ISTH

20

17 Poster presented at

ISTH2017 on:

Laura Alemanno, ScD1, Isabella Massimi, PhD1, Vanessa Klaus, ScD2, Maria Luisa Guarino, ScD1, Teresa Maltese, ScD1, Giulia Maria, ScD1, Dominick J. Angiolillo, MD, PhD3, Fabio M Pulcinelli, MD, PhD1

Department of Experimental Medicine, Sapienza University of Rome, Italy

TOPIC: 65.Platelets Function and Interactions

1 Department of Experimental Medicine, Sapienza University of Rome, Italy.

2 Julius-Maximilians-Universität Würzburg, Würzburg, Germany.

3 Division of Cardiology, University of Florida College of Medicine-Jacksonville, FL USA.

Platelet Multidrug resistance Protein 4 plays a modulating role on platelet function. 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.

Platelet aggregation (PA) evaluated as percent of aggregation observed after 4 min stimulation (%PA) in response to collagen (threshold concentration). ATP release (luciferin–luciferase assay) was measured to evaluate platelet secretion. VASP Phosphorylation to study cAMP dependent cilostazol effects . Populations: healthy volunteers (HV); patients under chronic aspirin treatment (N=122).

Collagen induced PA and secretion were inhibited when platelets were activated 10” after cilostazol addition (Figure 1). VASP phosphorylation was absent at this time, indicating that such inhibition is not cAMP-cGMP correlated (Figure 2). The effect of Cilostazol on PA is dependent on MRP4 inhibition, similar reduction was obtained using an MRP4 selective inhibitor, Ceefourin1 (Figure 3). In aspirin treated platelets Collagen induced PA and secretion were inhibited by cilostazol (Figure 4). Cilostazol treatment reduces collagen induced PA in patients with HARPR (Figure 5).

INTRODUCTION

METHODS

RESULTS

This study supports the role of MRP4 on platelet function which exerts its effects through cAMP independent mechanisms. Inhibition of MRP4 by cilostazol enhances aspirin-induced antiplatelet effects. Inhibition of MRP4 reduces HARPR, in patients under chronic aspirin treatment.

50 Kda

CTR T-10’’ VASP p – Ser239

TOTAL VASP

Assess the impact of cilostazol-induced inhibition of MRP4 mediated transport and assess aspirin-induced antiplatelet effects and rates of HARPR in humans.

OBJECTIVES

Inhibition of MRP4 Mediated Transport Reduces Platelet Function in a cAMP-cGMP Independent Manner

CONCLUSIONS

Figure 1 Collagen 0.5 µg/ml; cilostazol 5 µM

Mean ± SD 5 experiments performed

Figure 4 Aspirinated platelets (100µM, 20’ 37°C); collagen 8µg/ml; cilostazol 5 µM

Mean ± SD 5 experiments performed

Figure 2 WB representative of 3 independent

experiments performed

Figure 5 Collagen 4 µg/ml; cilostazol 20µM

Figure 3 Collagen 0.5 µg/ml; ceefourin1 50 µM Mean ± SD 3 experiments performed

References:

1. Lien LM, Chen ZC, Chung CL, Yen TL, Chiu HC, Chou DS, Huang SY, Sheu JR, Lu WJ, Lin KH. Multidrug resistance protein 4 (mrp4/abcc4) regulates thrombus formation in vitro and in vivo. Eur J Pharmacol. 2014;737:159-167

2. Decouture B, Dreano E, Belleville-Rolland T, Kuci O, Dizier B, Bazaa A, Coqueran B, Lompre AM, Denis CV, Hulot JS, Bachelot-Loza C, Gaussem P. Impaired platelet activation and camp homeostasis in mrp4-deficient mice. Blood. 2015;126:1823-1830

3. Cheepala SB, Pitre A, Fukuda Y, Takenaka K, Zhang Y, Wang Y, Frase S, Pestina T, Gartner TK, Jackson C, Schuetz JD. The abcc4 membrane transporter modulates platelet aggregation. Blood. 2015;126:2307-2319

4. Massimi I, Lotti LV, Temperilli F, Mancone M, Sardella G, Calcagno S, Turriziani O, Frati L, Pulcinelli FM. Enhanced platelet mrp4 expression and correlation with platelet function in patients under chronic aspirin treatment. Thrombosis and Haemostasis. 2016;116:1100-1110

1282--PBLaura Alemanno Tuesday, July 11DOI: 10.3252/pso.eu.ISTH2017.2017

Platelet Function and Interactions