Structural analysis of resting mouse platelets by 3D-EM reveals an unexpected variation in α-granule shape

Abstract

Mice and mouse platelets are major experimental models for hemostasis and thrombosis; however, important physiological data from this model has received little to no quantitative, 3D ultrastructural analysis. We used state-of-the-art, serial block imaging scanning electron microscopy (SBF-SEM, nominal Z-step size was 35 nm) to image resting platelets from C57BL/6 mice. α-Granules were identified morphologically and rendered in 3D space. The quantitative analysis revealed that mouse α-granules typically had a variable, elongated, rod shape, different from the round/ovoid shape of human α-granules. This variation in length was confirmed qualitatively by higher-resolution, focused ion beam (FIB) SEM at a nominal 5 nm Z-step size. The unexpected α-granule shape raises novel questions regarding α-granule biogenesis and dynamics. Does the variation arise at the level of the megakaryocyte and α-granule biogenesis or from differences in α-granule dynamics and organelle fusion/fission events within circulating platelets? Further quantitative analysis revealed that the two major organelles in circulating platelets, α-granules and mitochondria, displayed a stronger linear relationship between organelle number/volume and platelet size, i.e., a scaling in number and volume to platelet size, than found in human platelets suggestive of a tighter mechanistic regulation of their inclusion during platelet biogenesis. In conclusion, the overall spatial arrangement of organelles within mouse platelets was similar to that of resting human platelets, with mouse α-granules clustered closely together with little space for interdigitation of other organelles.

Keywords: 3D SBF-SEM; electron microscopy; mouse; organelles; platelets; α-granules.

FIGURE 1.. Serial block-face example image slice (A) and resulting 3D rendering of immediately fixed, resting mouse platelets (B), C57BL/6 strain.

Individual platelets were randomly chosen from image slices (A), validated for full platelet volume inclusion in the image stack and then 3D rendered (B) using Amira software as described in Methods. The 10 platelets in (B) are shown surface rendered and pseudocolored with shadowing to give a sense of 3D depth.

FIGURE 2.. Example single plane markup of intracellular organelles in resting mouse platelets.

Arrows point to individual organelles: α-G, α-granule; DG, dense granule; CS, canalicular system element; Mit, mitochondrion. Asterisk indicates an example of an apparent granule-granule fusion event.

FIGURE 3.. The spatial arrangement of α-granules, dense granules, canalicular system (open and closed), and mitochondria in mouse platelets.

Depicted are 3D renderings of individual platelets in which the organelles have been color-coded: α-granules (blue), dense granules (red), mitochondria (purple) and open and close canalicular system (yellow and turquoise, respectively). The relative distributions for given organelles are highlighted by direct comparison: A. α-Granules vs. Dense Granules; B. α-Granules vs. Mitochondria; C. α-Granules vs. Canalicular System; D. Mitochondria vs. Canalicular System. E-N. Depict combined color-coded images for 10 individual platelets rendered in 3D.

FIGURE 4.. Dispersion in mouse platelet number (A) and volume fraction per platelet (B) summarized in dot plots.

The data are from the 10 randomly chosen platelets 3D rendered and segmented. Volume fraction per platelet was computed from the summation of voxel volumes included within the segmented objects.

Figure 5.. Numbers and volume fractions of mouse platelet organelles plotted against platelet volume.

Numbers of α-granules (A), dense core granules (C), and mitochondria (E); and volume fractions of α-granules (B), dense core granules (D), and mitochondria (F), where dashed lines indicate ± one standard error of mean. α-granule and mitochondrial number per platelet correlate with platelet volume.

FIGURE 6.. Quantitative distribution of the axial shape parameters of 190 α-granules from 10 randomly chosen mouse platelets.

Total α-granule population plots. A. Binned minor (short) axis lengths, apparent normal distribution. B. Binned major (long axis) lengths of each α-granule. The α-granule axis ratio distribution (C) is calculated by dividing the width in A by the length in B and is a measure of the shape of the granules. In C, the x-axis scale of 1 is defined as a perfect sphere and anything less than 1 is elongate. The minor (short) and major (long) axis lengths were calculated using Amira software by creating a “Custom Measure” in the “Label Analysis” module after using the “Connected Components” module to assign a unique material classification to each α-granule in a platelet. Based upon skewness and kurtosis values calculated using Kaledagraph software (see Methods, Results), the distribution in (A) falls well within the statistical expectations for a normal Gaussian distribution while those in (B) and (C) do not.

FIGURE 7.. Qualitative and quantitative depiction of α-granule shape on an individual platelet basis.

A-J. α-Granules in individually rendered platelets are shown in blue while the rest of the platelet volume is depicted in dark gray. Granule heterogeneity was probed by measuring granule dimensions in each rendering (A-J) and plotting their distribution in each platelet (A’-J’). Histogram plots of α-granule axis ratio distributions: minor (short) axis divided by major (long) axis are shown.