Vitamin K (VK)dependent proteases are major players in blood coagulation, including both the initiation and the regulation of the cascade. Five different members of this protease family have been described, comprising the following coagulation factors: factor VII, FIX, FX, protein C (PC), and prothrombin (FII). FVII, FIX, FX and PC share a typical domain architecture, with an Nterminal carboxyglutamate (Gla) domain, two epidermal growth factorlike (EGF) domains, and a Cterminal trypsinlike serine protease (SP) domain.
We have identified uncharacterized proteins in snake genomes showing the typical GlaEGF1EGF2SP domain architecture but relatively low sequence conservation compared to known VKdependent proteases. On the basis of sequence analysis, we hypothesized that these proteins are functional members of the VKdependent protease family.
Using phylogenetic analyses, we confirmed the socalled ‘sirtilins as an additional VKdependent protease class. These proteases were found in several vertebrates, including jawless fish, cartilaginous fish, bony fish, reptiles, birds, and marsupials, but not in other mammals. The recombinant zymogen form of Thamnophis sirtalis sirtilin was produced by in vitro renaturation, and was activated with human activated FXI. The activated form of sirtilin proteolytically cleaved peptide and protein substrates, including prothrombin. Mass spectrometrybased substrate profiling of sirtilin revealed a narrower sequence specificity than those of FIX and FX.
The ubiquitous occurrence of sirtilins in many vertebrate classes might indicate an important functional role. Understanding the detailed functions of sirtilins might contribute to a deeper understanding of the evolution and function of the vertebrate coagulation system.