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)
Colman
R W
RW
Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA 19140, USA. colmanr@astro.temple.edu
Jameson
B A
BA
Lin
Y
Y
Johnson
D
D
Mousa
S A
SA
D000482:Allantois / Q000098:blood supply; D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D002633:Chemotaxis / Q000187:drug effects*; D002642:Chick Embryo; D002823:Chorion / Q000098:blood supply; D004730:Endothelium, Vascular / Q000166:cytology* / Q000187:drug effects / Q000502:physiology*; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D006801:Humans; D019679:Kininogen, High-Molecular-Weight / Q000737:chemistry / Q000494:pharmacology*; D008840:Microfilament Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D018919:Neovascularization, Physiologic / Q000187:drug effects* / Q000502:physiology; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000494:pharmacology*; D010455:Peptides / Q000138:chemical synthesis / Q000494:pharmacology; D011487:Protein Conformation; D015800:Protozoan Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000494:pharmacology; D016415:Sequence Alignment; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D013499:Surface Properties; D014471:Umbilical Veins
D002352:Carrier Proteins; D019679:Kininogen, High-Molecular-Weight; D008840:Microfilament Proteins; D010446:Peptide Fragments; D010455:Peptides; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D016222:Fibroblast Growth Factor 2; C058561:hisactophilin protein, Protozoan