Page 207 - Haematologica Vol. 107 - September 2022

ARTICLE - Platelet Biology & its Disorders
Specific inhibition of the transporter MRP4/ABCC4 affects multiple signaling pathways and thrombus formation
in human platelets
Robert Wolf,1,2 Sophie Grammbauer,1 Raghavendra Palankar,3 Céline Tolksdorf,1,4 Eileen Moritz,1,2 Andreas Böhm,1 Mahmoud Hasan,1 Annika Hafkemeyer,1 Andreas Greinacher,3 Mladen V. Tzvetkov,1 Bernhard H. Rauch1,2,4# and Gabriele Jedlitschky1#
1Department of General Pharmacology, Center of Drug Absorption and Transport (C_DAT), University Medicine Greifswald, Greifswald; 2German Center for Cardiovascular Research (DZHK), Partner Site Greifswald; 3Department of Immunology and Transfusion Medicine, University Medicine Greifswald, Greifswald and 4Department of Human Medicine, Section of Pharmacology and Toxicology, Carl von Ossietzky University of Oldenburg, Oldenburg, Germany
#BHR and GJ contributed equally as co-senior authors.
Abstract
The multidrug resistance protein 4 (MRP4) is highly expressed in platelets and several lines of evidence point to an impact on platelet function. MRP4 represents a transporter for cyclic nucleotides as well as for certain lipid mediators. The aim of the present study was to comprehensively characterize the effect of a short-time specific pharmacological inhibition of MRP4 on signaling pathways in platelets. Transport assays in isolated membrane vesicles showed a concentration- dependent inhibition of MRP4-mediated transport of cyclic nucleotides, thromboxane (Tx)B2 and fluorescein (FITC)- labeled sphingosine-1-phosphate (S1P) by the selective MRP4 inhibitor Ceefourin-1. In ex vivo aggregometry studies in human platelets, Ceefourin-1 significantly inhibited platelet aggregation by about 30-50% when ADP or collagen was used as activating agents, respectively. Ceefourin-1 significantly lowered the ADP-induced activation of integrin aIIbb3, indicated by binding of FITC-fibrinogen (about 50% reduction at 50 mM Ceefourin-1), and reduced calcium influx. Furthermore, pre-incubation with Ceefourin-1 significantly increased PGE1- and cinaciguat-induced vasodilator- stimulated phosphoprotein (VASP) phosphorylation, indicating increased cytosolic cAMP as well as cGMP concentrations, respectively. The release of TxB2 from activated human platelets was also attenuated. Finally, selective MRP4 inhibition significantly reduced both the total area covered by thrombi and the average thrombus size by about 40% in a flow chamber model. In conclusion, selective MRP4 inhibition causes reduced platelet adhesion and thrombus formation under flow conditions. This finding is mechanistically supported by inhibition of integrin aIIbb3 activation, elevated VASP phosphorylation and reduced calcium influx, based on inhibited cyclic nucleotide and thromboxane transport as well as possible further mechanisms.
Introduction
The multidrug resistance protein 4 (MRP4) (ABCC4) is a member of the MRP/CFTR subfamily (C-branch) of the ATP- binding cassette (ABC) transporters, a family of proteins that mediate an ATP-driven transmembrane transport of compounds. It represents a very versatile transporter, which is expressed in several tissues with high amounts in platelets.1-4 Its substrate spectrum covers several drugs, namely nucleoside-based antiviral and anti-cancer agents but interestingly also a number of endogenous signaling molecules. These include primarily the cyclic nucleotides cAMP and cGMP.5,6 MRP4 has been established as an inde-
pendent regulator of intracellular cAMP levels and of cell proliferation and differentiation in several cell types, in- cluding vascular smooth muscle cells as well as hemato- poietic cells.7,8 Furthermore, cyclic nucleotides play a major role in platelet activation and regulation. In Mrp4-deficient mice, dysregulation of platelet cAMP homeostasis was ob- served.9-11 This dysregulation may be due to reduced cAMP efflux and/or intracellular sequestration since the exact lo- calization of MRP4 in resting platelets has yet to be clari- fied. There is evidence for plasma membrane localization,10 but also for a partial intracellular localization of MRP4 in association with the dense granule markers.1-3,11 In addition, Cheepala et al. reported a reduced plasma membrane lo-
Haematologica | 107 September 2022
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Correspondence: G, Jedlitschky gabriele.jedlitschky@med.uni-greifswald.de
Received: Accepted: Prepublished:
August 4, 2021. March 9, 2022. March 17, 2022.
https://doi.org/10.3324/haematol.2021.279761
©2022 Ferrata Storti Foundation Published under a CC BY-NC license