MRP4 Expression and Platelet Activation in Children and Adolescents with Different Subtypes of Primary Thrombocythemia
Authors: Giona Fiorina , Laurino Marica , Massimi Isabella , Guarino Maria Luisa , Temperilli Flavia , Palumbo Giovanna , Mariani Sabrina , Marzella Deborah , Foà Robin , Pulcinelli Fabio Maria 
Published: October 2019

Background and aims. Essential thrombocythemia (ET) rarely occurs in children and adolescents. In our experience, 40% of pediatric patients with primary thrombocythemia (PT) have JAK2 V617F or CALR mutations, 24% have a diagnosis of ET without any known molecular markers while 36% have hereditary thrombocytosis (HT) with a MPLS505A mutation. Thrombotic events, frequent in adult ET presenting high-risk factors and rare in pediatric PT, have been observed in pediatric HT patients with MPLS505A mutation in treatment with aspirin (ASA). The multidrug resistance protein-4 (MRP4) is an ATP-binding cassette transporter involved in the efflux of several pharmacological and physiological compounds. MRP4 has been identified as a modulator of ASA action in platelets; in addition, it also influences platelet activation. Recent studies have shown that MRP4 over-expression has a role in reducing the effect of ASA in patients who have undergone a bypass surgical procedure and that ASA induces platelet MRP4 upregulation. Aims of this study were to evaluate and correlate MRP4 expression and platelet function in children and adolescents aged <20 years at diagnosis with different subtypes of PT.

Methods. MRP4 protein and mRNA expressions were evaluated in platelets obtained from healthy volunteers (HV) and from 41 PT patients: M/F ratio: 0.78; median age at diagnosis: 14.5 years; median platelet count at study: 671x109/L. Ten patients had MPLS505A mutation (HT), 10 were JAK2V617F-mutated (ET) and 6 harbored CALR mutations (ET) while 15 cases were JAK2, CALR and MPL wilde-type (ET). Expression of MRP4 protein and mRNA were analyzed by Western blot and RT-PCR, respectively, and the results were reported as ET/HV or HT/HV expression values. Platelet aggregation, using ADP at different concentration (0.8 to 2 μM) and collagen (1 μg/ml) as agonists, and platelet secretion, expressed as ATP release after U46619 + epinephrine (1 + 20 μM), using the luciferin-luciferase assay, were utilized as platelet function tests. This study was conducted in accordance with the Declaration of Helsinki.

Results. Protein MRP4 expression was higher both in HT (4.23+/-1.88) and in ET patients with CALR mutations (4.27+/-2.60) compared to the values found in wild-type ET patients (3.55+/-1.52). In JAK2V617F-mutated ET patients, the MRP4 protein expression (2.03+/-1.46) was significantly lower compared to the values observed in all other ET and HT patients (p .05), whereas the MRP4 mRNA expression was significantly higher (ΔΔCt 0.021+0.003) compared both to HT patients (0.009+/-0.0039 ΔΔCt) (p .01) and CALR-mutated patients (ΔΔCt 0.014+/- 0.006). Patients with HT showed a significantly higher response to ADP 0.8 µM (83+/-29 Mx%) compared to all subgroups of ET patients who showed a similar response (60+/-34 Mx%; p .01). A significantly shorter lag-phase in response to collagen (1 µg/ml) was observed in HT compared to ET patients (33+/-3 sec vs 51+/-4 sec, p .005). Among the ET population, JAK2-mutated patients showed a significantly shorter lag-phase in response to collagen compared to wild-type patients (39+/-12.5 sec vs 50+/-15 sec), p .011. Platelet secretion was significantly higher in HT compared to ET patients, p .001.

Conclusions. This study for the first time provides evidence that children and adolescents with MPLS505A-mutated HT show a higher platelet reactivity compared to age-matched patients with ET. Moreover, platelet reactivity correlates with MRP4 protein overexpression. These findings help to shed light into the thrombotic events observed in HT MPLS505A patients despite treatment with ASA.

Disclosures

No relevant conflicts of interest to declare.