PB1286 - Signaling Molecules Controlling Platelet Formation from IPSC-derived Megakaryocytes

Background: Megakaryocytes (MKs) derived from induced pluripotent stem cell (iPSC) of Bernard-Soulier syndrome (BSS) patients produced fewer and larger platelets in cultures. These changes were alleviated by myosin II inhibitor (blebbistatin) or ROCK inhibitor (Y27632) or pro-caspase-3 activator (PAC-1), but the molecular mechanisms of these corrections remain unclear.

Aims: To observe platelet formation in the presence of these pharmacological agents in normal MKs and MKs lacking glycoprotein (Gp) Ib.

Methods: Wild-type (WT), BSS-A (Lacking GpIα) and BSS-B (Lacking GpIbβ) iPSCs were differentiated into MKs and platelets. Blebbistatin (15 µM), ROCK inhibitor (10 µM) or PAC-1 (50 µM) were added on day 15, day 17 and day 20 of culture. Proplatelets were fluorescence stained for actin and tubulin for the structures. Additionally, WT and BSS MK cell lines containing the α-tubulin-GFP reporter were constructed to observe for real-time proplatelet formation under time-lapse video fluorescence microscopy. Cultured MKs were then classified as resting (MK), proplatelet formation (Thick vs. Thin proplatelets) and membrane budding.

Results: Small molecules showed minimal effects on WT-MK platelet formation. In BSS MKs, proplatelet formation did not increase after small molecule induction but MKs with membrane budding were increased (Figure1). Proplatelet formation in BSS after blebbistatin (Myosin inhibitor) or ROCK inhibitor (Y27632) increased proportions of proplatelets with thin shafts (Figure 2). Proplatelets from WT-MKs with procaspase-3 inhibitor (PAC-1) showed irregular clumping of the F-actin and produced more platelets but platelet CD42 expression was decreased suggesting abnormalities in MK differentiation by PAC-1.

Conclusion(s): The data suggest the roles of myosin and ROCK inhibition in MK budding and thinning of proplatelet processes. The knowledge may be applicable for BSS therapy and in vitro platelet generation in the future.