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  Topic: Blood-clotting, evolution, and Behe, References, links, and material on above< Next Oldest | Next Newest >  

Posts: 319
Joined: May 2002

(Permalink) Posted: Dec. 31 2002,17:37   

Please give us your informed opinion when you get a chance to read it.  A freely online 2001 PNAS article (from related articles) provides something of a preview:


Sheehan J, Templer M, Gregory M, Hanumanthaiah R, Troyer D, Phan T, Thankavel B, Jagadeeswaran P.

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

Proc Natl Acad Sci U S A 2001 Jul 17;98(15):8768-73

Department of Cellular and Structural Biology, South Texas Veteran's Health Care System, Audie Murphy Division, University of Texas Health Science Center, San Antonio, TX 78229, USA.

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-2/serine 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.


This investigation demonstrates the presence of coagulation factor VII in the zebrafish, providing molecular evidence for an extrinsic coagulation pathway in a nonmammalian species. Similarities between zebrafish and human primary protein sequence, domain organization (Gla-EGF-EGF-SP), and postulated three-dimensional structure suggest that this cDNA is orthologous to human factor VII. These sequence similarities include conservation of a single activation cleavage site, the cysteines participating in disulfide bonds, and residues that contribute to the formation of specific binding sites for sodium and calcium ions. The presence of the predicted protein was demonstrated in zebrafish plasma and liver with specific rabbit antisera vs. a unique zebrafish factor VII peptide. Immunodepletion of zebrafish plasma with the antipeptide antisera selectively inhibited thromboplastin-triggered thrombin generation. Finally, the zebrafish cDNA directed the expression of a secreted mature protein that was similar in size to human factor VII, which reconstituted thromboplastin-triggered thrombin generation in immunodepleted plasma. These structural and functional correlates reflect the conservation of factor VII gene function between zebrafish and man.

The coagulation serine proteases arose from the family of trypsin-like genes, characterized by protease domains that use the AGY codon for the catalytic serine {195}, and contain a specific sodium ion-binding motif, Tyr/Phe {225} (23, 27). The organization of the N terminus propeptide is unique to these proteases, composed of a 污-carboxylation (Gla) domain followed by two EGF or kringle domains. Although Gla, EGF, and SP modules exist in invertebrates such as Drosophila melanogaster, there is no evidence for this unique domain organization (28). Likewise, serine proteases responsible for hemolymph coagulation in Tachypleus tridentatus (Japanese horseshoe crab) and other invertebrates species differ significantly in N terminus domain content and organization from mammalian coagulation proteases (29每31). Evidence for prothrombin (which contains kringle domains) exists in the primitive jawless hagfish (Myxinidae) (11). However, zebrafish factor VII now provides the earliest known evolutionary appearance of the Gla-EGF-EGF-SP domain structure common to factors VII, IX, and X and protein C. Demonstration of both zebrafish prothrombin (18) and factor VII indicates that the relevant domain assembly for the coagulation proteases had occurred (presumably by exon shuffling) (32) at or before the time of the last common ancestor of humans and zebrafish, >450 million years ago (33).

Significant species specificity exists in the tissue factor每factor VIIa interaction between mammalian species and appears relatively complete between zebrafish and man (15). The human soluble tissue factor每factor VIIa structure demonstrates three major intermolecular contacts involving the Gla, EGF-1, and EGF-2/SP domains, respectively (24). Inspection of homologous surfaces on the zebrafish factor VIIa model suggests that intermolecular interactions are conserved largely for the Gla and EGF-1 contact regions. In contrast, surface residues in the EGF-2/SP contact region are poorly conserved, suggesting that the intermolecular interactions with human tissue factor are largely disrupted. This disruption includes the protease insertion loop Leu-371每Glu-385 {170每178}, which appears to undergo a conformational change between the bound and unbound structures of human factor VIIa (25). In the zebrafish protein, this loop demonstrates a 5-aa deletion and replacement of a neighboring conserved Met with Arg-348 {164}. These differences in the EGF-2/SP intermolecular contact region between zebrafish and man suggest a structural basis for the observed species specificity in the tissue factor每factor VIIa interaction. Hybrid recombinant human/zebrafish factor VII proteins in which the EGF-2/SP intermolecular contact regions are exchanged could directly test their contribution to the species specificity of this interaction.

Identification of zebrafish factor VII has important implications for the relevance of this powerful genetic model to the study of hemostasis and thrombosis. The ability to trigger (and selectively inhibit) tissue thromboplastin-dependent coagulation in a species-specific manner provides indirect evidence for tissue factor in the zebrafish. Functional data also suggest the presence of a contact-activated coagulation pathway that is independent of factor VII and factor X-like activity in the zebrafish (15). Factor X-like activity (based on RVV-X activator) is not affected by immunodepletion of factor VII, demonstrating that these activities are distinct. The degree of similarity between zebrafish and mammalian coagulation suggests that the zebrafish is a relevant animal model for the study of genes that affect hemostasis. Phenotypic screens of mutagenized zebrafish may identify novel genes that regulate the initiation of coagulation.

In conclusion, the structural features of factor VII in the zebrafish suggest that domain assembly for the coagulation proteases occurred before radiation of the ancestral Actinopterygii (ray-finned fishes) and Sarcopterygii (lungfish and tetrapods). The gene structure (intron/exon boundaries) of coagulation factors VII, IX, and X and protein C indicates that these genes are paralagous, suggesting a common origin via gene duplication (34). Thus, it appears likely that the vertebrate coagulation cascade arose rapidly during proposed genome duplications between ancestral chordates and the development of jawed vertebrates (35每37). Given the functional and structural similarities to mammalian coagulation demonstrated thus far, the zebrafish should be a powerful model to identify novel genes involved in vertebrate coagulation.

Doolittle and Patthy are referenced, unfortunately little of Doolittle's and none of Patthy's blood-clotting stuff is in widely available online journals...they all seem to be down at UCSD however.

  24 replies since May 17 2002,22:00 < Next Oldest | Next Newest >  


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