General structural features of activated platelets

The above described regulatory signals synergistically stimulate the platelets and in turn activate many complex internal signaling cascades in platelets. All of these together contribute to bringing about morphological and biochemical changes in the platelets whereby they dramatically change their shape from discoid to spherical, remodel their internal cytoskeleton, spread and release their granules. All these changes are collectively referred to as 'platelet activation'.

Most of these morphological changes can also occur in platelets when they come in contact with foreign surfaces like glass [79, 102, 123] and these changes also occur in platelets that are activated in suspension [94]. Such changes in the platelets have been visualized by electron microscopy studies (Figure 2.6A and B). In general, when platelets are activated, their peripheral MT coil which maintains their discoid shape contracts in the platelet center.

This contraction is brought about by the actomyosin cytoskeleton. The constriction of both the

23 acto-myosin cytoskeleton and the MT coil drive the platelet granules (like  granules and dense bodies) towards the platelet centers in a structure termed as the granulomere [79, 94, 102]. In the initial stages of activation, the platelets extend numerous filopodia and thin hyalomeres (Figure 2.6B). These lamellipodia and the granulomere are separated from the ring of cytoskeletal filaments that contracts into the platelet centers [94, 102]. Such morphological changes occur in the platelets when their stimulus is weak. If it is strong, the granules that have moved into the center are secreted to the platelet exterior surface through the channels of the OCS [94]. Furthermore, the MT coil may fragment and get distributed in the newly formed filopodia [102].

Figure 2.6: Transmission electron microscopy images of glass-adhered platelets

The TEM images of platelets adhered to glass are shown. A) Discoid platelet, incubated for 5 minutes before fixation shows peripheral MTC that stabilizes the discoid shape. Parts of the DTS line the margin and the  granules are randomly distributed. B) Activated platelet, incubated for 15 minutes before fixation has extended hyalomere. The MTC has contracted into the center and the  granules have also moved into the center in the granulomere. The granulomere and hyalomere distinctly appear to be separated by this MTC. The openings to the surface-connected OCS are also seen.

The figure caption information is adapted and the figure is reproduced from Neumüller J, Ellinger A, and Wagner T., 2015 originally published in Maaz K (ed) The Transmission Electron Microscope - Theory and Applications. InTech http://dx.doi.org/10.5772/60673 [102]. © 2015 (Neumüller et al).

This chapter is open-access under the Creative Commons Attribution License.

24 The transformation of platelets when they get activated by surfaces has also been imaged by differential interference contrast (DIC) microscopy (Figure 2.7). On adherence to glass the platelets change their shape from disc to sphere and undergo dramatic morphological transformation, spread completely and release their granules [7]. The platelets accomplish spreading within 10-12 minutes by first forming pseudopodia which extend and retract and then form hyalomeres that extend in between these pseudopodia and also laterally form from them. Furthermore, these platelets show granulomere hillocks in their center, during the early stages of spreading when pseudopodia form and which are described as being 'dome-shaped' [7]. The granules contained within these granulomeres are the dense bodies which can be seen with DIC. As these platelets begin to spread, the granulomeres slowly flatten and the dense bodies that are clustered inside are released from the granulomere and they leave 'craters' [7]

at the places in the granulomeres, from where they are released (Figure 2.7-4). The craters are suggested to be part of the OCS [7]. After full spreading, the hyalomeres show membrane ruffling (Figure 2.7-5) which is reminiscent of the lamellipodia in tissue cells [7]. The releasing of granules is referred to as 'exocytosis' or 'degranulation event' and occurs mostly after formation of hyalomeres (Figure 2.7-6, 7) when the spreading has advanced but can also occur during the entire transformation process. In fully spread platelets, the craters eventually disappear, and the granulomeres flatten, sometimes completely, and within 30 minutes (Figure 2.7-8, 9), most platelets have exocytosed as much as they can [7]. It has also been suggested that platelets activated by contact with glass may show only one exocytosis event that will go on during their entire morphological transformation, unlike several events shown by chemically activated platelets [7]. The terms pseudopodia and hyalomere are synonymous with the terms filopodia and lamellipodia [7, 127]. This platelet shape change and their transformation to a flat form, with the extension of filopodia, flattening via the lamellipodia and the squeezing of their granules into the center gives them the appearance of a fried-egg and these morphological changes are a result of the remodeling of their cytoskeleton [94]. In fact, ultrastructural studies have shown that these platelet protrusions contain F-actin [7, 173].

The platelet shape change is important for their ability to firmly attach to the ECM, and to each other in a platelet plug and also efficiently secrete their granular contents [106]. The platelets can spread rapidly and increase their surface area up to 420% [94]. This newly expanded membrane material is obtained by pulling out membrane material from within the invaginations of the OCS. The wrinkled surface (of the discoid platelet, described in section 2.3) too serves as an additional reservoir that contribute to the overall, 4-fold platelet plasma membrane expansion during their spreading [94, 166].

25 Figure 2.7: Differential interference contrast (DIC) microscopy of glass-adhered platelets

The DIC image of platelets in different spreading stages during their morphological transformation is shown. The numbering is in order of the spreading sequence seen in these platelets. 1) Platelet is not properly attached. 2) This platelet is in the late pseuodpodial stage and is beginning to spread. It shows the granulomere hillock (H). 3) Aggregate of platelets which are poorly attached. 4) This platelet is in the intermediate spreading stage and shows the hillock (H) granulomere which also shows a crater (Cr) formed presumably due to the release of dense bodies. 5) Another platelet showing the hillock with its hyalomere showing ruffles (R). 6 and 7) These platelets have flattened and so have their granulomeres, which have exocytosed particles (P) i.e. dense bodies (according to [7]), and the spaces where these particles were before show craters (Cr). 8 and 9) These are fully flattened platelets that have finished spreading.

The figure is adapted and the information in the caption is reproduced with permission from Allen RD, Zacharski LR, Widirstky ST, et al., 1979 originally published in The Journal of Cell Biology http://dx.doi.org/ 10.1083/jcb.83.1.126 [7]. © 1979 Rockefeller University Press, License number- 4003211252475, Licensed content publisher- Rockefeller University Press.