Early emergence of contractility and mechanotransduction during human megakaryopoiesis

Abstract

Abstract The ability to contract blood clots is a hallmark of functional platelets. Proteins that support agonist-induced platelet contraction are expressed in megakaryocytes (MKs) and transferred to platelets during thrombopoiesis. Here, we investigated when during MK development the functional machinery for contraction emerges. Human induced pluripotent stem cells (iPSCs) were differentiated into embryonic hematopoietic progenitor cells (HPCs) and further matured into MKs (iMKs) to map the emergence of contractility and mechanotransduction across differentiation. Expression of total (CD41) and activated (mAb PAC-1) αIIbβ3 integrin, the thrombin receptor PAR1, and the MK marker CD42b was quantified by flow cytometry. Cell contractility was assessed by optically tracking human plasma clot shrinkage following thrombin stimulation. Undifferentiated iPSCs lacked detectable αIIbβ3 and were non-contractile. Both HPCs and iMKs exhibited agonist-induced contractility, integrin activation, and force transmission to extracellular fibrin, although at different levels. Following thrombin stimulation, activated αIIbβ3 on the cell surface was approximately 5-fold lower in HPCs and restricted to the MK-committed CD42b⁺ subset, indicating that integrin activation is linked to MK commitment. Functionally, both HPCs and iMKs induced clot contraction but with distinct kinetics and magnitude, with HPCs exhibiting approximately 2.5-fold lower contraction rates and final extent. Depletion of CD42b⁺ cells from HPCs reduced contraction to baseline, identifying early MK-lineage progenitors as the principal contractile population at this stage. These findings demonstrate that cellular contractility and αIIbβ3-mediated mechanotransduction emerge early during MK lineage commitment, establishing contractility as an early functional hallmark of megakaryocyte differentiation.