Platelets are activated by and form aggregates on LECs in a SLP-76–dependent manner in vitro and in vivo. (A) Wild-type platelets bind and are activated on the surface of LECs but not BECs. Slp-76^−/− platelets adhere to LECs but do not form aggregates or express P-selectin. CD41 staining identifies platelets, and P-selectin expression identifies activated platelets. (B) Flow of heparinized wild-type blood at a shear level of 8 dynes/cm² results in platelet aggregate formation on LECs but not on BECs (left 2 panels). PDPN-deficient platelets adhere and form aggregates on LECs, but SLP-76–deficient platelets fail to form aggregates (right 2 panels). Platelets are visualized with Alexa Fluor 488–conjugated anti-CD41 antibody after 5 minutes of flow. (C) Identification of platelet aggregates on LYVE-1⁺ LECs in Slp-76^+/+ but not Slp-76^−/− developing embryos. Immunostaining of E11.5 transverse embryo sections with antibodies against platelets (red) and the LEC LYVE-1 protein (green) are shown. Arrows indicate platelet aggregates on the endothelial cell surface. CV indicates cardinal vein; and LS, lymph sac. (D-E) Injection of wild-type platelets into Slp-76^−/− animals results in the formation of platelet aggregates on the surface of LECs. Wild-type platelets were injected into the vitelline vein of E18 Slp-76^−/− embryos (D) or into the retroorbital sinus of P21 Slp-76^−/− animals (E). Platelet aggregates (red) and lymphatic endothelium (anti–LYVE-1, green) were detected by immunostaining of tissues harvested 12 hours after injection.