Platelets acquire TSP-1 through megakaryocytes. (A) Western blot of TSP-1 expression in platelets harvested from Tsp-1^−/− animals at indicated days after intravenous injection of 2 μg rTSP-1. GAPDH was probed as a loading control. (B) Left: Platelets from Tsp-1^−/− mice are immunostained with antitubulin (green) and anti–TSP-1 (red) after coincubation at 37°C for 2 hours in vitro with 2 μg rTSP-1. Right: Western blot analysis of wild-type platelets, Tsp-1^−/− platelets, and Tsp-1^−/− platelets after in vitro coincubation with rTSP-1. GAPDH was probed as a loading control. (C) Western blot analysis of megakaryocytes isolated from the bone marrow of Tsp-1^−/− mice after coincubation at 37°C for 2 hours in vitro with rTSP-1. β-Actin was probed as a loading control. (D) Immunostaining with anti-GPIBα (green) and anti–TSP-1 (red) of megakaryocytes isolated from the bone marrow of Tsp-1^−/− mice 24 hours after intravenous injection of saline or 2 μg rTSP-1. Bar represents 50 μm. (E) Western blot analysis of megakaryocytes isolated from Tsp-1^−/− mice 1 and 4 days after intravenous injection of 2 μg rTSP-1. GAPDH was probed as a loading control.