Snake venoms are a complex combination of neurochemical and blood hemostatic effector molecules including pro- and anti-coagulant proteins.  The Australian elapid family of venomous snakes contain prothrombin activators that strongly resemble coagulation factor Xa and can be divided into several groups based on their requirement for cofactors.  Group D prothrombin activators require calcium and factor Va from prey for maximal activity. We have expressed and characterized a recombinant Factor Xa ortholog from the Stephen’s Banded Snake (Hoplocephalus Stephensii), hopsarin.  Recombinant hopsarin binds to both human factor V and Va with a K_D of 15nM in the absence of membranes.  In the presence of human factor Va and phospholipids, hopsarin cleaves human prothrombin at a rate comparable to human factor Xa.  In the absence of membranes, hopsarin complexed with factor Va retains the ability efficiently cleave prothrombin.  In contrast to factor Xa, hopsarin shows specificity for the Arg320 cleavage site of prothrombin, generating meizothrombin, followed by cleavage at Arg271 to release fully activated thrombin.  These data suggest a new potential mechanism for the activation of prothrombin by the prothrombinase complex.