet mapping and validation by conventional aggregometry using arachidonic acid stimulation. J Am Coll Cardiol 2005; 46 (09) 1705-1709
    CrossrefPubMedGoogle Scholar
  • 23 Nagatsuka K, Miyata S, Kada A. , et al. Cardiovascular events occur independently of high on-aspirin platelet reactivity and residual COX-1 activity in stable cardiovascular patients. Thromb Haemost 2016; 116 (02) 356-368
    Article in Thieme ConnectPubMedGoogle Scholar
  • 24 Pulcinelli FM, Gresele P, Bonuglia M, Gazzaniga PP. Evidence for separate effects of U73122 on phospholipase C and calcium channels in human platelets. Biochem Pharmacol 1998; 56 (11) 1481-1484
    CrossrefPubMedGoogle Scholar
  • 25 Bai J, Lai L, Yeo HC, Goh BC, Tan TM. Multidrug resistance protein 4 (MRP4/ABCC4) mediates efflux of bimane-glutathione. Int J Biochem Cell Biol 2004; 36 (02) 247-257
    CrossrefPubMedGoogle Scholar
  • 26 Gaspari F, Locatelli M. Determination of aspirin and salicylic acid in uremic patients' plasma using reversed-phase high-performance liquid chromatography. Ther Drug Monit 1987; 9 (02) 243-247
    CrossrefPubMedGoogle Scholar
  • 27 Morita I, Schindler M, Regier MK. , et al. Different intracellular locations for prostaglandin endoperoxide H synthase-1 and -2. J Biol Chem 1995; 270 (18) 10902-10908
    CrossrefPubMedGoogle Scholar
  • 28 Borgognone A, Pulcinelli FM. Reduction of cAMP and cGMP inhibitory effects in human platelets by MRP4-mediated transport. Thromb Haemost 2012; 108 (05) 955-962
    Article in Thieme ConnectPubMedGoogle Scholar
  • 29 Born GVR. Aggregation of blood platelets by adenosine diphosphate and its reversal. Nature 1962; 194: 927-929
    PubMedGoogle Scholar
  • 30 Guarino ML, Massimi I, Mardente S. , et al. New platelet functional method for identification of pathogenic antibodies in HIT patients. Platelets 2017; 28 (07) 728-730
    CrossrefPubMedGoogle Scholar
  • 31 Rosenkranz B, Frölich JC. Plasma concentrations and anti-platelet effects after low dose acetylsalicylic acid. Prostaglandins Leukot Med 1985; 19 (03) 289-300
    CrossrefPubMedGoogle Scholar
  • 32 Shinohara Y, Katayama Y, Uchiyama S. , et al; CSPS 2 Group. Cilostazol for prevention of secondary stroke (CSPS 2): an aspirin-controlled, double-blind, randomised non-inferiority trial. Lancet Neurol 2010; 9 (10) 959-968
    CrossrefPubMedGoogle Scholar
  • 33 Fan ZG, Ding GB, Li XB, Gao XF, Gao YL, Tian NL. The clinical outcomes of triple antiplatelet therapy versus dual antiplatelet therapy for high-risk patients after coronary stent implantation: a meta-analysis of 11 clinical trials and 9,553 patients. Drug Des Devel Ther 2016; 10: 3435-3448
    CrossrefPubMedGoogle Scholar
  • 34 Chen KY, Rha SW, Li YJ. , et al; Korea Acute Myocardial Infarction Registry Investigators. Triple versus dual antiplatelet therapy in patients with acute ST-segment elevation myocardial infarction undergoing primary percutaneous coronary intervention. Circulation 2009; 119 (25) 3207-3214
    CrossrefPubMedGoogle Scholar
  • 35 Lee SW, Park SW, Kim YH. , et al; DECLARE-Long Study Investigators. Comparison of triple versus dual antiplatelet therapy after drug-eluting stent implantation (from the DECLARE-Long trial). Am J Cardiol 2007; 100 (07) 1103-1108
    CrossrefPubMedGoogle Scholar
  • 36 Angiolillo DJ, Capranzano P, Ferreiro JL. , et al. Impact of adjunctive cilostazol therapy on platelet function profiles in patients with and without diabetes mellitus on aspirin and clopidogrel therapy. Thromb Haemost 2011; 106 (02) 253-262
    Article in Thieme ConnectPubMedGoogle Scholar
  • 37 Lee SW, Park SW, Kim YH. , et al. Drug-eluting stenting followed by cilostazol treatment reduces late restenosis in patients with diabetes mellitus the DECLARE-DIABETES Trial (A Randomized Comparison of Triple Antiplatelet Therapy with Dual Antiplatelet Therapy After Drug-Eluting Stent Implantation in Diabetic Patients). J Am Coll Cardiol 2008; 51 (12) 1181-1187
    CrossrefPubMedGoogle Scholar