Domain 5 of high molecular weight kininogen (kininostatin) down-regulates endothelial cell proliferation and migration and inhibits angiogenesis

We have demonstrated that high molecular weight kininogen (HK) binds specif ically on endothelial cells to domain 2/3 of the urokinase receptor (uPAR). Inhibition by vitronectin suggests that kallikrein-cleaved HK (HKa) is ant iadhesive. Plasma kallikrein bound to HK cleaves prourokinase to urokinase, initiating cell-associated fibrinolysis. We postulated that HK cell bindin g,domains would inhibit angiogenesis. We found that recombinant domain 5 (D 5) inhibited endothelial cell migration toward vitronectin 85% at 0.27 mu M with an IC50 (concentration to yield 50% inhibition) = 0.12 mu M A D5 pept ide, G486-K502, showed an IC50 = 0.2 mu M, but a 25-mer peptide from a D3 c ell binding domain only inhibited migration 10% at 139 mu M (IC50 > 50 mu M ). D6 exhibited weaker inhibitory activity (IC50 = 0.50 mu M) D5 also poten tly inhibited endothelial cell proliferation with an IC50 = 30 nM, while D3 and D6 were inactive, Using deletion mutants of D5, we localized the small est region for full activity to H441-D474. To further map the active region , we created a molecular homology model of D5 and designed a series of pept ides displaying surface loops. Peptide 440-155 was the most potent (IC50 = 100 nM) in inhibiting proliferation but did not inhibit migration. D5 inhib ited angiogenesis stimulated by fibroblast growth factor FGF2 (97%) in a ch icken chorioallantoic membrane assay at 270 nM, and peptide 400-455 was als o inhibitory (79%), HK D5 (for which we suggest the designation, "kininosta tin") is a potent inhibitor of endothelial cell migration and proliferation in vitro and of angiogenesis in vivo. (Blood, 2000;95:543-550) (C) 2000 by The American Society of Hematology.