Page 215 - Haematologica Vol. 107 - September 2022

ARTICLE - Effects of specific inhibition of platelet MRP4 R. Wolf et al. AB
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Figure 6. Role of MRP4 in platelet adhesion and thrombus formation under flow. (A and B) Whole human blood with FITC-anti- CD42a-labeled platelets (from n=3 different donors) was pre-incubated with Ceefourin-1 (Ceef., 50 mM) or the respective solvent (Co, 0.5 % dimethyl sulfoxide [DMSO]) and perfused through collagen-coated microchannels under high arterial shear conditions (1,800-1) for 5 minutes. Micrographs were taken at the beginning, the end, and in the center of the channel using a confocal laser scanning microscope (Zeiss LSM 780) with a 40x objective. The average thrombus area (A, left panel) and surface area coverage by thrombi (A, right panel; means + standard error of the mean [SEM]) were analyzed with the ImageJ software. In (B) representative images are shown. (C and D) The effect of Ceefourin-1 was also evaluated in perfusions performed with whole blood from age- and sex-matched wild-type (WT) or Mrp4(-/-) mice (with X488-labeled platelets). The average thrombus area (C, means ± SEM and values of individual animals; n=4-8) was markedly reduced in Mrp4(-/-) and Ceefourin-1-treated WT mice with no significant additional Ceefourin-1 effect in Mrp4(-/-) animals. (D) Representative micrographs. Scale bars correspond to 50 μm.
thromboxane release seems to be a combined effect of a diminished thromboxane synthesis during activation and a direct impact on the transport across the plasma mem- brane, since not only the total amount of this mediator but also the relative percentage that was released, was reduced. The fact that we observed no significant effect of Ceefourin-1 on the aggregation induced by the synthetic thromboxane analog U46619 and only a tendency towards an additive effect with aspirin is in agreement with this assumption. However, the effect of Ceefourin-1 to reduce thromboxane release was rather small compared with the potent ability of aspirin to prevent thromboxane forma-
tion, which is required to effectively inhibit thromboxane- dependent platelet activation.25 Therefore, this effect of Ceefourin-1 can be an additional factor but is unlikely to fully account for the observed inhibition of platelet acti- vation in response to agonists such as ADP and collagen. We studied platelet aggregation after short-time exposure to Ceefourin-1 with different stimuli because the pub- lished aggregometry data are also inconsistent. While Cheepala et al.10 reported that diminished aggregation of platelets from Mrp4 knockout mice is specific for collagen and Mrp4 (-/-) platelets did not have any defect in aggre- gation with either ADP or thrombin, Decouture et al.9 ob-
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