High‐content, label‐free analysis of proplatelet production from megakaryocytes

Abstract

Background: The mechanisms that regulate platelet biogenesis remain unclear; factors that trigger megakaryocytes (MKs) to initiate platelet production are poorly understood. Platelet formation begins with proplatelets, which are cellular extensions originating from the MK cell body. Objectives: Proplatelet formation is an asynchronous and dynamic process that poses unique challenges for researchers to accurately capture and analyze. We have designed an open-source, high-content, high-throughput, label-free analysis platform. Methods: Phase-contrast images of live, primary MKs are captured over a 24-hour period. Pixel-based machine-learning classification done by ilastik generates probability maps of key cellular features (circular MKs and branching proplatelets), which are processed by a customized CellProfiler pipeline to identify and filter structures of interest based on morphology. A subsequent reinforcement classification, by CellProfiler Analyst, improves the detection of cellular structures. Results: This workflow yields the percent of proplatelet production, area, count of proplatelets and MKs, and other statistics including skeletonization information for measuring proplatelet branching and length. We propose using a combination of these analyzed metrics, in particular the area measurements of MKs and proplatelets, when assessing in vitro proplatelet production. Accuracy was validated against manually counted images and an existing algorithm. We then used the new platform to test compounds known to cause thrombocytopenia, including bromodomain inhibitors, and uncovered previously unrecognized effects of drugs on proplatelet formation, thus demonstrating the utility of our analysis platform. Conclusion: This advance in creating unbiased data analysis will increase the scale and scope of proplatelet production studies and potentially serve as a valuable resource for investigating molecular mechanisms of thrombocytopenia.

Sequence-specific 2'-O-methoxyethyl antisense oligonucleotides activate human platelets through glycoprotein VI, triggering formation of platelet-leukocyte aggregates

“…These images were analyzed for the percentage of megakaryocytes producing proplatelets, as well as the area containing proplatelets using Ilastik (version 1.3.0) and Cell Profiler (version 3.0.0) as described previously. 19 …”

Platelet Membrane: An Outstanding Factor in Cancer Metastasis

“…Platelet biogenesis begins during the maturational stage of megakaryocytes [17], within a cytoplasmic cavity with pseudopod-like structures called proplatelets where proteins and organelles will later become part of the platelets that are deposited [33,34]. The rearrangement of platelet cytoplasmic structures also begins in this cavity.…”

“…It is formed by an extensive network of membranous channels formed by flattened tubules and cisternae derived from tubular invaginations of the plasma membrane. This system is called the invaginated membrane system and is the origin of the proplatelet membrane [33][34][35][36]. The DMS allows both megakaryocytes and platelets to have more significant contact with the outside, and constitutes a membrane reservoir for forming and spreading proplatelets and platelets [35,36].…”

“…Thanks mainly to the continuous polymerization of the tubulin bundles at their free ends and to the sliding of superimposed microtubules driven by dynein, proplatelets can change their shape by extending or contracting [40]. When proplatelets acquire the components of functional platelets, they are excised from the megakaryocyte and released into the bloodstream as platelets with a size ranging from 2 to 3 µm [17,33].…”

“…Platelets modulate the shape and size of their membrane through processes such as pseudopod formation, adhesion, and platelet aggregation [42]. Various cellular structures, biomolecules, and mechanisms participate in these processes, including (1) the membrane reservoir in the DMS that is formed from the biogenesis of platelets and that is used in the extension and degranulation activities associated with platelet activation [33,36]; (2) the actin cytoskeleton of platelets [22]; (3) the activation of Rac1 and the redistribution and activation of the integrin αIIbβ3, which allow cells to change shape, mediated by the membrane and giving the platelets greater flexibility when spreading to small sites of the exposed subendothelium. Rac1 is an essential factor in the release of fibronectin and fibrinogen from the granules when α is activated by integrins [43].…”