Figure 3.
Ultrastructural features and functional analysis of culture-derived mouse platelets. Structure of platelets released into the megakaryocyte culture media. This representative micrograph shows released particles to be disc-shaped after rapid freezing, freeze drying, and metal coating. Particles have smooth, featureless surfaces except for periodic pits that delineate entrances to the open canalicular system, a hallmark feature of platelets. Pits are indicated with arrowheads. (b) Electron micrograph showing that a culture-derived platelet has a membrane skeleton similar in structure to that of a blood-derived platelet. Careful removal of the membranes with the detergent Triton X-100 reveals a dense fibrous lamina that coats the underside of the plasma membrane and obscures the underlying actin cytoskeleton. The membrane skeleton is composed of thin strands that interconnect forming triangular pores. (c) Electron micrograph showing the presence of a peripheral microtubule coil in the detergent insoluble cytoskeleton from a resting, culture-derived platelet. The microtubule coil retains the dimensions of the platelet. Actin fibers are also visible. (d) The cytoskeleton of a representative culture-derived platelet after treatment with thrombin. Culture-derived platelets grow both lamellipodia and filopodia from their edges after treatment with thrombin, and assemble a dense actin filament based cytoskeleton. (e) Morphology of a culture-derived platelet after treatment with thrombin. In response to thrombin, platelet-sized particles convert from discoid shapes in active forms having broad flat lamellae and thin filopodia. (f) After treatment with 1 U/ml thrombin, culture-derived platelets form microaggregates containing 5–10 particles. (g and h) Microtubule coils at the ends of proplatelets. Antitubulin immunofluorescent staining of megakaryocytes at the peak time of proplatelet production. Each representative micrograph shows megakaryocytes in culture to produce hundreds of microtubule coils within the proplatelet extensions emanating from a single megakaryocyte. Each microtubule coil is similar in size and structure to those observed in mouse blood platelets (inset in g). Bars, 1 μm.