STEM tomography reveals that the canalicular system and α‐granules remain separate compartments during early secretion stages in blood platelets

Abstract

SUMMARY Background Platelets 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. Objectives Our aim was to address how the structural organization of α-granules supports their dynamics. Methods To preserve the native state, we prepared platelets by high-pressure freezing and freeze-substitution; and to image almost entire cells, we recorded tomographic data in the scanning transmission electron microscope (STEM). Results and Conclusions In 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 more than 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 ~3-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 timed release of α-granule proteins.

Glucose Transporter 3 Potentiates Degranulation and Is Required for Platelet Activation

“…Under basal conditions, electron micrographs exhibited no differences in α-granule, δ-granule, or mitochondrial number relative to controls (Figure 5A–B). However, following stimulation with Par4 peptide, GLUT3-KO platelets displayed a significant accumulation of partially released α-granules (Figure 5A and 5C), which resemble decondensingα-granules 14 , suggesting a degranulation defect. To determine if these changes were accompanied by impaired α-granule cargo release, the releasate from PAR4 peptide stimulated platelets were evaluated for the protein content of angiogenic factors.…”

“…Immediately following stimulation, a small subset of α-granules fuse with the plasma membrane and release their cargo, potentially regulated by their proximity to the plasma membrane. The remaining α-granules are released through compound exocytosis, where granules fuse with each other to form intracellular networks following which α-granule content release is potentiated 13, 14 . Here we propose that GLUT3 translocation to the plasma membrane in the initial single exocytosis phase, fuels glucose uptake that generates the energy required for potentiation of exocytosis.…”

The cellular basis of platelet secretion: Emerging structure/function relationships

“…A second variation on frozen, hydrated samples is to take a frozen cryoprotected sample and either 1) section the sample as is done in typical immunogold labeling procedures in which the labeling and visualization is of thawed cryo-sections or 2) do the dehydration steps required for plastic embedding at a −90°C, a procedure referred to as freeze substitution. Platelets prepared by either of these frozen cell procedures display, in thin section, α-granules that are nearly devoid of nucleoids see Figure 2B [20, 23]. In the case of freeze substituted samples, the characteristic electron dense core seen in dense granules is preserved, an important control observation [23].…”

Golgi proteins in circulating human platelets are distributed across non-stacked, scattered structures

“…The human canalicular system in contrast to that of other species has been thought to be completely open, i.e., interconnected to the plasma membrane in resting human platelets. Recent work using high volume sampling electron microscopy strongly indicates that in immediately fixed human platelets a significant portion is closed [25]. Many of these closed elements could correspond to the disperse puncta we observed.…”