We have measured thrombin-induced secretion of platelet factor 4 antigen (PF4) and simultaneously followed its intracellular translocation by immunofluorescence. In permeable resting platelets, speckled intracellular immunofluorescent staining for PF4 was observed. Addition of thrombin to washed platelets at 22 degrees C resulted in secretion of PF4 and formation of large (approximately 0.5 micrometer) immunofluorescent masses. These masses moved to the cell periphery during secretion and were virtually absent at the conclusion of secretion. Ultrastructural examination of thrombin-treated platelets revealed vacuoles corresponding in size, shape, and time of occurrence to the large immunofluorescent masses of PF4. These vacuoles contained PF4 by immunoferritin staining of frozen thin sections; they therefore appear to represent the ultrastructural counterpart of the large PF4 masses. When intact cells were stained for PF4 after thrombin addition, only 5.6% of the large masses stained. Thus, during secretion, PF4 antigen is consolidated into large closed pools that appear as vacuoles in the electron microscope.

We have investigated the localization of thrombospondin (TSP), fibrinogen, fibronectin, and von Willebrand factor in human platelets by transmission electron microscopy of antibody-stained ultrathin frozen sections. In negatively stained thin sections, alpha granules were identified on the basis of their smooth, roughly spherical shape, size, single limiting electron-lucent 100 A membrane, and frequent presence of electron-dense nucleoid. In contrast, mitochondria exhibited characteristic double membranes and cristae. Sections were separately stained with affinity-purified polyclonal antibodies to these proteins as well as with three monoclonal anti-TSP antibodies. Antibody specificity was documented in radioimmunoassays, by immunofluorescent cross-blocking, and by staining of bands of appropriate mobility in Western blots of whole platelets. Bound antibody was visualized using a 5-nm colloidal gold-avidin conjugate. In resting cells, staining of virtually all alpha granules was observed for all four proteins. In contrast, consistent staining was absent from other organelles, including plasma membranes, mitochondria, and vacuolar structures that may represent the open canalicular system.