OC 48.4 - Loss of PP1β leads to abnormal megakaryocyte development, severe thrombocytopenia, osteosclerosis and myelofibrosis

Background: Serine/threonine (S/T) phosphorylation is fundamental in regulating megakaryocyte (MK) development and platelet function. However, the contribution of S/T dephosphorylation to these processes remains unclear. To date, studies examining genes encoding S/T phosphatases have not identified any with substantial effects on MK development or platelet count, highlighting a significant gap in our understanding.

Aims: To investigate the physiological consequences of ablating PP1β, a S/T phosphatase, on both MK development and platelet biology.

Methods: To achieve this, Ppp1cb-floxed mice were crossed with Pf4-Cre transgenic mice, creating PP1β conditional knockout (cKO) mice lacking of PP1β protein expression in MKs and platelets. MK numbers in bone marrow and spleen were assessed via immunofluorescence microscopy, and myelofibrosis was evaluated using reticulin silver staining. The size and pro-platelet formation capacity of in-vitro differentiated MKs, as well as glycoprotein expression and platelet activation, were analyzed using standard methods.

Results: PP1β cKO mice displayed severe macrothrombocytopenia with a 75% decrease in platelet count, accompanied by a 60% increase in platelet mean volume and an 85% rise in surface GPIbα and GPIX levels. Immunofluorescence microscopy demonstrated a notable decrease in both the number and size of MKs in the bone marrow. Additionally, in vitro-differentiated cKO MKs were significantly smaller and exhibited pronounced defects in proplatelet formation. Ablation of PP1β from MKs led to marked physiological changes in the mice, including the development of osteosclerosis, myelofibrosis, and splenomegaly. Mechanistically, PP1β deletion strongly increased MLC2 S19 phosphorylation in platelets, highlighting its pivotal role in the regulation of myosin activity, potentially explaining proplatelet formation defects. Preliminary data suggests the involvement of PP1β in a crucial differentiation pathway, which could contribute to developmental abnormalities.

Conclusion(s): Collectively, findings from this study establish PP1β as a critical phosphatase regulating MLC2 phosphorylation, MK development, and platelet biogenesis, illuminating the functional role of S/T dephosphorylation in these cells.