Imaging Platelet Processes and Function-Current and Emerging Approaches for Imaging in vitro and in vivo

Abstract

Platelets are small anucleate cells that are essential for many biological processes including hemostasis, thrombosis, inflammation, innate immunity, tumor metastasis, and wound healing. Platelets circulate in the blood and in order to perform all of their biological roles, platelets must be able to arrest their movement at an appropriate site and time. Our knowledge of how platelets achieve this has expanded as our ability to visualize and quantify discreet platelet events has improved. Platelets are exquisitely sensitive to changes in blood flow parameters and so the visualization of rapid intricate platelet processes under conditions found in flowing blood provides a substantial challenge to the platelet imaging field. The platelet's size (~2 μm), rapid activation (milliseconds), and unsuitability for genetic manipulation, means that appropriate imaging tools are limited. However, with the application of modern imaging systems to study platelet function, our understanding of molecular events mediating platelet adhesion from a single-cell perspective, to platelet recruitment and activation, leading to thrombus (clot) formation has expanded dramatically. This review will discuss current platelet imaging techniques in vitro and in vivo, describing how the advancements in imaging have helped answer/expand on platelet biology with a particular focus on hemostasis. We will focus on platelet aggregation and thrombus formation, and how platelet imaging has enhanced our understanding of key events, highlighting the knowledge gained through the application of imaging modalities to experimental models in vitro and in vivo. Furthermore, we will review the limitations of current imaging techniques, and questions in thrombosis research that remain to be addressed. Finally, we will speculate how the same imaging advancements might be applied to the imaging of other vascular cell biological functions and visualization of dynamic cell-cell interactions.

Keywords: interference; microscope; microscopy-brightfield; platelet; polarized light; receptors; scanning electron; thrombosis.

Figure 1

Platelet contributions to thrombus formation. Platelets circulate in the blood stream in a quiescent (resting) state. When exposed extracellular matrix proteins such as von Willebrand Factor (VWF) or collagen are detected at the site of injury, platelets are induced to roll, and then adhere. The GPIb-IX-V complex and GPVI receptors on platelets orchestrate this adhesion and activation process. Adherent platelets become activated, expose P-selectin and phosphatidylserine, and secrete secondary mediators such as ADP and thromboxane. This promotes platelet recruitment and activation of αIIbβ3 which mediates platelet aggregation by binding plasma fibrinogen. Coagulation is also activated resulting in fibrin formation following thrombin cleavage of fibrinogen, leading to the consolidation of the platelet aggregate into a thrombus and healing of the damaged area. Fibrinolytic processes eventually dissolve the formed thrombus, causing the thrombus to embolize. Thrombosis occurs when there is increased coagulation and exaggerated thrombus formation and/or reduction of fibrinolytic processes, potentially leading to occlusion of the blood vessel.

Figure 2

Platelet spreading. (A) Under resting conditions platelets normally are non-adherent. Upon exposure to an activating agonist, platelets change shape by reorganizing cytoskeletal elements, leading to the formation of filopodia, followed by lamellipodia and an increase in surface area. When platelets are exposed to immobilized ligands in experiments in vitro, this shape change is known as platelet spreading. Light microscopy images shows actin arrangement and morphology of phalloidin-treated platelets exposed to non-coated coverslips (left) or coverslips pre-coated with collagen (middle) or fibrin (right). Images were taken using an inverted bright-field fluorescence microscope. Scale bar = 20 μm. (B) Schematic of processes that can be imaged during platelet spreading in vitro include (1) Cytoskeletal protein rearrangement, such as formation of actin nodules, microtubule organization and generation of stress fibers; (2) super resolution microscopy (dSTORM, SIM) can capture GPVI clustering (purple dots) and alignment along collagen fibers (green lines); (3) microvesicle formation can be imaged using optical systems that provide resolution below 150 nm; discrete cytoskeletal rearrangement occurs alongside calpain-dependent processes, where calcium-sensitive proteases detach membrane proteins, allowing membrane blebbing required for microvesicle release from platelets and megakaryocytes.

Figure 3

Imaging modalities for visualizing platelets. Multiple imaging modalities can be used for platelet imaging depending on the process to be imaged and imaging environment. Epifluorescence and bright-field imaging are most commonly used for general assessment of thrombus size and the biochemical composition of platelets (195). Electron microscopy allows resolving fine physical structures of single or an aggregate of platelets but is limited to fixed samples (195). Where imaging of functional platelets is required, in vitro imaging using TIRF or confocal microscopy could reveal dynamic events of single platelet activity and thrombosis (196), with the option of employing super-resolution and single-molecule imaging techniques for nanometer resolution of fluorescently-tagged biomolecules (65). To recapitulate more physiological conditions, the use of microfluidics and label-free microscopy can provide physiological flow conditions and reduce the risk of phototoxicity incurred by photobleaching, respectively. Finally, in vivo platelet imaging has been realized by confocal and 2-photon microscopy, the latter which provides greater tissue penetration and less phototoxicity, but with a higher equipment cost (197). Microscopy images were obtained from https://doi.org/10.1038/s41467-019-09150-9, https://doi.org/10.1038/s41467-019-10067-6, https://doi.org/10.1038/ncomms8254, and https://doi.org/10.1371/journal.pone.0071447, under the Creative Commons license (CC BY 4.0, https://creativecommons.org/licenses/by/4.0/). Images were cropped and figure letters were removed for clarity.