Structural Analysis of Mouse Platelets using Serial Block-Face Scanning Electron Microscopy

Abstract

Platelets are small, anucleate cells in mammalian blood that maintain hemostasis and aggregate to seal leaks at sites of vascular injury, a vital element of wound healing. Upon detecting vascular lesions, blood platelets become activated and release components, such as the Von Willebrand factor, from at least three different storage compartments: alpha-granules, dense granules, and lysosomes. Abnormalities in platelet functions can contribute to thrombosis, atherosclerosis and other diseases. Obtaining cellular structure is critical for gaining better understanding of platelet physiology. We used Serial Block-Face Scanning Electron Microscope (SBF-SEM) to elucidate the 3-D ultrastructure of mouse blood platelets in both the activated and inactivated (resting) states. In SBF-SEM an in-situ ultramicrotome cuts slices of heavy-atom stained cells that are embedded in a resin block. The top surface of the block is then imaged using a back-scattered electron detector, and the process of slicing and imaging is repeated until the desired volume of cells is obtained. A stack of images can then be aligned, and ultrastructure visualized using segmentation software. Based on the segmentation and measurements on images from 4 different platelet activation states, we observed that rapid swelling of alpha-granules is accompanied by matrix decondensation that occurs first in peripheral granules, whereas condensed granules were situated more centrally. Decondensed granules appeared almost adjacent or connected to the plasma membrane. Based on these findings, we propose that the granule matrix organization is an important factor for platelet function, i.e., for timed-release properties of alpha-granule cargo, which could be physiologically important to thrombus formation, wound healing and angiogenesis. This work was supported by the Intramural Research Program of the National Institutes of Biomedical Imaging and Bioengineering, NIH (RDL) and NIH grants R01 HL119393 and R56 HL119393 (BS).