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 specifically on endothelial cells to domain 2/3 of the urokinase receptor (uPAR). Inhibition by vitronectin suggests that kallikrein-cleaved HK (HKa) is antiadhesive. Plasma kallikrein bound to HK cleaves prourokinase to urokinase, initiating cell-associated fibrinolysis. We postulated that HK cell binding domains would inhibit angiogenesis. We found that recombinant domain 5 (D5) inhibited endothelial cell migration toward vitronectin 85% at 0.27 microM with an IC(50) (concentration to yield 50% inhibition) = 0.12 microM. A D5 peptide, G486-K502, showed an IC(50) = 0.2 microM, but a 25-mer peptide from a D3 cell binding domain only inhibited migration 10% at 139 microM (IC(50) > 50 microM). D6 exhibited weaker inhibitory activity (IC(50) = 0.50 microM). D5 also potently inhibited endothelial cell proliferation with an IC(50) = 30 nM, while D3 and D6 were inactive. Using deletion mutants of D5, we localized the smallest 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 peptides displaying surface loops. Peptide 440-455 was the most potent (IC(50) = 100 nM) in inhibiting proliferation but did not inhibit migration. D5 inhibited angiogenesis stimulated by fibroblast growth factor FGF2 (97%) in a chicken chorioallantoic membrane assay at 270 nM, and peptide 400-455 was also inhibitory (79%). HK D5 (for which we suggest the designation, "kininostatin") is a potent inhibitor of endothelial cell migration and proliferation in vitro and of angiogenesis in vivo. (Blood. 2000;95:543-550)[1]References
- Domain 5 of high molecular weight kininogen (kininostatin) down-regulates endothelial cell proliferation and migration and inhibits angiogenesis. Colman, R.W., Jameson, B.A., Lin, Y., Johnson, D., Mousa, S.A. Blood (2000) [Pubmed]
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