Ancylostoma caninum Anticoagulant Peptide: A novel inhibitor of coagulation factor Xa with potent antimetastatic activity. • 650

Although complications related to activation of the coagulation cascade are common in patients with cancer, the role of individual clotting factors in tumorigenesis and metastasis remains unknown. We have identified a novel anticoagulant with potent antimetastatic activity. This molecule, named Ancylostoma caninum Anticoagulant Peptide (AcAP), is a specific inhibitor of coagulation factor Xa (K_i = 265 pM) and has been previously isolated from bloodfeeding hookworms. Subcutaneous injection of SCID mice with recombinant AcAP (rAcAP) (0.01-0.2 mg/mouse) prior to tail vein injection of 10⁵ highly metastatic LOX human melanoma cells resulted in a dose dependent reduction in pulmonary macro-metastases. In order to investigate the mechanism of this protection, as well as to further understand the role of coagulation proteases in tumor cell biology, LOX cells were assayed for the ability to bind factor Xa in vitro. Using biotinylated factor Xa, we found that the protease binds to LOX monolayers in a specific and saturable fashion, with a K_D of 15 nM. Increasing concentrations of rAcAP maximally inhibited labelled factor Xa binding by approximately 90%. The LOX cells also supported formation of the clot-forming prothrombinase complex, whose catalytic activity was effectively blocked in the presence of equimolar concentrations of rAcAP. Competition binding studies revealed that an excess of active site blocked factor Xa (EGR-Xa) successfully inhibited prothrombinase activity by 98%, while causing only a minimal reduction in total biotinylated Xa binding. These data suggest that the majority of factor Xa (>80%) binds to the surface of LOX cells at its active-site, rendering it unavailable for prothrombinase complex formation and thrombin generation. Therefore, in addition to its well recognized function in clot formation, factor Xa may play a role in promoting LOX metastasis via previously uncharacterized active site mediated interactions with the tumor cell membrane. rAcAP, by inhibiting both the binding of factor Xa as well as its catalytic activity, represents a potentially valuable therapeutic agent for the prevention of tumor metastasis.