PB0380 - Corrections of Bernard Soulier Syndrome Megakaryocyte Phenotypes by Small Molecules and Interleukin-1α

Background: We previously showed that megakaryocytes (MK) from Bernard Soulier syndrome (BSS) induced pluripotent stem cells (iPSCs) yielded reduced numbers but increased sizes of platelets. Various signaling molecules were involved. Furthermore, interleukin-1α (IL-1α) could cause ‘megakaryocyte rupture’ as a rapid mechanism producing large platelets distinct from proplatelet formation.

Aims: To determine if IL-1α and small molecules could rescue the BSS abnormalities in vitro.

Methods: Wild-type (WT) iPSCs and iPSCs from BSS patients with glycoprotein Ibα (BSS-A) or Ibβ (BSS-B) mutations were differentiated into MKs and platelets using the ES-SAC method. Platelets were quantified by flow cytometry with absolute counting beads. Platelet diameters were measured after staining to visualize tubulin rings. Besides IL-1α, a myosin II inhibitor (blebbistatin), ROCK inhibitor (Y27632) and procaspase-3 activator (PAC-1) were incubated. The iPSC lines containing tubulin-Green Fluorescence Protein (GFP) reporters were constructed to observe live MKs under time-lapse fluorescence microscopy for platelet production with or without IL-1α.

Results: BSS-derived MKs (BSS-MKs) yielded fewer but larger platelets compared to the wild-type. In the presence of blebbistatin, ROCK inhibitor or PAC-1, BSS-A and BSS-B MKs could generate more platelets with decreased sizes. These agents also increased the numbers and reduced the sizes of platelets from WT-MKs. On contrary, IL-1α did not increase platelet numbers from WT-MKs, but sizes were significantly larger. BSS-MKs yielded more platelets in IL-1α, but their sizes were still large (Table 1). The live MKs carrying tubulin-GFP were then observed in cultures for 18 hours. IL-1α increased membrane blebbing in both WT-MKs and BSS-MKs and caused BSS-MK ruptures. The proportions of proplatelet formation were reduced by IL-1α in the WT-MKs but not in BSS-MKs (Figure 1).

Conclusion(s): Signaling molecule modifications could enhance normal and BSS platelet formation in vitro. IL-1α promoted platelet production in the absence of glycoprotein Ib via MK budding and ruptures.