PART 11, ENDOTEELUL CELLS AND PLATELETS

ELUCIDATION OF THE PLATELET CYTOSKELETON *

Joan C. Mattson and Carol A. Zuiches

Department of Pathology Michigan State University

East Lansing, Michigan 48824

INTRODUCTION

Morphologic studies of the platelet cytoskeleton at the ultrastructural level have been hampered by the loss of detail that occurs in the process of plastic embedding and thin sectioning. Some of this loss has been ascribed to the action of osmium fixation on filamentous structures, most notably actin.’ However, the lack of resolution of cytoskeletal elements appears to be an inherent limitation in the approach of using ultrathin sections of material embedded in plastic.z The actual structure as well as the interrelationships of a three-dimensional cytoskeleton is difficult to appreciate in ultrathin sections since only perfectly oriented filaments that lie in the plane of the section will appear as filaments. All other orientations will produce oblique or cross- sectional views of filaments, which will appear in micrographs as spherical or ovoid structures. The technique is further hampered by the lack of density differential between the plastic embedding medium and cytoplasmic filaments, which results in almost total obscuration of filaments of finer dimensions. Wolosewick and Porter2 have demonstrated that if whole cells are processed for electron microscopy without an embedding medium, they can be examined at high voltages (1000 kV) with excellent resolution of minute cytoskeletal details. While these investigators utilized a 1 -million volt electron microscope not accessible to the majority of investigators for routine use, other investiga- tors 3, have demonstrated that excellent results can be obtained with whole cell preparations utilizing conventional accelerating voltages of 80 to 100 kV. The usefulness of whole cell preparations in studying cytoskeletal architecture has been amplified by the introduction of nonionic detergents to produce partial or complete removal of membranes to yield extracted “cytoskeletons.” 5 9

The study presented here was designed to overcome the inherent difficulties in demonstrating cytoskeletal elements in transmission electron micrographs of sectioned platelets by utilizing whole cell preparations and detergent-extracted cytoskeletons of contact-activated platelets.

MATERIALS AND METHODS

Zsolation of Platelets

Whole blood was collected by venipuncture from normal human volunteers who had previously been determined to have normal platelet function. Samples were anticoagulated with 3.8 percent sodium citrate in a ratio of 9 parts whole

*This work was supported by a grant from the Michigan Heart Association.

11 0077-8923/81/037(Mol1 $01.75/0 ‘0 1981, W A S

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blood to 1 part anticoagulant. Platelet-rich plasma (PRP) was prepared im- mediately by difFerentia1 centrifugation at 70 x g for 10 minutes. The resultant PRP was transferred to a clean plastic test tube and allowed to rest for at least 30 minutes, but no more than 1 hour before use in experiments. Whole blood and platelet rich plasma were maintained at room temperature throughout these procedures to avoid cold-induced morphologic alterations. All-plastic labware was used in handling whole blood and platelet-rich plasma to avoid platelet activation by glass or silicone.

Whole Cell Preparations of Contact-Activated Platelets

Whole cell mounts were prepared following a modification of the procedure outlined by Pudney and Singer.6 Platelets were allowed to settle and spread onto Formvar-coated, carbon-stabilized copper grids that had been briefly pre- treated with 0.1 percent polylysine. This was accomplished by placing a drop of PRP onto each prepared grid and allowing the platelets to settle out for 1 hour at room temperature in a moist chamber. Grids were then washed free of nonadherent platelets by briefly rinsing in phosphate-buffered saline (PBS) solution. They were then fixed for 2 hours in 2.5 percent glutaraldehyde in 0.1 M cacodylate buffer pH 7.2, rinsed briefly in distilled water, placed in 30 percent acetone for 10 minutes, stained with 1 percent uranyl acetate in 50 percent acetone for 3 minutes, dehydrated in graded acetones, and dried from CO, by the critical point method.

Cytoskeletal Preparations

Cytoskeletal preparations were prepared in an identical fashion, except that a nonionic detergent (Nonedet P40 or Triton X-100) in concentrations from 0.1 to 2 percent was introduced either immediately before fixation for a 2- minute digestion or simultaneously with fixation by placing the appropriate detergent directly into the fixative. In the latter instance, grids containing whole platelets were fixed in the detergent-glutaraldehyde mixture for 15 minutes and then transferred to fresh 2.5 percent glutaraldehyde without detergent for an additional 15 minutes. Grids were then processed as described for whole cell preparations.

