Summary
We evaluated the in vivo anti-metastatic activity of recombinant Ancylostoma caninum Anticoagulant Peptide (rAcAP), a potent (Ki
= 265 pM) and specific active site inhibitor of human coagulation factor Xa originally
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 rAcAP’s anti-metastatic activity, experiments were carried out to identify
specific interactions between factor Xa and LOX. Binding of biotinylated factor Xa
to LOX monolayers was both specific and saturable (Kd
= 15 nM). Competition experiments using antibodies to previously identified factor
Xa binding proteins, including factor V/Va, effector cell protease receptor-1, and
tissue factor pathway inhibitor failed to implicate any of these molecules as significant
binding sites for Factor Xa. Functional prothrombinase activity was also supported
by LOX, with a half maximal rate of thrombin generation detected at a factor Xa concentration
of 2.4 nM. Additional competition experiments using an excess of either rAcAP or active
site blocked factor Xa (EGR-Xa) revealed that most of the total factor Xa binding
to LOX is mediated via interaction with the enzyme’s active site, predicting that
the vast majority of cell-associated factor Xa does not participate directly in thrombin
generation. In addition to establishing two distinct mechanisms of factor Xa binding
to melanoma, these data raise the possibility that rAcAP’s antimetastatic effect in
vivo might involve novel non-coagulant pathways, perhaps via inhibition of active-site
mediated interactions between factor Xa and tumor cells.
