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

Acknowledgements

The authors thank Carl Zeiss, Inc (Thornwood, NY) for performing the FIB-SEM imaging and Joel Mancusco of Zeiss for arranging this.

Disclosure Statement

The authors have no conflict of interest to declare.

Authorship

Contribution: I.D.P., M.T., R.D., S.J., J.A.K., and G.Z. performed different aspects of the individual experiments and the analysis of the data; B.S., M.A.A., and R.D.L. were involved in the experimental design; B.S. and I.D.P. wrote the manuscript; B.S. and I.D.P. edited the manuscript drafts; B.S., I.D.P., M.A.A., S.J., and S.W.W. did final editing and J.A.K. supported the electron microscopy efforts at the University of Arkansas for Medical Sciences.

Additional information

Funding

The Storrie laboratory was supported in part by National Institutes of Health grants [R01 HL119393 and R56 HL119393]. The Whiteheart laboratory was supported in part by National Institutes of Health grants [HL56652 and HL138179], American Heart Association grant [AHA16GRNT27620001], and a Veterans Affairs Merit Award to SWW and an American Heart Association predoctoral grant [AHA15PRE25550020] to SJ. The Leapman laboratory was supported by the intramural program at NIBIB at the National Institutes of Health, Bethesda, MD