Page 216 - Haematologica Vol. 107 - September 2022

ARTICLE - Effects of specific inhibition of platelet MRP4
R. Wolf et al.
served a significant decrease in ADP- and PAR-4-activating peptide-induced aggregation in their knockout model. In human platelets, an impact of MRP4 inhibition was re- ported mainly on collagen-induced platelet aggre- gation.10,15,16 However, in ABCC4/MRP4-negative individuals a significant decrease in platelet aggregation was not ob- served with collagen and ADP at high concentration (10 mM), but at lower ADP concentrations of 2.5 mM and 5 mM.4 With Ceefourin-1, we observed a significant effect on ag- gregation when collagen (5 mg/mL) was used, but also with ADP (5 mM) or PAR1-AP (30 mM). Detection of fibrinogen binding to the platelets as a measure for integrin aIIbb3 activation affirmed the observation that MRP4 inhibition affects platelet reactivity most effectively when the acti- vating stimuli were used at a low submaximal dose, while it does not interfere at maximal activating conditions. At low ADP concentrations, an effect on degranulation was also observed indicated by the reduced surface exposure of P-selectin (CD62P).
Inconsistent observations regarding the role of cGMP in the context of MRP4-mediated effects on platelet func- tion have been reported. ATP-dependent transmembrane transport and export of cGMP in platelets were shown to be affected by MRP4 inhibitors.1,19 However, Decouture et al.9 reported that MRP4 appears not to interfere with pla- telet cGMP homeostasis in their murine model since they observed no difference in total and secreted cGMP in WT or Mrp4-deficient platelets pre-incubated with sodium nitroprusside and stimulated by a PAR4-activating pep- tide. A rise in platelet cGMP levels, e.g., induced by NO- mediated activation of the soluble guanylate cyclase, results in a downregulation of platelet-activating signal- ing pathways. In this study, we used the phosphorylation of VASP at ser-239 as an indicator of platelet cytosolic cGMP levels and cinaciguat26 as an activator of the sol- uble guanylate cyclase. Here, Ceefourin-1 was able to sig- nificantly increase the cinaciguat-stimulated VASP phosphorylation as well as to enhance markedly the ci- naciguat-induced inhibitory effects on platelet aggre- gation. Ceefourin-1 analogously increased the phosphorylation at ser-157 induced by the cAMP-elevating agent PGE1. These results indicate that MRP4 inhibition can intensify both cAMP- and cGMP-mediated effects in platelets and thus the response to several endothelium- derived vasodilators such as cAMP-elevating prosta-glan- dins as well as the cGMP-elevating nitric oxide, even though Ceefourin-1 alone only slightly elevated the back- ground levels of these mediators. VASP phosphorylation is one key factor in the inhibition of platelet aggregation, while Gαi signaling leads to activation. Ca2+-dependent signaling pathways synergize with the Gai signaling in the activation of integrin aIIbb3 and also play a key role in granule secretion from activated platelets. Therefore, we examined the effect of Ceefourin-1 on the free calcium
concentrations in platelets, both in the presence and the absence of extracellular calcium, to discriminate between calcium entry across the plasma membrane and release from intracellular stores in the dense tubular system. The results indicate that blocking MRP4 mainly affects the agonist-induced calcium influx but also to some extent the intracellular calcium mobilization through direct or indirect mechanisms.
It was also the question if these relatively moderate ef- fects of a short-time pharmacological MRP4 inhibition on platelet activation are sufficient to affect platelet ad- hesion and thrombus formation under blood flow. There- fore, we also tested the impact of Ceefourin-1 in a microfluidic flow chamber model and perfused whole blood through collagen-coated microchannels under high arterial shear conditions. Such devices have been recog- nized as a valuable tool to mimic the anatomy of healthy and stenotic blood vessels.27,28 Here, we could also dem- onstrate that spiking human or WT murine blood with the MRP4 inhibitor significantly reduced the average thrombus size and the surface area covered by thrombi.
In conclusion, pharmacological inhibition of MRP4 affects several signaling pathways in platelets mechanistically based on the transport inhibition not only of cAMP but also cGMP as well as of the lipid mediators thromboxane and S1P. However, additional direct effects on alternative biochemical pathways cannot be excluded. MRP4-selec- tive platelet inhibitors may perspectively prove advantage- ous, especially in cases of platelet hyperreactivity that may be associated with MRP4 overexpression. Besides Ceefourin-1, other effective MRP4-inhibiting compounds have been recently published.29 These were developed pri- marily for the reversal of drug resistance in MRP4-over- expressing cancer cells. Since tumors are often associated with thrombosis and aspirin has been recently recognized as a promising cancer-preventive agent probably based on anti-platelet-mediated effects,30 one may speculate that MRP4 inhibitors may provide dual benefits in some tumor patients. Other compounds such as the phosphodieste- rase-3 and MRP4 inhibitor cilostazol may affect platelet reactivity by a dual-action.10,16 Further studies are required to evaluate which MRP4 inhibitors may be best suitable for an in vivo application.
Disclosures
No conflicts of interests to disclose.
Contributions
RW and SG conducted and designed the main experiments and data analyses. RP, CT, EM, AB, MH, and AH provided ad- ditional analyses and methodological expertise. AG and MVT contributed to the study design, BHR and GJ designed and supervised the study. RW and GJ drafted and all authors edited the manuscript.
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