Platelet activation and cytoskeletal reorganization: high voltage electron microscopic examination of intact and Triton-extracted whole mounts.

Abstract

The sequential changes in the three-dimensional organization of the filamentous components of human platelets following surface activation were investigated in whole-mount preparations . Examination of intact and Triton-extracted platelets by high voltage electron microscopy provides morphological evidence of increased polymerization of actin into the filamentous form and an increased organization of the cytoskeletal elements after activation . The structure of resting platelets consists of the circumferential band of microtubules and a small number of microfilaments randomly arranged throughout a dense cytoplasmic matrix. Increased spreading is accompanied by cytoskeletal reorganization resulting in the development of distinct ultrastructural zones including the peripheral web, the outer filamentous zone, the "trabecular-like" inner filamentous zone, and the granulomere . These zones are present only in well-spread platelets during the late stages of surface activation and are retained following Triton extraction . Extraction of the less stable cytoplasmic components provides additional information about the underlying structure and filament interactions within each zone .The morphological transformation that accompanies platelet activation has been attributed to alterations involving both the distribution and the degree of polymerization of the various contractile and structural proteins within platelets. Contractile proteins have been implicated in many of the events ofactivation including centralization and secretion of granules, aggregation, and clot retraction (1,7,8,35,48). Recent biochemical studies of thrombin-induced platelet activation have demonstrated a time-dependent incorporation of actin into the filamentous form and an increase in the amount of myosin associated with the cytoskeleton (5, 20, 37). The organization of these platelet contractile proteins, particularly actin and myosin, has been studied at the light microscopic level through immunocytochemical techniques (9,11,12,33,35). These studies have indicated that there are temporal changes in the whole-cell distribution of actin and myosin within platelets. While they are initially uniformly distributed, actin and myosin are segregated to selective areas as spreading progresses. Other studies employing transmission THE JOURNAL OF CELL BIOLOGY " VOLUME 98 JUNE 1984 2019-2025 ® The Rockefeller University Press -0021-9525/84/06/2019/07 $1 .00 electron microscopy (TEM)' have yielded considerable information regarding the types of filaments within platelets and overall platelet structure (22,23,42,(44)(45)(46)(47)(48) . Only a very small part of the platelet cytoskeleton is visible in conventional TEM thin sections hindering an accurate determination of the overall three-dimensional organization and in situ relationships ofthe filamentous cytoskeletal elements. Wholemount preparations have therefore been increasingly utilized to study platelet ultrastructure (2,22,23,25,26,29,42). However, masking ofthe cytoskeletal elements by the exten...

Assembly of a fibronectin matrix by adherent platelets stimulated by lysophosphatidic acid and other agonists

“…64 Fully spread platelets have 4 distinct ultrastructural zones: a peripheral web of a densely packed meshwork of fine microfilaments, an outer filamentous zone of a loosely interwoven array of microfilaments, an inner filamentous zone of a densely packed network of anastomotic discrete filaments surrounding the granulomere region, and the granulomere region. Staining of platelets with rhodamine-phalloidin demonstrated the various zones in both unstimulated and LPA-stimulated platelets adherent to FN or VN (Figure 1): an outer band of rhodamine-phalloidin-stained microfilaments (the terminal web), a loose network of microfilaments (the outer filamentous zone), and a dense polygonal microfilament network (the inner filamentous zone) that surrounds the core region containing granules (the granulomere region).…”

“…64,87,88 Our experiments indicate that agonists generated during blood coagulation stimulate adherent platelets to deposit a FN matrix and that such deposition is modulated by the nature of the adhesive substrate. We speculate that such an FN matrix may overlap functionally with FN incorporated into fibrin clots to provide a substrate for cellular ingrowth during the repair process.…”

Platelet activation.

“…15 In support of this conclusion, the'cytoplasm of purely discoid platelets does not appear to be replete with fully formed, long microfilaments, but rather to contain short fragments of such filaments and amorphous ground cytoplasm. 16 ' 17 The amorphous component could be composed of actin complexed with profilin, a protein present in platelets in amounts sufficient to complex part, but not all, of the actin (see Table 1J. 18 - 50 However, some investigators, using different methods, 49 .…”

Granule exocytosis is required for platelet spreading: differential sorting of α-granules expressing VAMP-7

“…34 However, alternative methods of imaging, such as differential interference contrast (DIC) microscopy, atomic force microscopy, and scanning electron microscopy demonstrate organelles in the periphery of the spread platelet. 35,36 We postulated that a subpopulation of ␣-granules were among the peripheral organelles in spread platelets and that platelets actively sort this subpopulation of ␣-granules to the periphery during spreading.…”