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In vivo proplatelet formation
previously noted by data showing increased cPPT elonga- tion velocity under flow compared to static conditions,11,51 and from our observations that nPPT always align in the direction of flow, especially visible upon inverse flows (Online Supplementary Video S8) or when flow stopped (Online Supplementary Video S6; Figure 7). Even with low blood flow velocities as in sinusoids, ranging from about ten to several hundred mm/s42,43 (Figure 7E), we calculated that the Stokes’ force exerted on the nPPT end would be sufficient to stretch the membrane.37-39 The same force, applied all along the PPT shaft, would further increase the overall driving force and promote its extension. Taking into account the fact that DMS fuses with the plasma membrane all along the nPPT shaft as shown by Brown et al.,4 this continuous membrane replenishment most prob- ably considerably decreases membrane tension as demon- strated in other systems,52,53 thus even further lowering the forces required for nPPT elongation.
Our findings do not exclude a key role of microtubules in the final platelet formation. We may speculate that this final nPPT remodeling into barbell platelets and final platelets could occur through microtubule-based mecha- nisms, similar to those previously established in liquid cul- ture,6,11 leading to microtubule coils prefiguring the margin- al band. In favor of this idea, Lefrançais et al. recently pub- lished some videos of free MK fragment remodeling in lung vessels that produced extensions strikingly resembling branched cPPT extended by MK in culture.5 In addition, we could observe that in situ barbell platelets and free nPPT fragments share a microtubule organization similar to cPPT including microtubules coils (personal observation and 54).
It is apparent from the present work added to earlier data, that the cytoplasmic processes of MK are structurally different in vivo and in vitro due to the different mecha- nisms leading to their extension. The question then arises as to the respective nomenclature of these extensions. Should the term PPT be used for extensions within the bone marrow or for the extensions observed in in vitro sys- tems, or even for MK fragments released into the circula- tion and which are truly “pro”-platelets? Defining distinct nomenclatures for each of these structures might help to get a clearer picture of thrombopoiesis in pathophysiology compared to the in vitro platelet production process. We propose that large MK fragments extended in vivo are named “nPPT” for native PPT, as opposed to “cPPT” for cultured PPT present in vitro.
We therefore propose a model for the nPPT formation in the bone marrow that differs from the one established in vitro. In vivo, while microtubules may contribute to the
initial cytoplasmic extension of the nascent nPPT, they appear to be far less important for the elongation process once the nPPT is inside the blood flow. Microtubules rather play a role as a backbone to prevent nPPT retrac- tion mediated by actomyosin contraction. nPPT elonga- tion per se could proceed through blood drag forces that stretch nPPT plasma membrane as it is fueled by the DMS. We propose that this in vivo mechanism which occurs in the native and complex marrow environment, is bypassed in the liquid culture conditions. The in vitro microtubule-based mechanisms previously described potentially takes place at a later second time point, once nPPT have been released inside the blood circulation (Figure 8). Taken together, these data may explain why in some cases, strong discrepancies were observed between the capacity to extend cPPT, quantified in vitro, compared to the moderately decreased circulating platelet count.33,55,56 Our work may thus help to understand the mechanisms of thrombocytopenia in patients especially when mutations occur in cytoskeletal proteins.
Disclosures
No conflicts of interest to disclose.
Contributions
AB: conducted all intravital experiments and analyses; JB and CS: performed in vitro experiments; FP: developed the experi- mental intravital set up; AE: performed electron microscopy; DS: provided important key reagents; CG and FL: wrote the manu- script; CL: designed and analyzed experiments and wrote the manuscript.
Acknowledgments
The authors would like to thank Florian Gaertner (IST, Austria) for his expert advice on two-photon microscopy experi- ments and Yves Lutz at the Imaging Center IGBMC (Illkirch, France) for his expertise and help with the two-photon micro- scope. We thank Josiane Weber for excellent technical help and Jean-Yves Rinkel for help in 3D reconstructions. We thank Ramesh Shivdasani for his generous gift of Tubb1-/- mice. We thank Juliette Mulvihill for language editing. We thank ARME- SA (Association de Recherche et Développement en Médecine et Santé Publique) for support in the acquisition of the two-photon microscope.
Funding
AB was supported by a fellowship from EFS (APR2016). JB was a recipient of a FRM (foundation pour la Recherche Médicale) fellowship.
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