Transmission Electron Microscopy ( T E M )

The “critical-point-dried’’ whole platelets were examined on a Philips 201 microscope using a 20-pm objective aperture and operated at an accelerating voltage of 80 kV. Cytoskeletal preparations were examined on the same instrument operated at an accelerating voltage of 60 kV. Selected specimens were photographed as stereoscopic pairs using tilts of & 6 O from the horizontal to allow study of filament interrelationships. Filaments were measured from photographic prints taken immediately after instrument calibration with a germanium-shadowed carbon replica having 28,800 lines per inch.

Mattson & Zuiches: Platelet Cytoskeleton 13

Scanning Electron Microscopy ( S E M )

Occasional whole-mount preparations were first examined by TEM and then coated with 100 A of gold and examined by scanning electron microscopy on an IS1 Super 111 scanning electron microscope at an accelerating voltage of 15 kV.

FIGURE 1. Whole cell preparation of platelets allowed to spread for 1 hour prior to fixation. Polygonal cell has centralized granules (G) with closely associated micro- tubule coil (MT). Fan-shaped cell at upper left also contains aggregated granules (G), but these remain close to the unspread margin of the cell. Clear channels of the surface connecting system (SCS) are visible in close proximity to granules. Occa- sional channels of SCS extend peripherally into the cytoplasm (arrows). The platelet hyalomere contains faintly discernible bundles of filaments (F) interspersed with unstructured areas of cytoplasm having a mat-like appearance (M). (80 kV; original magnification x 11,550; reduced by 15 percent.)

RESULTS

The general features of contact-activated platelets could be appreciated in both whole cell preparations examined at 80 kV and detergent-extracted cyto- skeletons examined at 60 kV. Both types of preparations demonstrated that after 1 hour of contact with the substrate, activated platelets spread to produce circular, fan-shaped, polygonal, or angulated configurations , (FIGS. 1-6).

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FIGURE 2. Scanning electron micrograph of platelets. The platelet is fully spread with a roughly circular profile. The aggregated granule mass (G) is seen as a hillock in the cell center. Openings of the surface connecting system (SCS) are seen en- circling the centralized granules with a few openings extending into the peripheral cytoplasm (arrows). Note that fully spread platelets do not contain long pseudopodia or dendritic processes. (15 kV; original magnification x 15,000, reduced by 15 percent. )

Pseudopodia were absent. In 25 randomly selected cells and cytoskeletons, the longest diameters ranged from 5.2 to 10.2 pm. Approximately 25 percent of the platelets were unevenly spread (that is, long and short dimensions differed by 2 pm or more), producing a clearly distinguishable long axis. The mean length along the long axis in this group was 7.8 pm as compared with a. mean longest dimension of 6.9 pm in more evenly spread platelets.

In the majority of platelets, granules were found aggregated centrally (FIGURES 1 through 4). However in fan-shaped cells, the aggregated granule mass was found at the unspread, stable margin of the cell (FIG. 5 ) . The morphologic configuration of granules was similar to that seen in thin-sectioned material. They appeared as electron-dense ovoid or spheroid structures that ranged in size from 0.15 to 0.28 pm. However, the density of nonsectioned granules did not allow appreciation of any substructure. In addition, it was not possible to distinguish mitochondria from granules in these preparations.

In both whole cells and in cytoskeleton preparations, channels of the surface connecting system (SCS) were found concentrated near the central granulomere (FIGS. 1 , 3, and 5). Surface openings to this system could be seen by scanning electron microscopy to encircle the central granule mass (FIG. 2). In several

Mattson & Zuiches: Platelet Cytoskeleton 15

platelets a few channels of the surface connecting system appeared to extend toward the cell periphery (FIGS. 1 and 3 ) .

Some limited details of the platelet cytoskeleton could be appreciated in whole cell preparations (FIGS. 1-3). Bundles of elongated filaments could be identified interspersed with areas having a more mat-like appearance. At the cell surface, hair-like projections of the glycocalyx were visible in some prepara- tions (FIG. 3, inset). Whole cell preparations examined at an accelerating voltage of 80 kV did not, however, reveal sufficient detail to allow either sue of filaments or complex interrelationships to be defined; for this, detergent extraction was required.

