) High molecular weight kininogen (HK) is an abundant plasma glycoprotein that plays a central role in contact activation. HK is cleaved to two-chain high molecular weight kininogen (HKa) on the endothelial cell surface. Cleavage of HK to HKa leads to the release of bradykinin, and is accompanied by a dramatic structural arrangement. Although binding of bradykinin to endothelial cells leads to well-defined responses, functional consequences that result from binding of HKa have not been reported. We have observed that HKa, but not HK, inhibits endothelial proliferation and induces endothelial apoptosis in a Zn2+-dependent manner. Proliferation is inhibited by 50% at an HKa concentration of 10 nM. In contrast, HKa does not inhibit the proliferation of aortic smooth muscle or trophoblast cells, or tumor-derived cell lines. In mice, HKa inhibits the neovascularization of basic FGF-containing Matrigel plugs. Modeling of HKa domain 5 (D5), which also induces endothelial cell apoptosis in a Zn2+-dependent manner, reveals that the structure of the heparin and zinc-binding pharmacophores within this domain closely resemble those within the antiangiogenic polypeptide, endostatin. We have also identified peptides from the endothelial cell binding regions of HKa domains 3 and 5 that inhibit endothelial cell proliferation at low micromolar to nanomolar concentrations; D5, but not D3-derived peptide exhibit Zn2+ dependence. Further insight into the mechanism of HKa is provided by the observations that HKa causes apoptosis of only subconfluent, proliferative endothelial cells, and that culture of endothelial cells on collagen I, IV or a smooth muscle cell-derived matrix protects against HKa-induced apoptosis. In this application, we propose to further define the mechanism of Hka-induced endothelial cell apoptosis, and to use site-directed mutagenesis to determine whether the Zn2+-binding pharmacophore(s) in domain 5 are important in antiangiogenic activity. We will also characterize the in viva antiangiogenic activity of HKa domains 3 and 5, as well as peptides derived from them. Finally, we propose to identify the endothelial cell receptor that mediate the antiangiogenic effects of Hka. These studies should provide new information concerning the physiologic regulation of angiogenesis, and may lead to identification of new antiangiogenic agents.
