Urokinase plasminogen activator (uPA) belongs to a family of proteins that contains kringle domain and plays an important role in inflammation, tissue remodeling, angiogenesis, and tumor metastasis by pericellular plasminogen activation. Kringle domains of plasminogen have been shown to demonstrate anti-angiogenic and anti-tumor activities. Here, we report our investigation of the kringle domain of uPA for anti-angiogenic activity and a possible cellular mechanism of action. The recombinant kringle domain of uPA (Asp 45 -Lys 135 ) (UK1) inhibited endothelial cell proliferation stimulated by basic fibroblast growth factor, vascular endothelial growth factor (VEGF), or epidermal growth factor. It also inhibited migration of endothelial cells induced by VEGF or uPA, and in vivo angiogenesis on the chick chorioallantoic membrane. It did not block plasminogen activation by activated uPA in clot lysis and chromogenic substrate assays. Neither binding of UK1 to immobilized uPA receptor nor competitive inhibition of uPA binding were confirmed by real-time interaction analysis. However, internalization of UK1 followed by translocation from cytosol to nucleus was determined to be specific to endothelial cells. It also elicited a transient increase of Ca 2؉ flux of more than 2-fold within 2 min of exposure in an endothelial cell-specific manner. These results suggest that the kringle domain of uPA exhibits anti-angiogenic activity and that its anti-angiogenic activity may occur through a different mechanism from inhibition of uPA-uPA receptor interaction or uPA proteolytic activity and may be associated with endothelialcell specific internalization not mediated by the uPA receptor.

Abbreviations: bFGF, basic fibroblast growth factor; CAM, chorioallantoic membrane; HUVE, human umbilical vein endothelial; scu-PA, single-chain uPA; u-ATF, amino-terminal fragment of uPA; UK1; kringle domain of uPA; uPA, urokinase plasminogen activator; uPAR, uPA receptor A bstractThe serine protease urokinase-type plasm inogen activator (uPA ) is im plicated in pericellular proteolysis in a variety of physiological and pathological processes including angiogenesis and tum or m etastasis. The kringle dom ain of uPA (UK1) has proven to be an anti-angiogenic m olecule with unknown m echanism and am ino term inal fragm ent of uPA (u-ATF) w ith additional grow th factor-like dom ain can be used for blocking interaction of uPA and uPA receptor. Here, w e com pared anti-angiogenic activities of these two m olecules in vitro and in vivo. The recom binant u-ATF from E. coli and refolded in vitro w as found to bind to uPA R w ith high affinity, w hereas E. coli-derived UK1 show ed no binding by Biacore analysis. In contrast to UK1 having potent inhibitory effect, u-ATF exhibited low inhibitory effect on bovine capillary endothelial cell grow th (ED 50 320 nM ). Furtherm ore, u-ATF inhibition of VEG F-induced m igration of hum an um bilical vein endothelial cell was far less sensitive (IC 50 = 600 nM) than those observed with UK1, and angiogenesis inhibition was marginal in chorioallantoic m em brane. These results suggest that kringle dom ain alone is sufficient for potent antiangiogenic activity and additional growth factor-like dom ain diverts this m olecule in undergoing different m echanism such as inhibition of uPA/uPAR interaction rather than undergoing distinct antiangiogenic m echanism driven by kringle dom ain.