Abstract Number: PB1841
Meeting: ISTH 2020 Congress
Background: The multidrug resistance protein 4 (MRP4) is a versatile efflux transporter highly expressed in platelets and transports cyclic nucleotides as well as certain lipid mediators including sphingosine-1-phosphate (S1P).
Aims: Aim of the present study was to comprehensively characterize the effects of the selective MRP4 inhibitor Ceefourin-1 on different signaling pathways in platelets.
Methods: Platelet activation was determined by light transmission aggregometry and flow cytometry. Thrombus formation in whole blood was analyzed in collagen-coated microchannels under high arterial shear conditions. Phosphorylation of vasodilator-stimulated phosphoprotein (VASP) was quantified by flow cytometry; calcium influx in Fura-2 loaded platelets. Transport of 3H-cGMP, fluorescein (FITC)-labelled S1P and thromboxane B2 (TXB2) was studied in membrane vesicles from MRP4-overexpressing SF9 cells and human platelets.
Results: Ceefourin-1 concentration-dependently inhibited the MRP4-mediated membrane transport of cyclic nucleotides, TXB2 and S1P. In ex-vivo aggregometry studies in human platelets, Ceefourin-1 significantly inhibited platelet aggregation by about 30 to 50% when ADP or collagen were used as activating agents, respectively. Furthermore, Ceefourin-1 significantly lowered the ADP-induced activation of integrin αIIbβ3, indicated by binding of FITC-fibrinogen (about 50% reduction at 50 µM Ceefourin-1). Platelets incubated with Ceefourin-1 (50 µM) showed a reduced Calcium influx and a 1.5-fold and 1.7-fold increase in PGE1- and Cinaciguat-induced VASP phosphorylation, indicating increased cytosolic concentrations of cAMP and cGMP, respectively. The release of TXB2 and S1P from activated human platelets was also attenuated. Moreover, selective MRP4 inhibition significantly reduced both the total area covered by thrombi and the average size of thrombi by about 40% in the flow chamber model.
Conclusions: Selective MRP4 inhibition causes reduced platelet adhesion and thrombus formation under flow conditions. This is reflected by an inhibition of integrin αIIbβ3 activation, elevated VASP phosphorylation and reduced calcium influx. Beside inhibition of cyclic nucleotide transport, release of pro-inflammatory mediators such as S1P is also affected.
To cite this abstract in AMA style:Wolf R, Grammbauer S, Palankar R, Moritz E, Greinacher A, Tzvetkov M, Jedlitschky G, Rauch BH. Specific Inhibition of MRP4 Affects Several Signaling Pathways and Thrombus Formation in Human Platelets [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/specific-inhibition-of-mrp4-affects-several-signaling-pathways-and-thrombus-formation-in-human-platelets/. Accessed March 4, 2024.
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