Further studies of the secretory pathway in thrombin-stimulated human platelets

The pathway followed by secretory products stored in platelet alpha granules during the release reaction remains controversial. Tannic acid has been used in the present study as an electron-dense stain to follow the secretory process in thrombin-stimulated platelets. Preliminary experiments demonstrated that tannic acid precipitates fibrinogen, and binds osmium tetroxide to fibrinogen and fibrin strands. Examination of platelets fixed at short intervals after exposure to thrombin and incubated in solutions containing tannic acid revealed electron-dense deposits of osmium not apparent in resting platelets. Granules and lumina of channels making up the open canalicular system (OCS) were unstained in discoid cells. However, exposure to thrombin at concentrations of 1 to 5 U/mL for thirty seconds or more resulted in intense staining of alpha granules by osmium. Some granules communicated directly with dilated channels of the OCS, and several were frequently connected to the same canaliculus. The electron-dense substance in swollen granules and channels appeared to be in the process of extrusion through narrow or dilated openings of the OCS onto the platelet surface. Granule-to-granule fusion and formation of sealed vacuoles of fused granule products unstained by tannic acid-osmium were not observed. The findings support the concept that secretion by stimulated human platelets results from development of direct communications between granules and channels of the OCS and subsequent extrusion of products through channel pores to the surrounding medium.