Angiostatin Inhibits Endothelial and Melanoma Cellular Invasion by Blocking Matrix-Enhanced Plasminogen Activation

Angiostatin,a kringle-containing fragment of plasminogen,is a potent inhibitor of angiogenesis. The mechanism(s) responsible for the anti-angiogenic properties of angiostatin are unknown. We now report that human angiostatin blocks plasmin(ogen)-enhanced in vitro invasion of tissue plasminogen activator (t-PA)-producing endothelial and melanoma cells. Kinetic analyses demonstrated that angiostatin functions as a non-competitive inhibitor of extracellular-matrix (ECM)-enhanced,t-PA-catalysed plasminogen activation,with a Ki of 0.9+/-0.03 microM. This mechanism suggests that t-PA has a binding site for the inhibitor angiostatin,as well as for its substrate plasminogen that,when occupied,prevents ternary complex formation between t-PA,plasminogen and matrix protein. Direct binding experiments confirmed that angiostatin bound to t-PA with an apparent Kd [Kd(app)] of 6.7+/-0.7 nM,but did not bind with high affinity to ECM proteins. Together,these data suggest that angiostatin in the cellular micro-environment can inhibit matrix-enhanced plasminogen activation,resulting in reduced invasive activity,and suggest a biochemical mechanism whereby angiostatin-mediated regulation of plasmin formation could influence cellular migration and invasion.