Rapamycin inhibits retraction of fibrin clots by activated human platelets. Washed human platelets were pretreated with buffer, rapamycin (10 nM), or the vehicle for rapamycin for 1 hour. Exogenous labeled fibrinogen was then added to the cells, and they were left unstimulated or activated with thrombin (1.0 U/mL) for 2 hours. Appearance of the platelet suspensions under each experimental condition is shown in the middle panel. The stir bar in each tube is indicated by an arrow. The panels labeled A to D are photomicrographs from tubes shown in the same order in the middle panel. Actin was stained in unactivated and activated platelets (green fluorescence). Red fluorescence indicates fibrin strands. The platelet suspension in the first tube (control) is opaque, indicating dispersed, single unactivated platelets as seen by microscopy (A). No polymerized fibrin is present. Thrombin-stimulated platelets in the second tube have formed a tight fibrin clot adherent to the stir bar at the bottom, with clear media containing few residual platelets above. By microscopy, the clot consisted of platelet-fibrin complexes (B). After pretreatment with rapamycin, thrombin-stimulated platelets in the third tube formed a loose, gelatinous clot that was only slightly retracted, revealing clear media just below the meniscus. Polymerized fibrin was present but was much more dispersed (C). Activated platelets treated with vehicle for rapamycin in the fourth tube formed a tightly retracted fibrin clot around the stir bar at the bottom with clear media above, equivalent to the response of control platelets. Microscopically, the clot consisted of aggregated platelets and tightly retracted polymerized fibrin (D), similar to the clot in the control incubations (B). This figure is representative of 4 separate experiments.