Antibody-directed targeting of angiostatin's receptor annexin II inhibits Lewis Lung Carcinoma tumor growth via blocking of plasminogen activation: possible biochemical mechanism of angiostatin's action.

Angiostatin, the N-terminal four kringles (K1-4) of parent molecule plasminogen, is reported to block Lewis Lung Carcinoma (LLC) tumor growth and metastasis. However, angiostatin's mechanism of action is unclear. We earlier reported that angiostatin binds to cell surface annexin II through the lysine-binding domain (kringles 1-4) [Tuszynski, G.P., Sharma, M., Rothman, V.L., Sharma, M.C., 2002. Angiostatin binds to tyrosine kinase substrate annexin II through the lysine-binding domain in endothelial cells. Microvasc. Res. 64:448-462.]). We now show that annexin II on the cell surface of LLC cells regulates conversion of plasminogen to plasmin. Activation of plasminogen to plasmin is time-dependent, with the linear activation lasting up to 120 min. Monoclonal antibodies to annexin II reduced plasminogen activation by 92.6%, suggesting a specific role of annexin II in plasmin generation. Angiostatin also reduced plasmin generation by 81.6%, suggesting that angiostatin may be competing with plasminogen through lysine-binding domain. epsilon-Aminocaproic acid, a lysine analogue, effectively blocked plasminogen activation indicating that, indeed, the lysine-binding site of the kringles domain is required for activation. These data suggest that annexin II may be a receptor target for angiostatin's action. Therefore, we tested the effect of high affinity monoclonal antibody to annexin II in mouse model of LLC. A single dose of antibody treatment inhibited LLC tumor growth almost 70% with concomitant inhibition of circulating plasmin generation and its proteolytic activity. Taken together, it is possible that inhibition of LLC tumor growth and metastasis reported by angiostatin therapy may be due to blocking of annexin-II-dependent plasmin generation. Plasmin is known to influence angiogenic, invasive and metastatic capability of tumors.