Identification of the mechanobiological factors involved in platelet formation – PlatForMechanics

Task1. MK matrix stiffness/ECM sensing and impact on platelet formation.
In vitro analysis are performed using MK differentiated in vitro from Lin- progenitor, during 3 days. They are deposited onto fibronectin-coated substrate with stiffness ranging from <1kpa up to >90 kpa. The extent of adhesion is determined after 2h, the extent of spreading after 5h, and the capacity of proplatelet formation after 24h. The type of adhesion is analyzed using antibodies directed against integrin and components of focal adhesion complex. Traction force experiments will be performed. Transcriptomic analyses have been performed with MK adhered onto soft (<1kPa) and stiff (90 kPa) matrices. Finally, the behavior of MK from WT mice or mice deficient for mechanosensitive receptors are compared.
Task 2: Deciphering mechanotransduction pathways.
The mechanotransduction pathways are evaluated by determining the relocalization of mechanosensitive transcription factors (MKL1, Yap, Taz) and the potential involvement of the small Rho GTPases upon adhesion onto the surface of various stiffness. Piezo1/2 and Yap/Taz double deficient mice are being studied regarding their capacity for (pro)platelet formation in homeostatic state and following conditions of bone marrow regeneration (5-FU, TPO), MK number and ploidy and ultrastructure.

We showed that megakaryocytes are true mechanosensitive cells and behave differently depending on the stiffness of the matrix. Accordingly, they spread more on fibronectin-coated stiffer surface. This spreading depends on b3-type integrins but not b1.
Yap/Taz deficient mice have been generated and they do not exhibit platelet production defects at the homeostatic state. However under conditions of marrow regeneration induced by 5-FU treatment, the reactive thrombocytosis is increased compared to WT mice.
Transcriptomic analysis showed no difference in gene expression between MK adhered onto soft vs. stiff surface. However strong variability was observed between replicate, which probably hamper a definitive conclusion, and such experiments will need to be reproduced under conditions that minimize variability.

Megakaryocytes are true mechanosensitive cells and upon adhesion onto fibronectin, integrin b3, but not b1, would play the role of mechanosensor. How this interaction modulates intracellular signaling is currently under evaluation.
Yap and Taz could be involved in the negative regulation of platelet formation upon marrow regeneration following myeloablation.

Orales présentation:
Thao Nguyen, Alicia Bornert, Christian Gachet, François Lanza, Catherine Léon. Integrin ß3 regulates fibronectin rigidity sensing by megakaryocytes and proplatelet formation. 2nd Mechanobiology meeting in Vietnam: when physics meets biology. Quy Nhon, Vietnam, 2019

Thao Nguyen, Alicia Bornert, Christian Gachet, François Lanza, Catherine Léon. L’intégrine ß3 régule la sensibilité des mégacaryocytes à la rigidité du substrat et leur capacité à former des proplaquettes. Club Français des Plaquettes et Mégacaryocytes. Toulouse 2019

Platelets play an essential role in daily life by preventing bleeding. A decrease in the number of circulating platelets as a result of trauma, chemotherapy or hereditary disease is a life-threatening condition. Platelet transfusion is the only treatment option in most cases. The ageing of the population and the increase in aggressive chemotherapy are increasing the demand, which could lead to shortages. Currently, platelet availability is exclusively dependent on donors, associated with limited shelf life and safety concerns about product quality and complications from immunological and infectious diseases. For these reasons, in vitro platelet production has attracted considerable attention over the last decade, and represents a major therapeutic and economic challenge. Yet, despite the efforts of several laboratories around the world, in vitro platelet yields remain insufficient to offer a realistic alternative to transfusion.
Platelets are naturally produced in the bone marrow by megacaryocytes (MKs), cells derived from the differentiation of hematopoietic stem cells and maturation of progenitors. When mature, MKs extend long extensions called platelets through the blood vessel wall before they are released as platelets. Today, although they can be grown in vitro in liquid media, the maturation of MKs is far from being optimal. While significant strides have been made in our understanding of the process of platelet development from MKs in suspension, there is still limited knowledge related to cellular mechanisms regulating this process in the context of bone marrow matrices. Our project aims to understand the role of the cellular environment in the differentiation and maturation of MKs, by focusing on physical and mechanical components.
The marrow is a soft tissue, where containment generates strong mechanical stresses during megakaryocyte maturation stages, and then very weak stress during the release of platelets into the bloodstream. However, the impact of these forces on platelet formation has been little studied. Our work has recently shown that growing in a 3D semi-rigid environment improves the maturation of MKs and the formation of proplatelets compared to the liquid medium. As a logical continuation, in this proposed research program we will explore how the MKs sense these forces (study of integrins and Piezo mechanoreceptors) and adapt to them (mechanotransduction through the role of MKL1 and YAP/TAZ) to optimize platelet formation. This work is guided by unpublished preliminary data showing that 1) MKs preferentially form platelets on semi-rigid surfaces, 2) integrin and Piezo-type mechanoreceptors play a role in regulating platelet formation in vivo, and 3) Mechanosensitive MKL1 and YAP transcription factors are activated when MKs are grown in a semi-rigid environment. The project is based on the complementary knowledge and approaches of the three partners, together offering expertise in MKs biology and mechanobiology. This project combines innovative microscopy imaging techniques (confocal and two-photon fluorescence microscopy/transmission electron microscopy) and cellular (mechano)biology approaches (adhesion on variable stiffness surface, micropatterns, traction force measurement) and molecular approaches (transcriptome, signalling pathways, primary cell transduction). An additional strength is the ability to combine in vitro and in vivo studies in mouse and human biological systems. Together, the proposed studies will provide an understanding of mechanisms previously neglected, but still at play in key stages of platelet biogenesis. The data obtained may generate new strategies to improve platelet production for transfusion.