Differential Binding of Plasminogen, Plasmin, and Angiostatin4.5 to Cell Surface β-Actin: Implications for Cancer-Mediated Angiogenesis

Angiostatin4.5 (AS4.5) is the product of plasmin autoproteolysis and consists of kringles 1 to 4 and ∼85% of kringle 5. In culture, cancer cell surface globular β-actin mediates plasmin autoproteolysis to AS4.5. We now show that plasminogen binds to prostate cancer cells and that the binding colocalizes with surface β-actin, but AS4.5 does not bind to the cell surface. Plasminogen and plasmin bind to immobilized β-actin similarly, with a Kd of ∼140 nmol/L. The binding is inhibited by ε-aminocaproic acid (εACA), indicating the requirement for a lysine-kringle domain interaction. Using a series of peptides derived from β-actin in competitive binding studies, we show that the domain necessary for plasminogen binding is within amino acids 55 to 69 (GDEAQSKRGILTLKY). Substitution of Lys61 or Lys68 with arginine results in the loss of the ability of the peptide to block plasminogen binding, indicating that Lys61 and Lys68 are essential for plasminogen binding. Other actin peptides, including peptides with lysine, did not inhibit the plasminogen-actin interaction. AS4.5 did not bind actin at concentrations up to 40 μmol/L. Plasminogen, plasmin, and AS4.5 all contain kringles 1 to 4; however, kringle 5 is truncated in AS4.5. Isolated kringle 5 binds to actin, suggesting intact kringle 5 is necessary for plasminogen and plasmin to bind to cell surface β-actin, and the truncated kringle 5 in AS4.5 results in its release from β-actin. These data may explain the mechanism by which AS4.5 is formed locally on cancer cell surfaces and yet acts on distant sites. (Cancer Res 2006; 66(14): 7211-5)