In Vitro Characterization of Peripheral Blood Progenitor Cell Differentiation and Platelet Function in Essential Thrombocythemia (ET)

Bleeding and thrombotic events are major clinical complications of ET, together with progression to myelofibrosis and leukemic transformation (AML). The JAK2 V617F mutation, the thrombopoietin receptor mutation MPL W515K/L and the most recently discovered calreticulin (CALR) mutations are mutually exclusive in ET, account for up to 80-90 % of ET cases and support a novel molecular categorization of ET. However, the link between these driver mutations and ET pathogenesis and complications remains elusive.

We have studied megakaryocyte (MK) differentiation of peripheral blood progenitor cells in vitro as well as the platelet aggregation capacity in ET patients. Progenitors cells were grown under standard conditions in serum-free media supplemented with stem cell factor (SCF), FLT3 ligand, erythropoietin (Epo) and thrombopoietin (TPO), and gradually were switched to TPO only media after almost a week of expansion of the hematopoietic progenitors. ET MKs are larger in size, they are more granular and they display higher ploidy levels when compared to healthy MKs. They also co-express the megakaryocytic and erythroid markers, i.e. CD41 and CD71 at higher levels suggesting aberrant lineage specification from multipotent progenitors that can give rise to both the megakaryocytic and erythroid lineage. Remarkably, ET peripheral blood progenitors have a prolonged survival in vitro under the tested culture conditions when compared to healthy MKs. Megakaryocytic cultures from healthy donors last for 12-14 days in vitro while the ET MK cultures can be maintained for up to two extra weeks. Electron microscopy analysis of cultured MKs is consistent with the morphological observations mentioned above regarding size and granularity, which is higher in ET MKs, and hence support aberrant differentiation pathways. However, no differences were found between the molecular subtypes of ET. Therefore, we performed transcriptional analysis by RNA sequencing of megakaryocytic and erythroid cultures of progenitors from JAK2 V617F ET, CALR mutant ET or triple negative ET in order to identify the molecular cues that characterize abnormal megakaryopoiesis in ET subgroups. We have analyzed three biological replicates per genotype and we are currently confirming the results by RT-PCR and functional assays in primary cells and cell lines.

In parallel, we analyzed the aggregation capacity of JAK2 V617F, CALR mutant and triple negative ET platelets by a novel flow cytometry-based aggregation assay (De Cuyper IM et al. Blood 2013) in order to dissect potential signaling defects due to single receptor malfunction. We were able to pinpoint a significant defect in the response to collagen mediated by integrin a2b1 in ET platelets, regardless of the driver mutation, in comparison with platelets from healthy donors. However, convulxin-stimulated aggregation mediated by GPVI was normal. We also explored the degranulation capacity of ET platelets upon stimulation with thrombin peptide PAR-1. Platelets from CALR mutant or triple negative ET exhibited a reduced response as measured by the change of surface expression of the alpha granule protein CD62P compared to WT (healthy donors) and ET JAK2 V617F platelets. This was correlated with increased basal CD62P expression, suggesting that platelets in CALRmutant or triple negative ET degranulate spontaneously.

In summary, we have developed in vitro assays to explore peripheral blood progenitor cell differentiation and platelet function in ET. Our findings indicate functional differences between JAK2 V617F ET as compared with CALR mutant or triple negative ET. The correlation between these functional differences and the clinical behavior of the molecular subtypes of ET needs to be further investigated.

References

Cuyper IM, Meinders M, van de Vijver E, de Korte D, Porcelijn L, de Haas M, Eble JA, Seeger K, Rutella S, Pagliara D, Kuijpers TW, Verhoeven AJ, van den Berg TK, Gutiérrez L. A novel flow cytometry-based platelet aggregation assay. Blood 2013 Mar 7;121(10):e70-80