Isolation of an angiostatin receptor from the membranes of human umbilical vein endothehal cells

Angiostatin, a kringle-containing fragment of plasminogen, is a potent antagonist of angiogenesis, resulting from its ability to inhibit endothelial cell migration and proliferation. To test the hypothesis that alteration of the biologic properties of endothelial cells requires interaction of angiostatin with the vascular cell surface, binding of angiostatin to human umbilical vein endothelial cells (HUVEC) was assessed. Binding of radiolabelted angiostatin to HUVEC was concentration dependent and saturable, with a Kd of approximately 250 nM and 4 x 104 receptors/cell. In control experiments, plasminogen bound HUVEC with similar affinity (160 nM), but with 9 x Vf receptors/cell. As it is unknown whether the mechanism by which angiostatin inhibits endothelial cell migration and/or proliferation involves an interaction with the plasminogen receptor, competition binding experiments were performed. These experiments demonstrated that plasminogen binding to HUVEC was not inhibited by up to a 100-fold excess of angiostatin, suggesting the possibility of distinct binding sites for angiostatin and plasminogen on endothelial cells. Intact HUVEC were surface biotin-labelled and plasma membrane extracts were subjected to affinity chromatography on plasminogen- or angiostatin-Sepharose. A 44 kDa protein was eluted from plasminogen-Sepharose. This protein cross-reacted on Western blots with an antibody directed against annexin u, a known plasminogen binding protein. In contrast, a 55 kDa protein was eluted from angiostatin-Sepharose which did not cross-react with annexin E antibody. Amino terminal sequence analysis is currently underway to identify this angiostatin-binding cell membrane component. Together these data suggest that binding of angiostatin and plasminogen to distinct sites on endothelial cell membranes may result in differential regulation of biologic properties including cellular migration and proliferation.

Duke Scholars

Author Salvatore Vincent Pizzo Pathology