KRINGLE DOMAINS OF HUMAN ANGIOSTATIN - CHARACTERIZATION OF THE ANTIPROLIFERATIVE ACTIVITY ON ENDOTHELIAL-CELLS

Recently we have identified angiostatin, an endogenous angiogenesis in hibitor of 38 kDa which specifically blocks the growth of endothelial cells (O'Reilly, M. S., Holmgren, L., Shing, Y., Chen, C., Rosenthal, R. A., Moses, M., Lane, W. S., Cao, Y., Sage, E. H., and Folkman, J. ( 1994) Cell 79, 315-328; Folkman, J. (1995) Nat. Med. 1, 27-31). Angios tatin was shown to represent an internal fragment of plasminogen conta ining the first four kringle structures. We now report on the inhibito ry effects of individual or combined kringle structures of angiostatin on capillary endothelial cell proliferation. Recombinant kringle 1 an d kringle 3 exhibit potent inhibitory activity with half-maximal conce ntrations (ED(50)) of 320 nM and 460 nM, respectively. Also, recombina nt kringle 2 displays a significant inhibition, although decreased com pared with both kringle 1 and kringle 3. In contrast, kringle 4 is an ineffective inhibitor of basic fibroblast growth factor-stimulated end othelial cell proliferation. Among the tandem kringle arrays, the reco mbinant kringle 2-3 fragment exerts inhibitory activity similar to kri ngle 2 alone. However, relative to kringle 2-3, a marked enhancement i n inhibition is observed when individual kringle 2 and kringle 3 are a dded together to endothelial cells. This implies that it is necessary to open the cystine bridge between kringle 2 and kringle 3 to obtain t he maximal inhibitory effect of kringle 2-3. An increased (<2-fold) in hibitory activity is observed for the kringle 1-3 fragment (ED(50) = 7 0 nM) compared with kringle 1-4 (ED(50) = 135 nn). These data indicate that the anti-proliferative activity of angiostatin on endothelial ce lls is shared by kringle 1, kringle 2, and kringle 3, but probably not by kringle 4 and that more potent inhibition results when kringle 4 i s removed from angiostatin. Thus, in view of the variable lysine affin ity of the homologous domains, it would appear that lysine binding cap ability does not correlate with the relative inhibitory effects of the kringle-containing constructs. However, as we also demonstrate, appro priate folding of kringle structures is essential for angiostatin to m aintain its full anti-endothelial activity.