The lymphangiogenic factors VEGFC and VEGFD are proteolytically activated by thrombin and plasmin. (A-D) Proteolytic release of VEGFC VHD and VEGFD VHD by plasmin, thrombin, and ADAMTS3 proteases. Conditioned supernatant containing VEGFD VHD-FLAG or VEGFC VHD-FLAG was incubated with the indicated concentrations of thrombin and plasmin for 1, 2, or 18 hours, and VHD-FLAG detected by western blotting with anti-FLAG antibodies. The protein domains within the detected bands are shown schematically on the right. Note that VEGFD is efficiently cleaved by plasmin and, to a lesser extent, thrombin, but not by ADAMTS3, whereas VEGFC is efficiently cleaved by ADAMTS3 and thrombin, but not by plasmin. (E) Thrombin cleavage of VEGFC is inhibited by addition of thrombin-specific inhibitor dabigatran. (F) Thrombin-activated washed platelets drive VEGFR3 phosphorylation in cultured LECs. Washed human platelets were exposed to the indicated concentrations of thrombin, added to cultured LECs and phospho-VEGFR3 measured by ELISA. Each color represents results obtained using platelets from a single donor. (G) LEC exposure to thrombin alone does not increase phospho-VEGFR3. Error bars indicate SEM. **P < .01; ns, not significant.