Angiostatin binds ATP synthase on the surface of human endothelial cells

Angiostatin, a proteolytic fragment of plasminogen, is a potent antagonist of angiogenesis and an inhibitor of endothelial cell migration and prolifer ation. To determine whether the mechanism by which angiostatin inhibits end othelial cell migration and/or proliferation involves binding to cell surfa ce plasminogen receptors, we isolated the binding proteins for plasminogen and angiostatin from human umbilical vein endothelial cells. Binding studie s demonstrated that plasminogen and angiostatin bound in a concentration-de pendent, saturable manner. Plasminogen binding was unaffected by a 100-fold molar excess of angiostatin, indicating the presence of a distinct angiost atin binding site. This finding was confirmed by ligand blot analysis of is olated human umbilical vein endothelial cell plasma membrane fractions, whi ch demonstrated that plasminogen bound to a 44-kDa protein, whereas angiost atin bound to a 55-kDa species. Amino terminal sequencing coupled with pept ide mass fingerprinting and immunologic analyses identified the plasminogen binding protein as annexin II and the angiostatin binding protein as the a lpha/beta-subunits of ATP synthase, The presence of this protein on the cel l surface was confirmed by flow cytometry and immunofluorescence analysis, Angiostatin also bound to the recombinant alpha-subunit of human ATP syntha se, and this binding was not inhibited by a 2,500-fold molar excess of plas minogen. Angiostatin's antiproliferative effect on endothelial cells was in hibited by as much as 90% in the presence of anti-alpha-subunit ATP synthas e antibody. Binding of angiostatin to the alpha/beta-subunits of ATP syntha se on the cell surface may mediate its antiangiogenic effects and the down- regulation of endothelial cell proliferation and migration.