In cytoskeletons prepared by partial detergent extraction, the filamentous components were clearly visible (FIGS. 4-8). It was immediately obvious that the cytoskeletons of fully spread platelets have a highly structured organization. All platelets in detergent-treated preparations contained loose bundles of fila-

FIGURE 3. Whole cell preparation. Detail from the peripheral hyalomere of a fully spread platelet. A bundle of filaments (F) is seen at the lower right. Resolution of individual filaments in this buodle is insufficient to allow measurements or to judge interrelationships. At the cell periphery, channels of the surface connecting system render the cell more permeable to the electron beam. In this area a network of thin filaments (arrowheads) can be distinguished in random array. These become compact at the cell cortex (C). A single microtubule curves through the filamentous network (MT). Irregularly shaped opaque tubular structures (circle) are suspended in the interconnecting filaments. On the external surface of the platelet, hair-like projections extend at a 90" angle from the cell surface (inset). (80 kV; original magnification x 32,500; reduced by 15 percent.)

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FIGURE 4. Detergent-extracted cytoskeletons produced by partial dissolution of membranes with 2 percent Nonedet P40 in 2.5 percent glutaraldehyde. Details of the cytoskeleton revealed by this technique include prominent loose bundles of elongated filaments (F). I n angulated cells, filament bundles coalesce at the apices of cell projections (arrows). Note that granules (G) usually remain in place in this partially extracted preparation. As in whole-cell preparations, granules either coalesce centrally or along the unspread margin of incompletely spread platelets. No microtubular coil is identified surrounding granulomeres. (60 kV; original magnification x 4750; re- duced by 15 percent.)

ments, which encircled the central granule mass and which were usually oriented in the long axis of the cell (FIGS. 5-7). Individual elongated filaments appeared to come together to form thicker fibrils, then to separate for short distances only to re-form lateral associations with the same or different adjacent filaments (FIG. 7). In this manner an interwoven effect is created within the bundles of long filaments. In angulated cells with short cytoplasmic projections, bundles of filaments often converged and coalesced at the vortex of each projection (FIG. 4). Individual filaments within these bundles ranged in diameter from 60 to 120 A with occasional larger filaments measuring up to 180 A. The larger measurements may represent indistinguishable coalescence of two or more thin filaments.

At high magnifications, a filamentous network of thinner, shorter filaments

Mattson & Zuiches: Platelet Cytoskeleton 17

could be seen both interacting with the bundles of long filaments (FIGS. 7 and 8) and extending to the periphery of cells, where it formed the major cytoskeletal framework in areas that contained no filament bundles (FIGS. 5 and 6). This latticework appeared to condense at the cell cortex (FIGS. 3 and 5 ) . Filaments in this network measured from 30 to 85 A in diameter with occasional thicker segments measuring up to 150 A. Within this three- dimensional lattice, numerous ovoid or spherical particles measuring 140 to 170 A could be identified intimately associated with both short and long filaments (FIG. 8). Larger particles measuring up to 280 A were also found suspended within the filamentous network.

A tight coil of microtubules was rarely found surrounding the central granule mass in any of the whole cells or cytoskeletons examined. Usually, single microtubules were seen coursing in a gently curved fhshion through the periphery of the cytoplasm (FIG. 6).

$IGURE 5. Detergent-extracted cytcskeleton. As in whole-cell preparations, chan- nets of the surface connecting system (SCS) appear as clear spaces in close association with the granulomere (G). Filaments are oriented in elongated bundles (F) on the long axis of the cell. Peripheral to the filament bundles is a loosely structured filamentous network (N) which appears to condense at the platelet margin. Note the absence of a microtubular coil surrounding the granulomere. (60 kV; original magnification x 11,375; reduced by 15 percent.

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FIGURE 6. Detergent-extracted cytoskeleton. The central granule mass has become dislodged to reveal a set of channels or spaces (S). Loosely structured bundles of elongated filaments (F) surround this central area. Again, filaments (F) are oriented with the long axis of the platelet. At the cell periphery, the cytoskeleton is composed of a more loosely structured filamentous network (N). Single straight and curved microtubules (MT) course through the cell periphery, but these do not appear to be elements of a continuous coil since the ends of some individual microtubules can be identified (arrow). 60 kV; original magnification x 12,150; reduced by 15 percent.)

