Shear enhances thrombopoiesis and formation of microparticles that induce megakaryocytic differentiation of stem cells

Abstract

• Physiological shear stress promotes megakaryocytic maturation, DNA synthesis, phosphatidylserine exposure and caspase-3 activation.• Shear enhances the production and function of PLPs and Mk-derived microparticles possessing a novel function.In vivo visualization of thrombopoiesis suggests an important role for shear flow in platelet biogenesis. In vitro, shear stress was shown to accelerate proplatelet formation from mature megakaryocytes (Mks). Yet, the role of biomechanical forces on Mk biology and platelet biogenesis remains largely unexplored. In this study, we investigated the impact of shear stress on Mk maturation and formation of platelet-like particles (PLPs), pro/preplatelets (PPTs), and Mk microparticles (MkMPs), and furthermore, we explored a physiological role for MkMPs. We found that shear accelerated DNA synthesis of immature Mks in an exposure time-and shear stress level-dependent manner. Both phosphatidylserine exposure and caspase-3 activation were enhanced by shear stress. Exposure to physiological shear dramatically increased generation of PLPs/PPTs and MkMPs by up to 10.8 and 47-fold, respectively. Caspase-3 inhibition reduced shear-induced PLP/PPT and MkMP formation. PLPs generated under shear flow displayed improved functionality as assessed by CD62P exposure and fibrinogen binding. Significantly, coculture of MkMPs with hematopoietic stem and progenitor cells promoted hematopoietic stem and progenitor cell differentiation to mature Mks synthesizing a-and dense-granules, and forming PPTs without exogenous thrombopoietin, thus identifying a novel and unexplored potential physiological role for MkMPs. (Blood. 2014;124(13):2094-2103

Megakaryocytic Maturation in Response to Shear Flow Is Mediated by the Activator Protein 1 (AP-1) Transcription Factor via Mitogen-activated Protein Kinase (MAPK) Mechanotransduction

“…In circulation, MKs might experience different levels of biomechanical forces. Notably, MKs may experience higher levels of shear and for longer periods of time in the lung vasculature as detailed in our 2014 article (8). It has been shown that shear stress promotes MK morphological maturation, increased DNA synthesis, and extensive plateletlike particle (PLP), PPT, and MK-microparticle production (7,8).…”

“…Mature MKs may squeeze through the incomplete endothelial lining of bone marrow capillaries to shed pro/preplatelets (PPT) into circulation under shear forces generated by the blood flow (6 -8). Less mature MKs may enter circulation and mature and shed PPT in the lung vasculature, thus being exposed to a wide spectrum of shear and other biomechanical forces (8). The evidence for the physiological importance of shear forces in platelet biogenesis has been established based on a series of studies and notably in Refs.…”

“…The evidence for the physiological importance of shear forces in platelet biogenesis has been established based on a series of studies and notably in Refs. 6 and 7, as reviewed in our 2014 article (8). Briefly, MKs are exposed to shear forces of different intensity and duration depending on the location in the body.…”

“…On day 8, MKs were then seeded into von Willebrand factor-coated (Hematologic Technologies) rectangular flow slides (-Slide I 0.6 Luer, Ibidi) (to be used for exposing MK cells to shear in "fluidic" flow chambers (Ibidi)) at 300,000 cells per slide (8). On day 9, cells were subjected to shear flow that results in shear stress levels of either 1.0 dyn/ cm 2 for 2 h or 2.5 dyn/cm 2 for 30 min using a syringe pump system (Dual NE-4000 Double Syringe Pump; New Era Pump Systems) established by Jiang et al (8). The remaining MKs were cultured to day 12 using the current condition at day 9.…”

“…PLP Particle Analysis-PLPs were harvested from d12 MKs as previously described (8). PLPs were obtained from MKs following 2.5 h (30 min of MAPK inhibition in static culture followed by 120 min of shear flow or static culture with MAPK inhibitor) of MAPK inhibition or vehicle control (dimethyl sulfoxide).…”