in-situ-exploration-murine-megakaryopoiesis-using-transmission

In Situ Exploration of Murine Megakaryopoiesis using Transmission Electron Microscopy

Differentiation and maturation of megakaryocytes occur in close association with the cellular and extracellular components of the bone marrow. These ...

proplatelet-formation-dynamics-of-mouse-fresh-bone-marrow-explants

Proplatelet Formation Dynamics of Mouse Fresh Bone Marrow Explants

The last stage of megakaryopoiesis leads to cytoplasmic extensions from mature megakaryocytes, the so-called proplatelets. Much has been learned about the ...

in-vitro-and-in-vivo-methods-to-explore-megakaryopoiesis

In Vitro and In Vivo Methods to Explore Megakaryopoiesis

ARTICLES DISCUSSED:
1. Pongerard, A., Mallo, L., Gachet, C., Lanza, F., Strassel, C. Leukodepletion filters-derived CD34+ cells as a cell source to study ...

Université de Strasbourg, INSERM, EFS Grand Est, BPPS UMR-S 1255, FMTS

Université de Strasbourg, INSERM, EFS Grand Est, BPPS UMR-S 1255, FMTS, F-67065 Strasbourg, France

In this chapter, we describe the study of bone marrow megakaryocytes (MKs) using a high-resolution 3D imaging approach known as focused ion beam-scanning electron microscopy (FIB-SEM). The apparatus consists of a scanning electron microscope equipped with a focused gallium ion beam, used to sequentially mill away the sample surface, and an electron beam, used to image the milled surfaces. This produces a series of ultrastructural images which can be computationally reconstructed into three-dimensional (3D) volume images. Using this approach it is possible to characterize the 3D ultrastructure of MKs in their native bone marrow environment, to study subcellular organelle interactions in the context of a complete cell and to quantify specific features. This chapter provides protocols for sample preparation, image acquisition and 3D reconstruction, the whole procedure requiring about 7-8 days. It also describes a method combining light microscopy (LM) with FIB-SEM, a procedure called correlative light electron microscopy (CLEM), which allows the site-specific 3D imaging of MKs in tissues.

High-Resolution 3D Imaging of Megakaryocytes Using Focused Ion Beam-Scanning Electron Microscopy.

Electron microscopy (EM) has a long history in megakaryocyte (MK) cellular biology. This chapter shows how the electron microscope, since its first appearance almost 90 years ago, has occupied center stage in the studies of MK morphology and function. It describes some of the more productive EM techniques that have shaped our understanding of the physiology of thrombopoiesis. These include the standard transmission and scanning EM techniques as well as the new imaging methods, correlative microscopy and volume EM which provide information on the 3D organization of MKs on different scales: single organelles, whole cells and tissues. For each technique, we list the advantages and limitations, the resolution that can be achieved, the technical difficulties and the applications in MK biology.

Use of electron microscopy to study megakaryocytes.

Platelets are produced by bone marrow megakaryocytes through cytoplasmic protrusions, named native proplatelets (nPPT), into blood vessels. Proplatelets also refer to protrusions observed in megakaryocyte culture (cPPT) that are morphologically different. Contrary to cPPT, the mechanisms of nPPT formation are poorly understood. We show here in living mice that nPPT elongation is in equilibrium between protrusive and retraction forces mediated by myosin-IIA. We also found, using WT and β1-tubulin-deficient mice, that microtubule behavior differs between cPPT and nPPT, being absolutely required in vitro, while less critical in vivo. Remarkably, microtubule depolymerization in myosin-deficient mice did not affect nPPT elongation. We then calculated that blood Stokes'forces may be sufficient to promote nPPT extension, independently of myosin and microtubules. Together, we propose a new mechanism for nPPT extension that might explain contradictions between severely affected cPPT production and moderate platelet count defects in some patients and animal models.

Cytoskeletal-based mechanisms differently regulate  and  proplatelet formation.

Blood platelets are anucleate cell fragments that prevent bleeding and minimize blood vessel injury. They are formed from the cytoplasm of megakaryocytes located in the bone marrow. For successful platelet production, megakaryocyte fragments must pass through the sinusoid endothelial barrier by a cell biology process unique to these giant cells as compared with erythrocytes and leukocytes. Currently, the mechanisms by which megakaryocytes interact and progress through the endothelial cells are not understood, resulting in a significant gap in our knowledge of platelet production.

Megakaryocytes use in vivo podosome-like structures working collectively to penetrate the endothelial barrier of bone marrow sinusoids.

