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3D ultrastructural analysis of ?-granule, dense granule, mitochondria, and canalicular system arrangement in resting human platelets.

ABSTRACT: Background:State-of-the-art 3-dimensional (3D) electron microscopy approaches provide a new standard for the visualization of human platelet ultrastructure. Application of these approaches to platelets rapidly fixed prior to purification to minimize activation should provide new insights into resting platelet ultrastructure. Objectives:Our goal was to determine the 3D organization of ?-granules, dense granules, mitochondria, and canalicular system in resting human platelets and map their spatial relationships. Methods:We used serial block face-scanning electron microscopy images to render the 3D ultrastructure of ?-granules, dense granules, mitochondria, canalicular system, and plasma membrane for 30 human platelets, 10 each from 3 donors. ?-Granule compositional data were assessed by sequential, serial section cryo-immunogold electron microscopy and by immunofluorescence (structured illumination microscopy). Results and Conclusions:?-Granule number correlated linearly with platelet size, while dense granule and mitochondria number had little correlation with platelet size. For all subcellular compartments, individual organelle parameters varied considerably and organelle volume fraction had little correlation with platelet size. Three-dimensional data from 30 platelets indicated only limited spatial intermixing of the different organelle classes. Interestingly, almost 70% of ?-granules came within ?35 nm of each other, a distance associated in other cell systems with protein-mediated contact sites. Size and shape analysis of the 1488 ?-granules analyzed revealed no more variation than that expected for a Gaussian distribution. Protein distribution data indicated that all ?-granules likely contained the same major set of proteins, albeit at varying amounts and varying distribution within the granule matrix.

SUBMITTER: Pokrovskaya ID

PROVIDER: S-EPMC6971324 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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3D ultrastructural analysis of α-granule, dense granule, mitochondria, and canalicular system arrangement in resting human platelets.

Pokrovskaya Irina D ID Yadav Shilpi S Rao Amith A McBride Emma E Kamykowski Jeffrey A JA Zhang Guofeng G Aronova Maria A MA Leapman Richard D RD Storrie Brian B

Research and practice in thrombosis and haemostasis 20191025 1

<h4>Background</h4>State-of-the-art 3-dimensional (3D) electron microscopy approaches provide a new standard for the visualization of human platelet ultrastructure. Application of these approaches to platelets rapidly fixed prior to purification to minimize activation should provide new insights into resting platelet ultrastructure.<h4>Objectives</h4>Our goal was to determine the 3D organization of α-granules, dense granules, mitochondria, and canalicular system in resting human platelets and ma ...[more]

PMID: 31989087

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Project description:UnlabelledESSENTIALS: How platelets organize their α-granule cargo and use their canalicular system remains controversial. Past structural studies were limited due to small sampling volumes or decreased resolution. Our analyses revealed homogeneous granules and a closed canalicular system that opened on activation. Understanding how platelets alter their membranes during activation and secretion elucidates hemostasis.SummaryBackgroundPlatelets survey the vasculature for damage and, in response, activate and release a wide range of proteins from their α-granules. Alpha-granules may be biochemically and structurally heterogeneous; however, other studies suggest that they may be more homogeneous with the observed variation reflecting granule dynamics rather than fundamental differences.ObjectivesOur aim was to address how the structural organization of α-granules supports their dynamics.MethodsTo preserve the native state, we prepared platelets by high-pressure freezing and freeze-substitution; and to image nearly entire cells, we recorded tomographic data in the scanning transmission electron microscope (STEM).Results and conclusionsIn resting platelets, we observed a morphologically homogeneous α-granule population that displayed little variation in overall matrix electron density in freeze-substituted preparations (i.e., macro-homogeneity). In resting platelets, the incidence of tubular granule extensions was low, ~4%, but this increased by > 10-fold during early steps in platelet secretion. Using STEM, we observed that the initially decondensing α-granules and the canalicular system remained as separate membrane domains. Decondensing α-granules were found to fuse heterotypically with the plasma membrane via long, tubular connections or homotypically with each other. The frequency of canalicular system fusion with the plasma membrane also increased by about three-fold. Our results validate the utility of freeze-substitution and STEM tomography for characterizing platelet granule secretion and suggest a model in which fusion of platelet α-granules with the plasma membrane occurs via long tubular connections that may provide a spatially limited access route for the timed release of α-granule proteins.

Project description:In addition to haemostasis, platelets mediate inflammation and clearance of bacteria from the bloodstream. As with platelet-platelet interactions, platelet-bacteria interactions involve cytoskeletal rearrangements and release of granular content. Stimulation of the immune Toll-like receptor 2 (TLR2) on the platelet surface, activates phosphoinositide-3-kinase (PI3K) and causes platelet activation and platelet-dependent thrombosis. It remains unknown if platelet activation by immune versus thrombotic pathways leads to the differential regulation of signal transduction, protein-protein interactions, and alpha-granule release, and the physiological relevance of these potential differences. We investigated these processes after immune versus thrombotic platelet stimulation. We examined selected signalling pathways and found that phosphorylation kinetics of Akt, ERK1/2 and p38 differed dramatically between agonists. Next, we investigated platelet protein-protein interactions by mass spectrometry (MS)-based proteomics specifically targeting cytosolic factor XIIIa (FXIIIa) because of its function as a cytoskeleton-crosslinking protein whose binding partners have limited characterisation. Four FXIIIa-binding proteins were identified, two of which are novel interactions: FXIIIa-focal adhesion kinase (FAK) and FXIIIa-gelsolin. The binding of FAK to FXIIIa was found to be altered differentially by immune versus thrombotic stimulation. Lastly, we studied the effect of thrombin versus Pam(3)CSK(4) stimulation on alpha-granule release and observed differential release patterns for selected granule proteins and decreased fibrin clot formation compared with thrombin. The inhibition of PI3K caused a decrease in protein release after Pam(3)CSK(4)- but not after thrombin-stimulation. In summary, stimulation of platelets by either thrombotic or immune receptors leads to markedly different signalling responses and granular protein release consistent with differential contribution to coagulation and thrombosis.

Dataset Information

3D ultrastructural analysis of ?-granule, dense granule, mitochondria, and canalicular system arrangement in resting human platelets.

Publications

3D ultrastructural analysis of α-granule, dense granule, mitochondria, and canalicular system arrangement in resting human platelets.

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