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

Abstract

It is not known whether the mammalian mechanism of coagulation initiation is 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 Western blot analysis and immunohistochemistry. Immunodepletion of factor VII from zebrafish plasma selectively inhibited thromboplastin-triggered thrombin generation. Heterologous expression of zebrafish factor VII demonstrated a secreted protein (50 kDa) that reconstituted thromboplastin-triggered thrombin generation in immunodepleted zebrafish plasma. These results suggest conservation of the extrinsic coagulation pathway between zebrafish and humans 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 VIIa. However, amino acid disparities were found in the epidermal growth factor-2͞serine protease regions that are present in the human tissue factor-factor VIIa contact surface, suggesting a structural basis for the species specificity of this interaction. In addition, zebrafish factor VII demonstrates 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 significantly narrows the evolutionary window for development of the vertebrate coagulation cascade and provides insight into the structural basis for species specificity in the tissue factor-factor VIIa interaction.

The evolution of vertebrate blood coagulation as viewed from a comparison of puffer fish and sea squirt genomes

“…As such, the events involving the thrombin-catalyzed conversion of fibrinogen to fibrin and its subsequent cross-linking and lysis were not an issue. The exact pathways of thrombin generation were still unclear, however, and it remained uncertain whether all of the vitamin K-dependent factors are present in lower vertebrates, although it should be noted that factor VII, as well as prothrombin, has been identified in zebrafish (34,35).…”

Zebrafish: A new model on the pharmaceutical catwalk

“…Blood from aspirinexposed adult zebrafish demonstrated inhibition of arachidonic acid-induced aggregation and agonist-induced ATP release, consistent with a partial dependence on the cyclooxygenase pathway. (9) Since zebrafish hemostatic proteins, like their mammalian orthologues, depend on vitamin K-dependent g-carboxylation, warfarin, an inhibitor of this process, also had strong anti-thrombotic effects. Warfarin, added to the tank water, induced spontaneous visible bleeding in adult zebrafish and prolongation of modified prothrombin and partial thromboplastin times, which are common diagnostic markers for coagulation disorders.…”

Mechanism of blood coagulation in common carp (Cyprinus carpio)

“…We arrived at this conclusion on the basis of experimental results whereby the coagulation times for carp MgSO 4 plasma were longer when Ca 2+ + H 2 O rather than just Ca 2+ was added to the samples (Table 1). A possible explanation for this phenomenon could be that the extrinsic clotting pathways of carp blood are activated by materials promoting clotting that are released from hemolyzed red blood cells, and that the pathway whereby prothrombin is activated by the thrombokinase released from broken thrombocytes is enhanced by water (Sheehan et al 2001).…”

“…However, for fish, the initial process and the complete pathways of blood coagulation have not been well characterized (Jagadeeswaran and Sheehan 1999;Sheehan et al 2001).…”