Page 211 - Haematologica Vol. 107 - September 2022

ARTICLE - Effects of specific inhibition of platelet MRP4 R. Wolf et al.
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Figure 2. Effect of Ceefourin-1 on platelet aggregation ex vivo. (A and B) Human platelet-rich plasma (PRP) was pretreated with either only the solvent (control, (-)), or Ceefourin-1 (Ceef.; 10 mM for 20 minutes), or aspirin (30 mM; for 5 minutes), or the combination of both, and then stimulated with the following agonists: collagen (5 mg/mL), ADP (5 mM), PAR1-AP (30 mM) or U46619 (2 mM). Platelet aggregation was determined by light transmission aggregometry. Representative curves of platelet aggregation stimulated by collagen and ADP are given in (A). The inhibition of the maximal aggregation is summarized in (B) (means + standard error of the mean [SEM]; n=6-11; *P<0.05 vs. control). (C) PRP (diluted 1:2 with Tyrode’s buffer) from age- and sex- matched wild-type (WT) or Mrp4-deficient (Mrp4(-/-)) mice was pretreated with either only the solvent (control, (-)) or Ceefourin-1 (Ceef.; 10 mM) and then stimulated with collagen (10 mg/mL). Aggregation curves were monitored and the maximal extent of aggregation (%) was calculated (mean + SEM; n=5-7). Ceefourin-1 led to a significant reduction of the aggregation only in WT platelets (*P<0.05).
determine cytosolic cyclic nucleotide levels in human platelets, we measured the phosphorylation of VASP at two different serine residues (ser-157 and ser-239) by flow cytometry. The cAMP-elevating agent PGE1 pro- foundly increased VASP phosphorylation at ser-157, the preferred phosphorylation site of PKA. Pre-incubation with Ceefourin-1 (50 mM) significantly increased this ef- fect (310.4±25.3% vs. 211.3±14.7% with 1 mM PGE1) (Figure 5A, left panel). Note that Ceefourin-1 alone only tends to
elevate background VASP phosphorylation. The phos- phorylation of ser-239, the preferred substrate of PKG, is similarly elevated in the presence of cinaciguat, an acti- vator of soluble guanylate cyclase. As shown in Figure 5A, right panel, the specific inhibition of MRP4 resulted in a 1.8-fold increase in the cinaciguat-stimulated VASP phosphorylation.
In order to investigate whether MRP4 inhibition can en- hance cGMP-mediated effects in platelets, we measured
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