Abstract
The impact of inhibition of multidrug resistance protein 4 (MRP4) on nitric oxide (NO) resistance and on ADP-induced platelet aggregation is unknown. The aim of this investigation was to verify whether platelet NO resistance correlates with MRP4 expression and evaluate whether this can be reduced by in vitro MRP4 inhibition mediated by cilostazol. Moreover, we assessed if inhibition of MRP4-mediated transport reduces ADP-induced platelet reactivity. The inhibitory effect of sodium nitroprusside (SNP), a NO-donor that enhances cyclic guanosine monophosphate (cGMP) cytosolic concentration, was assessed in platelets obtained from aspirin treated patients and in a control population. The inhibitory effect of SNP was evaluated by ADP-induced aggregation in SNP-treated platelets. The impact of MRP4 on ADP-induced platelet aggregation was performed in high on aspirin residual platelet reactivity (HARPR) patients and compared to healthy volunteers (HV), and a control cohort (CTR). In aspirin-treated patients with high levels of MRP4, reduced SNP inhibition was found compared to those with low levels of MRP4. MRP4 inhibition by cilostazol significantly reduced ADP-induced platelet aggregation in HARPR population, and to a lesser extent in HV and CTR populations. In conclusion, cilostazol can mitigate the hyper-reactive platelet phenotype of HARPR patients by reducing residual ADP-induced platelet aggregation and increasing NO-dependent endothelial antiplatelet effects.
Highlights
Platelets play a key role in haemostatic processes [1]
In order to confirm a role of multidrug resistance protein 4 (MRP4) in reducing sodium nitroprusside (SNP) effects, platelets obtained from 8 patients with ADP-induced Platelet aggregation (PA) > 20% after SNP treatment were incubated with two MRP4 inhibitors cilostazol (20 μM)
nitric oxide (NO) plays an essential role in the process of haemostasis by enhancing cytosolic concentration of cyclic guanosine monophosphate (cGMP) and inhibiting platelet activation and thrombus formation [20]
Summary
To limit excessive platelet activation and thrombus formation, platelets are regulated by endothelial cell-derived inhibitory signals such as nitric oxide (NO). Sassi et al demonstrated secretion of cyclic nucleotides from the cytoplasm through the multidrug-resistance protein MRP4/ABCC4 [5]. MRP4 is mainly located on the membrane of dense granules and to a lesser extent on the plasma membrane [6] where it acts as a negative regulator of cyclic nucleotides hens limiting their inhibitory effects [6, 7]. The role of MRP4 in modulating platelet function by regulating intracellular concentration of cyclic nucleotides was demonstrated in a MRP4 knock-out mouse model showing prolonged bleeding times, defective platelet aggregation and in vivo thrombus formation [8, 9]. Most recently, impaired platelet aggregation in response to ADP was observed in ABCC4 null individuals [10]
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