Page 209 - Haematologica Vol. 107 - September 2022

ARTICLE - Effects of specific inhibition of platelet MRP4
R. Wolf et al.
were analyzed with ImageJ22 software. Image segmenta- tion was performed in Bitplane Imaris version 7.65. (Oxford Instruments, Abingdon, UK) using the surfaces creation wizard algorithm.23 All flow experiments were performed according to International Society on Thrombosis and Hemostasis Scientific and Standardization Committee (ISTH SSC) recommendations.24
Results
Ceefourin-1 effectively inhibits MRP4-mediated transport of several signaling compounds in vitro
In order to evaluate how effectively Ceefourin-1 interferes with the direct ATP-dependent transport of several signal- ing compounds, transport assays using inside-out mem- brane vesicles containing recombinant human MRP4 were performed. ATP-dependent transport of 3H-labeled cGMP was inhibited with a half maximal inhibitory concentration (IC50) value of 5.7 mM, indicating that the transport of cyclic nucleotides is affected by Ceefourin-1 with high affinity (Online Supplementary Figure S1A). Also the more lipophilic substrates TxB2 and S1P were actively transported by MRP4, confirming previous studies.13,14 TxB2 transport could be blocked by Ceefourin-1 with nearly the same in vitro potency (IC50: 3.6 mM) as cGMP transport (Online Supple- mentary Figure S1B). However, higher concentrations of Ceefourin-1 were required to interfere with S1P transport (IC50 of about 50 mM) (Online Supplementary Figure S1C).
MRP4 inhibition reduces the release of thromboxane from human platelets
Since the ATP-dependent TxB2 transport was potently in- hibited by Ceefourin-1 in the membrane vesicle assay, the impact of MRP4 inhibition on thromboxane release by stimulated human platelets ex vivo was further investi- gated. For this, incubation of the platelets with Ceefourin- 1 was performed prior to the addition of CRP-XL or PAR1-AP, which activate collagen or thrombin receptors, respectively. Subsequently, concentrations of TxB2 (the stable metabolite of TxA2) were measured in platelet supernatants and the respective pellets by LC-MS/MS. In order to validate the results, we compared this method with an established enzyme-linked immunosorbent assay (ELISA) and found only a negligible inter-method variability (Online Supplementary Figure S2). Basal TxB2 release in control platelets was 2.27±0.39 pg/106 platelets and in- creased strongly upon stimulation with CRP-XL (1 mg/mL) and PAR1-AP (50 mM) to 5.96±1.56 pg/106 and 5.91±1.64 pg/106 platelets, respectively. In comparison, pre-treat- ment with Ceefourin-1 led to a significant decrease in basal as well as CRP-XL- and PAR1-AP-induced TxB2 re- lease (Figure 1A). In addition, also the total amount of TxB2 (supernatant and pellet combined) was significantly re-
duced by Ceefourin-1 in CRP-XL- and PAR1-AP-treated samples (Figure 1B). This indicates that TxB2 formation is also affected by MRP4 inhibition. However, further ana- lyses of our data revealed that the relative TxB2 release, which was calculated from the fraction released divided by the total amount, was still significantly diminished. In platelets stimulated with CRP-XL (1 mg/mL) 35.8±3.3% were released in the presence of Ceefourin-1 (vs. control: 45.0±1.5%) and with PAR1-AP (50 mM) 36.4±4.7% (vs. con- trol: 48.0±7.8%) (Figure 1C). This suggests that Ceefourin- 1 attenuates platelet TxB2 release via a diminished TxB2 synthesis during activation combined with a direct effect on the TxB2 transport across the plasma membrane.
Ceefourin-1 treatment impairs platelet aggregation in human and murine PRP
Light transmission aggregometry with different stimuli was performed to investigate, whether short-time expo- sure of PRP to Ceefourin-1 leads to impaired platelet ag- gregation. MRP4 inhibition resulted in a reduction of maximum platelet aggregation, with the most prominent effect (about 50% inhibition at 10 mM Ceefourin-1) being observed with the strong agonist collagen (5 mg/mL) (38.3± 10.3% aggregation vs. 77.3±4.0% for the solvent control) (Figure 2). Although less pronounced, a significant effect on aggregation was also observed in ADP- and PAR1-AP- stimulated platelets (27% and 13% reduction at 10 mM Cee- fourin-1, respectively), while the synthetic thromboxane analog U46619 had no significant effect. In comparison, aspirin was used to block thromboxane synthesis, leading, as expected, to a reduced aggregation with the most pro- nounced effect also with collagen-induced activation (63% at 30 mM aspirin). When both compounds were combined, only a tendency towards an additive effect was observed, which, however, was not statistically significant. These re- sults further substantiate the finding of MRP4 being in- volved in the release of thromboxane, not excluding its potential role in intrinsically controlling platelet activation and, therefore, thromboxane production.
Since several MRP4-inhibiting compounds show signifi- cant off-target effects, we performed platelet aggregation experiments with PRP from WT and Mrp4-deficient mice to verify the selectivity of Ceefourin-1. In line with previous studies,9,10 the Mrp4 knockout led to an impaired aggre- gation response to collagen stimulation (30.7± 5.7% vs. 46.7±3.8% aggregation with 10 mg/mL collagen) (Figure 2C). A similar reduction was achieved by the treatment with Ceefourin-1 in WT platelets (31.3±3.9% vs. 46.7±3.8%). However, Ceefourin-1 resulted in no further attenuation of aggregation in the Mrp4-deficient platelets, indicating that the effect of Ceefourin-1 on platelet function is only due to MRP4 inhibition. Ceefourin-1 also reduced platelet ag- gregation after stimulation with ADP only in the WT pla- telets (Online Supplementary Figure S3).
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