Page 213 - Haematologica Vol. 107 - September 2022

ARTICLE - Effects of specific inhibition of platelet MRP4 R. Wolf et al. A
B
C
Figure 4. Effect of MRP4 inhibition on intracellular calcium levels in platelets. Cytosolic free calcium was measured spectrophotofluorometrically in Fura-2-loaded washed platelets. Measurements were performed either in Ca2+ medium (Tyrode’s buffer with 2 mM CaCl ) (left panels) or Ca2+-free medium (Tyrode’s buffer with 0.2 mM EGTA) (right panels). In (A) the
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time-dependent increase in cytosolic calcium (indicated by R (340/380 nm)) after stimulation with ADP (1 mM) and the effect of
Ceefourin-1 (50 mM) in representative experiments are depicted. In (B) the area under the fluorescence curve (area under the curve [AUC]) as a measure for the total change in calcium concentration was calculated (means + standard error of the mean from n=3 different donors, each measured in duplicates or quadruplicates). The reduction after Ceefourin-1 treatment is also given in % of the respective solvent control for each donor (each separate experiment) in (C) (significant differences with *P<0.05, and **P<0.01).
Discussion
An impact of MRP4 on platelet function is indicated by several lines of evidence, including studies in Mrp4 knock- out mice9,10 and recent data from human individuals with a defect in the ABCC4/MRP4 gene.4 MRP4 may affect dif- ferent signaling pathways in human platelets through the transport of several compounds. In the present study, we used Ceefourin-1, which has been described as a selective MRP4 inhibitor21 to comprehensively characterize the ef- fects on the function of human platelets that could be an- ticipated when applying a short-time pharmacological
inhibition of MRP4. It has been shown that Ceefourin-1 is highly selective for MRP4 over several ABC transporters, including other members of the MRP family such as MRP1 and MRP3 (ABCC1 and ABCC3), which are also expressed in platelets.2 However, this does not exclude off-target ef- fects of Ceefourin-1 on other structures in platelets. Therefore, we compared the effect of the compound in WT and Mrp4-deficient mice in the classic aggregometry studies and in the flow chamber experiments and ob- served significant effects on platelet function only when MRP4 is expressed. Possible unspecific effects of Cee- fourin-1 on platelet viability were also tested and ruled
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