Platelet aggregation is driven by podoplanin and linked to separation of the lymph sacs from the cardinal veins. (A-B) Still frames of movies showing coincubation under flow conditions of isolated normal mouse platelets with monolayers of NIH-3T3 cells that transgenetically express podoplanin (A) or NIH-3T3 cells that were transfected with an empty vector (B). While large platelet aggregates form on the surfaces of podoplanin-expressing NIH-3T3 cells (red arrowheads), NIH-3T3 cells that lack podoplanin fail to interact with platelets even after 20 minutes. Imaged on an Olympus IX50 using a 40×/0.65 air LCAch (objective lens and a FViewII) camera (Olympus). (C-F) Direct demonstration in E12.5 and E13.5 podoplanin^+/+ embryos of platelet thrombi (labeled for integrin α_IIbβ₃ in red) at the junction of cardinal veins and lymphatic sacs that are marked by podoplanin (green; C,E) or Lyve-1 (green; D,F). By contrast, platelets are absent from the lymph sac's orifice in E13.5 podoplanin^−/− embryos (G-H) as seen on serial sections (220 μm apart) stained for Lyve-1 (green) and the platelet integrin α_IIbβ₃ (red). Note the still persisting connection (G) between lymph sac and vein. In the section 220 μm apart (H), the connection between lymph sac and cardinal vein is no longer visible; in panels G and H, erythrocytes/reticulocytes are present in the lymph sacs (Ter119 staining not shown). Cell nuclei are stained with DAPI (blue); Olympus AX70, 10×/0.40 air UPlanApo. Scale bars in panels A and B equal 10 μm; in panels C through H, 50 μm.