Arterial thrombosis causes heart attacks and most strokes and is the most common cause of death in the world. Platelets are the cells that form arterial thrombi, and antiplatelet drugs are the mainstay of heart attack and stroke prevention. Yet, current drugs have limited efficacy, preventing fewer than 25% of lethal cardiovascular events without clinically relevant effects on bleeding. The key limitation on the ability of all current drugs to impair thrombosis without causing bleeding is that they block global platelet activation, thereby indiscriminately preventing platelet function in hemostasis and thrombosis. Here, we identify an approach with the potential to overcome this limitation by preventing platelet function independently of canonical platelet activation and in a manner that appears specifically relevant in the setting of thrombosis. Genetic or pharmacological targeting of the class II phosphoinositide 3-kinase (PI3KC2α) dilates the internal membrane reserve of platelets but does not affect activation-dependent platelet function in standard tests. Despite this, inhibition of PI3KC2α is potently antithrombotic in human blood ex vivo and mice in vivo and does not affect hemostasis. Mechanistic studies reveal this antithrombotic effect to be the result of impaired platelet adhesion driven by pronounced hemodynamic shear stress gradients. These findings demonstrate an important role for PI3KC2α in regulating platelet structure and function via a membrane-dependent mechanism and suggest that drugs targeting the platelet internal membrane may be a suitable approach for antithrombotic therapies with an improved therapeutic window.

Disrupting the platelet internal membrane via PI3KC2α inhibition impairs thrombosis independently of canonical platelet activation.

Platelet dense-granules are small organelles specific to the platelet lineage that contain small molecules (calcium, adenyl nucleotides, serotonin) and are essential for the activation of blood platelets prior to their aggregation in the event of a vascular injury. Delta-storage pool diseases (δ-SPDs) are platelet pathologies leading to hemorrhagic syndromes of variable severity and related to a qualitative (content) or quantitative (numerical) deficiency in dense-granules. These pathologies appear in a syndromic or non-syndromic form. The syndromic forms (Chediak-Higashi disease, Hermansky-Pudlak syndromes), whose causative genes are known, associate immune deficiencies and/or oculocutaneous albinism with a platelet function disorder (PFD). The non-syndromic forms correspond to an isolated PFD, but the genes responsible for the pathology are not yet known. The diagnosis of these pathologies is complex and poorly standardized. It is based on orientation tests performed by light transmission aggregometry or flow cytometry, which are supplemented by complementary tests based on the quantification of platelet dense-granules by electron microscopy using the whole platelet mount technique and the direct determination of granule contents (ADP/ATP and serotonin). The objective of this review is to present the state of our knowledge concerning platelet dense-granules and the tools available for the diagnosis of different forms of δ-SPD.

Platelet δ-Storage Pool Disease: An Update.

Deterioration in quality of platelet concentrates (PCs) during storage results from the appearance of storage lesions affecting the hemostatic functions and posttransfusion survival of platelets. These lesions depend on the preparation and pathogen inactivation methods used, duration of storage, and platelet additive solutions (PASs) present in storage bags.

Removal of citrate from PAS-III additive solution improves functional and biochemical characteristics of buffy-coat platelet concentrates stored for 7 days, with or without INTERCEPT pathogen reduction.

The production of platelet concentrates (PCs) is evolving, and their survival capacity needs in vivo evaluation. This requires that the transfused platelets (PLTs) be distinguished from those of the recipient. Labeling at various biotin (Bio) densities allows one to concurrently trace multiple PLT populations, as reported for red blood cells.

Human platelets labeled at two discrete biotin densities are functional in vitro and are detected in vivo in the murine circulation: A promising approach to monitor platelet survival in vivo in clinical research.

Primary hemostasis consists in the activation of platelets, which spread on the exposed extracellular matrix at the injured vessel surface. Secondary hemostasis, the coagulation cascade, generates a fibrin clot in which activated platelets and other blood cells get trapped. Active platelet-dependent clot retraction reduces the clot volume by extruding the serum. Thus, the clot architecture changes with time of contraction, which may have an important impact on the healing process and the dissolution of the clot, but the precise physiological role of clot retraction is still not completely understood. Since platelets are the only actors to develop force for the retraction of the clot, their distribution within the clot should influence the final clot architecture. We analyzed platelet distributions in intracoronary thrombi and observed that platelets and fibrin co-accumulate in the periphery of retracting clots in vivo. A computational mechanical model suggests that asymmetric forces are responsible for a different contractile behavior of platelets in the periphery versus the clot center, which in turn leads to an uneven distribution of platelets and fibrin fibers within the clot. We developed an in vitro clot retraction assay that reproduces the in vivo observations and follows the prediction of the computational model. Our findings suggest a new active role of platelet contraction in forming a tight fibrin- and platelet-rich boundary layer on the free surface of fibrin clots.

Anita Eckly

Université de Strasbourg, INSERM, EFS Grand Est, BPPS UMR-S 1255, FMTS

In Situ Exploration of Murine Megakaryopoiesis using Transmission Electron Microscopy

Proplatelet Formation Dynamics of Mouse Fresh Bone Marrow Explants

In Vitro and In Vivo Methods to Explore Megakaryopoiesis