The inhibition of tube formation in a collagen–fibrinogen, three-dimensional gel by cleaved kininogen (HKa) and HK domain 5 (D5) is dependent on Src family kinases

Cleaved high molecular weight kininogen (HKa), as well as its domain 5 (D5), inhibits migration and proliferation induced by angiogenic factors and induces apoptosis in vitro. To study its effect on tube formation we utilized a collagen–fibrinogen, three-dimensional gel, an in vitro model of angiogenesis. HKa, GST–D5 and D5 had a similar inhibitory effect of tube length by 90 ± 4.5%, 86 ± 5.5% and 77 ± 12.9%, respectively. D5-derived synthetic peptides: G440–H455 H475–H485 and G486–K502 inhibited tube length by 51 ± 3.7%, 54 ± 3.8% and 77 ± 1.7%, respectively. By a comparison of its inhibitory potency and its sequences, a functional sequence of HKa was defined to G486–G496. PP2, a Src family kinase inhibitor, prevented tube formation in a dose-dependent manner (100–400 nM), but PP3 at 5 μM, an inactive analogue of PP2, did not. HKa and D5 inhibited Src 416 phosphorylation by 62 ± 12.3% and 83 ± 6.1%, respectively. The C-terminal Src kinase (Csk) inhibits Src kinase activity. Using a siRNA to Csk, expression of Csk was down-regulated by 86 ± 7.0%, which significantly increased tube length by 27 ± 5.8%. The addition of HKa and D5 completely blocked this effect. We further showed that HKa inhibited Src family kinase activity by disrupting the complex of uPAR, αvβ3 integrin and Src. Our results indicate that the anti-angiogenic effect of HKa and D5 is mediated at least in part through Src family kinases and identify a potential novel target for therapeutic inhibition of neovascularization in cancer and inflammatory arthritis.