Fibrinogen-activating enzymes, widely distributed in Crotalidae and Viperidae venoms, are single-chain glycosylated serine proteases that display high macromolecular selectivity and are often referred to as thrombin-like enzymes (TLEs). TLEs serve as structural models to extend our understanding of the structure–function relationships of blood coagulation factors, have been clinically used for the treatment of thrombotic diseases, and are used as tools in clinical assays. The combination of gel filtration and ion-exchange chromatography proved to be successful in obtaining milligram quantities of pure samples of TLEs from the venoms of Crotalus durissus terrificus (white venom) and Crotalus durissus collilineatus (yellow venom). Functional characterization indicates that both enzymes preferentially degrade the Bβ chain of bovine fibrinogen and possess edema-inducing and coagulant activities. However, the TLE from C. d. collilineatus venom shows twofold higher coagulant activity with a minimum coagulant dose (MCD) of 0.6 µg/μl, whereas the enzyme isolated from C. d. terrificus indicated an MCD of 1.5 µg/μl. Molecular modeling of gyroxin and structural comparisons with other highly conserved snake venom serine proteases, underlines the key role played by the surface charge distribution and the double insertion in the 174-surface loop in macromolecular substrate recognition by TLEs.