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

Abstract

The blood coagulation scheme for the puffer fish, Fugu rubripes, has been reconstructed on the basis of orthologs of genes for mammalian blood clotting factors being present in its genome. As expected, clotting follows the same fundamental pattern as has been observed in other vertebrates, even though genes for some clotting factors found in mammals are absent and some others are present in more than one gene copy. All told, 26 different proteins involved in clotting or fibrinolysis were searched against the puffer fish genome. Of these, orthologs were found for 21. Genes for the "contact system" factors (factor XI, factor XII, and prekallikrein) could not be identified. On the other hand, two genes were found for factor IX and four for factor VII. It was evident that not all four factor VII genes are functional, essential active-site residues having been replaced in two of them. A search of the genome of a urochordate, the sea squirt, Ciona intestinalis, did not turn up any genuine orthologs for these 26 factors, although paralogs and/or constituent domains were evident for virtually all of them.

Fig. 1.

Diagrammatic depiction of genes for factor VII and factor X on FUGU scaffold 2859 (32,129 nt) from the puffer fish (F. rubripes) genome. The section denoted by ψF7 lacks the catalytic site characteristic of serine proteases and is likely not a functional entity. In F7A, the serine at the active site has been replaced by an aspartic acid.

Fig. 2.

Phylogenetic tree constructed from serine protease portions of the vitamin K-dependent proteases (prothrombin, protein C, factor VII, factor IX, and factor X) of human (HU), bovine (BO), and puffer fish (FU). Puffer fish entries are also marked with asterisks. The puffer fish entry denoted FUNFU (from scaffold 6546) does not fall into any of the usual groups.

Fig. 3.

Schematic comparison of human and puffer fish factors V and VIII. The regions denoted B are variable from species to species; numbers indicate residues in the B segment.

Fig. 4.

(A) Alignment of FN1 sequences from human t-PA and factor XII with two homologous sequences from puffer fish. No homologs were found in the sea squirt genome. (B) Alignment of FN2 sequences from human factor XII, puffer fish scaffold 1677, and sea squirt scaffold 883.

Fig. 5.

Putative evolution of nine proteases involved in generation and destruction of fibrin clots by a small number of domainal exchanges. The proposed root protein is a four-kringle protease, prototypical genes for which are found in the sea squirt genome. A three-kringle protease root could equally well have been used. G, GLA; E, EGF; K, kringle; P, PAN domain; F1, fibronectin domain 1; F2, fibronectin domain II; SP, serine protease. All of these domains are found in the sea squirt genome, but not associated in the arrangements found in clotting factors. Factor XII does not appear in the puffer fish genome and likely appeared after the divergence of fish and tetrapods.