Abstract
Platelets are specialized anucleate cells that play a major role in hemostasis following vessel injury. More recently, platelets have also been implicated in innate immunity and inflammation by directly interacting with immune cells and releasing proinflammatory signals. It is likely therefore that in certain pathologies, such as chronic parasitic infections and myeloid malignancies, platelets can act as mediators for hemostatic and proinflammatory responses. Fortunately, murine platelet function ex vivo is highly analogous to human, providing a robust model for functional comparison. However, traditional methods of studying platelet phenotype, function and activation status often rely on using large numbers of whole isolated platelet populations, which severely limits the number and type of assays that can be performed with mouse blood. Here, using cutting edge 3D quantitative phase imaging, holotomography, that uses optical diffraction tomography (ODT), we were able to identify and quantify differences in single unlabeled, live platelets with minimal experimental interference. We analyzed platelets directly isolated from whole blood of mice with either a JAK2V617F-positive myeloproliferative neoplasm (MPN) or Leishmania donovani infection. Image analysis of the platelets indicates previously uncharacterized differences in platelet morphology, including altered cell volume and sphericity, as well as changes in biophysical parameters such as refractive index (RI) and dry mass. Together, these data indicate that, by using holotomography, we were able to identify clear disparities in activation status and potential functional ability in disease states compared to control at the level of single platelets.
Highlights
Maintaining blood flow in basal states and preventing excessive blood loss following injury relies on an orchestrated response from different cell types and non-cellular components, such as clotting factors
The HT-2H microscope is based on a Mach–Zehnder interferometer equipped with a digital micromirror device (DMD) (Figure 1A)
Using a coherent monochromatic laser (λ = 532 nm) divided into a sample and reference beam, 2D holographic QPI images were generated at multiple illumination angles (Figure 1B) where the incidence light is accurately controlled by the DMD (Shin et al, 2015)
Summary
Maintaining blood flow in basal states and preventing excessive blood loss following injury relies on an orchestrated response from different cell types and non-cellular components, such as clotting factors. Platelets are key cells in this process, becoming rapidly activated following injury and form a platelet plug to reduce blood loss, as well as initiating secondary hemostasis to promote the formation of a stable fibrin-rich thrombus (Machlus and Italiano, 2013). In addition to being essential for hemostasis, there is compelling evidence suggesting key roles for platelets in other processes including wound healing, angiogenesis, inflammation, and innate immunity (Machlus and Italiano, 2013; van der Meijden and Heemskerk, 2019). Platelets express a range of receptors that allow them to interact with and respond to pathogens and assist in regulating an immune response (Assinger, 2014; Hamzeh-Cognasse et al, 2015; van der Meijden and Heemskerk, 2019). Platelets assist the formation of the traps, and concurrently the nets contribute to platelet activation, linking inflammation to thrombosis (Gros et al, 2015; Hamzeh-Cognasse et al, 2015)
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