Wolf, Robert and Grammbauer, Sophie and Palankar, Raghavendra and Tolksdorf, Céline and Moritz, Eileen and Böhm, Andreas and Hasan, Mahmoud and Hafkemeyer, Annika and Greinacher, Andreas and Tzvetkov, Mladen V. and Rauch, Bernhard H. and Jedlitschky, Gabriele (2022) Specific inhibition of the transporter MRP4/ABCC4 affects multiple signaling pathways and thrombus formation in human platelets. Haematologica. pp. 1-9. ISSN 1592-8721 - 0390-6078

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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 to 50% when ADP or collagen was used as activating agents, respectively. Ceefourin-1 significantly lowered the ADP-induced activation of integrin αIIbβ3, indicated by binding of FITCfibrinogen (about 50% reduction at 50 μM 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 αIIbβ3 activation, elevated VASP phosphorylation and reduced calcium influx, based on inhibited cyclic nucleotide and thromboxane transport as well as possible further mechanisms.

Item Type:	Article
Subjects:	Technology, medicine, applied sciences > Medicine and health
Divisions:	Faculty of Medicine and Health Sciences > Department of Human Medicine
Date Deposited:	07 Jun 2022 07:03
Last Modified:	07 Jun 2022 07:03
URI:	https://oops.uni-oldenburg.de/id/eprint/5438
URN:	urn:nbn:de:gbv:715-oops-55197
DOI:	10.3324/haematol.2021.279761
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