DISCUSSION

By using whole cell preparations and detergent-extracted cytoskeletons, the present work has shown that the spread platelet produced by contact activation contains a well-organized cytoskeleton composed of loose filament bundles and single microtubules intimately associated with a network of short, thin inter- connecting filaments. The highly structured organization of the platelet cyto- skeleton in these preparations appears to be related to and perhaps responsible for the nonrandom distribution of organelles. Platelet granules were, without exception, found as an aggregate surrounded, by filament bundles. The micro- tubular coil, on the other hand, was generally absent from this location.

Several features of the platelet cytoskeleton as demonstrated in this study are similar to the cytoskeletal components described in other cell systems. Most notably, the fine filamentous latticework, which appears to support organelles and interact with bundles of long filaments and with microtubules, appears to

Mattson & Zuiches: Platelet Cytoskeleton 19

be comparable to the microtrabecular network revealed by high-voltage electron microscopy of cells in culture.** 7+ Similarly the bundles of elongated filaments have the same dimensions and orientation as the stress fibers of cultured cells.8 It would be reasonable to suppose that such stress fibers might be a common feature of all cells that are adherent to a substrate. It is also tempting to postulate that the peculiar arrangement of filament bundles around granules in platelets may be critical to the generation of forces that modulate the granule extrusion known to occur during platelet ~preading .~

It is interesting to note that spread platelets characteristically do not contain a microtubular coil surrounding the granulomere, as is commonly seen in platelets activated in suspension. Instead, individual microtubules were found curving through the peripheral cytoplasm of fully spread platelets. Nach- mias 101 11 has seen microtubular coils in platelet cytoskeletons treated with Triton 45 seconds after contact activation, but has also demonstrated that fully

FIGURE 7. Detail of Nament bundles from the platelet shown in FIGURE 6. Note that individual thin Naments in these bundles do not run in precise parallel array. Instead, several filaments come together for a short distance, then separate for a distance, and reassociate with the same or different filaments, always maintaining a general orientation along the same axis. This produces an interwoven effect. In the spaces created as bundles separate, thin filaments interconnect long filaments at a roughly 90" angle (arrowheads). A similar network of short, thin filaments is also seen within the central space. (60 kV; original magnification ~ 3 1 , 5 0 0 ; reduced by 15 percent.)

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spread platelets may not contain this coil. Our interpretation is that microtubule reorientation occurs as platelet spreading progresses.

Further studies are required to identify the precise composition of the platelet cytoskeleton. The platelet cytoskeleton is an extremely complex system that contains multiple components including actin, myosin, tropomyosin, actin- binding protein, a-actinin, and calcium-binding proteins.l2, l3 The adaptation of techniques that allow preservation and visualization of the platelet cyto- skeleton is, therefore, an important step toward the ultimate localization of these components and the understanding of their interrelationships. The

FIGURE 8: High magnification through filament bundles seen in FIGURE 7 shows a detail of the underlying filamentous network. Small groupings of long filaments are encircled. In the spaces between groups of long filaments, short filaments can be seen to cross at right angles (arrows). Many long filaments appear to have small spherical or ovoid globules (140 to 180 A) attached along their surface (circles). (60 kV; original magnification ~ 7 8 , 0 0 0 ; reduced by 15 percent.)

excellent preservation of cytoskeletal architecture obtained in this study suggests that this model will provide an appropriate method for biochemical and im- munocytochemical studies.

ACKNOWLEDGMENTS

We wish to thank Belinda Oxender for clerical assistance with this manu- script and Dee Jakubiak and Donna Craft for technical assistance in the preparation of the electron micrographs.

Mattson & Zuiches: Platelet Cytoskeleton 21

[Note added in proof: Subsequent to the submission of this manuscript, Lewis et al.” reported a similar reorientation of platelet microtubules during adhesion and spread- ing. These authors observed loss of the circumferential band of microtubules during early activation, radially oriented microtubules in the dendritic platelets found in the intermediate stages of spreading, and finally reorientation of microtubules to the cytoplasmic margin in fully spread platelets. These findings support our observations that, in the fully spread platelet, microtubules are seldom found in close association with the central granule mass, but are present in the peripheral cytoplasm where they are oriented parallel to the platelet membrane.]

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