ANCYLOSTOMA-CANINUM ANTICOAGULANT PEPTIDE BLOCKS METASTASIS IN-VIVO AND INHIBITS FACTOR XA BINDING TO MELANOMA-CELLS IN-VITRO

We evaluated the in vivo anti-metastatic activity of recombinant Ancyl ostoma caninum Anticoagulant Peptide (rAcAP), a potent (K-i = 265 pM) and specific active site inhibitor of human coagulation factor Xa orig inally isolated from bloodfeeding hookworms. Subcutaneous injection of SCID mice with rAcAP (0.01-0.2 mg/mouse) prior to tail vein injection of LOX human melanoma cells resulted in a dose dependent reduction in pulmonary metastases. In order to elucidate potential mechanisms of r AcAP's anti-metastatic activity, experiments were carried out to ident ify, specific interactions between factor Xa and LOX. Binding of bioti nylated factor Xa to LOX monolayers was both specific and saturable (K -d = 15 nM). Competition experiments using antibodies to previously id entified factor Xa binding proteins, including factor V/Va, effector c ell protease receptor-1, and tissue factor pathway inhibitor failed to implicate any of these molecules as significant binding sites for Fac tor Xa. Functional prothrombinase activity was also supported by LOX, with a half maximal rate of thrombin generation detected at a factor X a concentration of 2.4 nM. Additional competition experiments using an excess of either rAcAP or active site blocked factor Xa (EGR-Xa) reve aled that most of the total factor Xa binding to LOX is mediated via i nteraction with the enzyme's active site, predicting that the vast maj ority of cell-associated factor Xa does nor participate directly in th rombin generation. In addition to establishing two distinct mechanisms of factor Xa binding to melanoma, these data raise the possibility th at rAcAP's antimetastatic effect in vivo might involve novel non-coagu lant pathways, perhaps via inhibition of active-site mediated interact ions between factor Xa and tumor cells.