Demonstration of the extrinsic coagulation pathway in teleostei: Identification of zebrafish coagulation factor VII

It is not known whether the mammalian mechanism of coagulation initiation i s conserved in fish. Identification of factor VII is critical in providing evidence for such a mechanism. A cDNA was cloned from a zebrafish (teleost) library that predicted a protein with sequence similarity to human factor VII. Factor VII was shown to be present in zebrafish blood and liver by Wes tern blot analysis and immunohistochemistry. Immunodepletion of factor VII from zebrafish plasma selectively inhibited thromboplastin-triggered thromb in generation. Heterologous expression of zebrafish factor VII demonstrated a secreted protein (50 kDa) that reconstituted thromboplastin-triggered th rombin generation in immunodepleted zebrafish plasma. These results suggest conservation of the extrinsic coagulation pathway between zebrafish and hu mans and add credence to the zebrafish as a model for mammalian hemostasis. The structure of zebrafish factor VIIa predicted by homology modeling was consistent with the overall three-dimensional structure of human factor VII a. However, amino acid disparities were found in the epidermal growth facto r-2/serine protease regions that are present in the human tissue factor-fac tor VIIa contact surface, suggesting a structural basis for the species spe cificity of this interaction. In addition, zebrafish factor VII demonstrate s that the Gla-EGF-EGF-SP domain structure, which is common to coagulation factors VII, IX, X, and protein C, was present before the radiation of the teleosts from the tetrapods. Identification of zebrafish factor VII signifi cantly narrows the evolutionary window for development of the vertebrate co agulation cascade and provides insight into the structural basis for specie s specificity in the tissue factor-factor VIIa interaction.