(453h) Shear, Microparticles and Platelet-Like Particles in the Interplay Between Platelet Biogenesis and Megakaryopoiesis

Shear stress has been shown to be an important differentiation cue for platelet biogenesis (Baruch et al., Blood 114:1975 (2009)). Yet, the mechanisms by which shear flow enables platelet biogenesis and megakaryopoietic (Mk) maturation remains largely unexplored. Separately, there has been an enormous activity in exploring the role of microparticles (MPs; also referred to as microvesicles) in the biology and differentiation of a variety of tissues and organs. Yet, little is known on the biogenesis and role of MPs from megakaryocytes or platelets. Here we report our studies on the role of shear stress on the generation of platelet-like particles (PLPs), and Mk-derived MPs (MKMPs) from mature Mks in vitro, and their role in Mk maturation.

Human Mks were differentiated from primary stem and progenitor cells (SPCs; CD34⁺ cells) and seeded into Von Willebrand Factor-coated parallel flow slides. The continuous laminar flow was applied to Mks at defined shear stress level for different duration times. Cell fragments and particles were harvested after the flow application. When medium flow at shear stress level of 1 dyn/cm² was applied, 5.5-fold higher numbers of PLPs (diameter of 2-3 µm) were generated after 2 hr compared to static conditions. 30 min exposure to 4 dyn/cm² (the upper limit of physiological shear range) increased the generation of PLPs by 180% compared to1 dyn/cm² for 2 hrs. The quality and functionality of these PLPs were also studied based two standard platelet functionality assays: P-selectin exposure and fibrinogen binding assays. After activation, the % P-selectin⁺ PLPs from flow conditions was 2.35-fold higher than in PLPs from static conditions. Similar results were found regarding to fibrinogen binding. These results show that PLPs generated from flow condition have better quality than PLPs generated under static condition.

Mature Mks can also generate CD41⁺ MKMPs with diameter < 1 µm (Flaumenhaft et al., Blood 113:1112 (2009)), but a biological role for these MKMPs remains to be discovered. We found that shear stress enhances MKMP generation at day 10 of culture. When Mks were exposed to shear flow at 2.5 dyn/cm², 30-50 times more MKMPs were generated after 2 hrs of flow application. To explore the physiological function of MKMPs that is unknown, MKMPs were cultured together with hematopoietic progenitor cells (HPCs) under cytokine-free condition. Our results show that there were more 2N, 4N and 8N or higher polyploidy Mks in cultures with MKMPs than in control cultures. These data suggest that MKMPs can communicate with HPCs to promote their differentiation to Mks in the absence of Mk-specific cytokines. Data regarding the mechanisms by which this communication takes place will be presented and discussed in the context of the in vivo milieu.