Angiogenesis regulators are taken up by platelets in vitro. (A) PRP was incubated with increasing concentrations of recombinant human bFGF (rhbFGF) or recombinant human endostatin (rh endostatin) for an hour. The platelets were then isolated by sequential centrifugation, washed, treated with 1% Triton X to remove the membrane, and lysed for SDS-PAGE analysis. Standard Western blots using antihuman endostatin and antihuman bFGF antibodies reveal a dose-dependent increase in the respective proteins in the cytoplasmic fraction of fresh platelets. (B) To establish the localization of proteins taken up by platelets, the platelets were incubated with His-tag labeled endostatin, fixed using paraformaldehyde, and anti-His antibody was used to separate the platelet endogenous endostatin (not labeled) from the endostatin “loaded” into platelets (fluorescent label). (Left) DIC image of the platelets. (Middle) The fluorescent label of the His tag. (Right) The overlay of the 2 images. The pattern of the fluorescent signal indicates that endostatin is taken up into the granules of platelets rather than remaining membrane-associated.