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

Abstract

Platelet activation has long been known to be accompanied by secretion from at least three types of compartments. These include dense granules, the major source of small molecules; α-granules, the major protein storage organelle; and lysosomes, the site of acid hydrolase storage. Despite ~60 years of research, there are still many unanswered questions about the cell biology of platelet secretion: for example, how are these secretory organelles organized to support cargo release and what are the key routes of cargo release, granule to plasma membrane or granule to canalicular system. Moreover, in recent years, increasing evidence points to the platelet being organized for secretion of the contents from other organelles, namely the dense tubular system (endoplasmic reticulum) and the Golgi apparatus. Conceivably, protein secretion is a widespread property of the platelet and its organelles. In this review, we concentrate on the cell biology of the α-granule and its structure/function relationships. We both review the literature and discuss the wide array of 3-dimensional, high-resolution structural approaches that have emerged in the last few years. These have begun to reveal new and unanticipated outcomes and some of these are discussed. We are hopeful that the next several years will bring rapid advances to this field that will resolve past controversies and be clinically relevant.

Keywords: Alpha-granules; microscopy; platelets, platelet release reaction; secretion.

Figure 1. Electron microscopy image of human platelets showing (A) immediate fixed and (B) washed platelets

Platelets in the blood stream are poised to activate. When immediately fixed upon blood draw as shown in A), the platelets are discoid in shape and typically appear elongated in electron micrographs of thin sectioned material [23]. Here examples of α-granules, putative canalicular system elements, dense granules, and dense tubular network are indicated by 1–3 letter abbreviations. In B), platelets drawn into citrate were purified in the presence of activation suppressor [43]. These platelets are referred to as “washed” platelets and such platelet preparations are a typical starting point for platelet release reaction studies. Note that washed platelets are more rounded and extend pseudopods. The α-granules have not released their contents. Scale bar = 500 nm.

Figure 2. Representative electron microscope images of human platelets prepared in two different ways (conventional dehydration (A) versus freeze substitution dehydration (B)

Arrows and arrowheads point to example α-granules within a human platelet. The α-granules in these thin section images are frequently round to ovoid and are approximately 200–300 nm in diameter/length. With conventional room temperature dehydration (A), the α-granules commonly display a relatively central electron dense staining area referred to as a nucleoid. Using the newer, state-of-the-art dehydration technique freeze substitution in which the fixed platelets are first exposed to organic solvent at −90°C, the platelet α-granules as shown in (B) still maintain an electron dense protein matrix. However, there is now no dense nucleoid suggesting that the nucleoid may be an artefact of dehydration. White arrows point to α-granules containing electron dense nucleoids in (A) and white arrowheads in (A) point to examples of condensation of the α-granule protein matrix away from the limiting granule membrane, another indication of possible artefact from the conventional dehydration procedure. Note that the α-granules are spaced apart within the human platelets at sufficiently small distances that the widefield or confocal fluorescence microscope will not be able to resolve the individual granules one from another (see legend to Figure 3 for more details). Scale bar = 500 nm.

Figure 3. Appearance of cargo protein stained α-granules in human platelets visualized by either confocal microsopy or 3D–structured illumination microscopy (SIM)

Images are MIPs, maximum intensity projections, that include all images in the 3D image stack. (A) Field of 6 human platelets imaged by confocal microscopy. The platelets are stained for fibrinogen (green) and VWF (red). These proteins are content/cargo markers for α-granules. There is little correspondence in the distribution of the two proteins and the number of stained puncta is small, ~15 for each protein. (B) A representative individual platelet stained for fibrinogen (green) and VWF (red) and imaged by 3D–SIM, a super resolution light microscopy technique. Again, there is little correspondence in the distribution of each of the two α-granule cargo markers. However, now the number of stained puncta is much larger, ~50–80 for each protein. The difference in number between the two approaches to fluorescence microscopy can be readily explained by the difference in resolution between the two techniques. Confocal microscopy like widefield microscopy is diffraction limited with a resolution of 235 nm for green light and 260 nm for red light. In comparison, the resolution of the 3D–SIM microscope is 2-fold better in XYZ, ~120 nm XY in simple terms. Considering the spacing of α-granules in human platelets, frequently <100 nm or so apart [Figure 2], confocal microscopy is then unable to resolve such adjacent granules from one another. The number of apparent granules by confocal or widefield fluorescence microscopy will be low. However, by 3D–SIM fluorescence microscopy, adjacent α-granules will frequently be resolved one from another because of the two-fold better resolution and the number of apparent α-granules will be larger. Note that the distribution of fibrinogen or VWF fluorescence shown in these micrographs is a direct measure of the mass distribution of these proteins within the cell. It does not directly indicate the dimensions of any given α-granule. Scale bar = 2 µm.