Purification of Procoagulant Proteins

Hoplocephalus stephensi, an Australian elapid, produces a strongly procoagulant venom. A prothrombin activator, hopsarin D (MW = 46,102 Da), has been recently characterized. This study attempts to isolate and characterize other, smaller clotting factors present in the venom. Gel filtration of the crude venom produced six fractions, two of which (4GF and 5 GF) were subjected to either reverse-phase high pressure liquid chromatography (RP-HPLC)/cation exchange chromatography. The effect of the fractions 4GF and 5GF on the blood coagulation pathway, its homogeneity and composition were analyzed. The data obtained suggest that 4GF contains at least two proteins: a 13.2kDa and a 6.7kDa protein/s, and that 5GF contains a 13.0kDa and a 2.3kDa protein. Both the ~13kDa proteins show anticoagulant effects by itself. The smaller proteins are hemostatically inactive. In both, the smaller proteins convert the anticoagulant activity of the larger ones through an unknown mechanism, resulting in a procoagulant species. The interaction between the two proteins is most probably non-covalent. Sequencing the pure samples would greatly aid further analysis.