Step-by-step evolution of vertebrate blood coagulation.

The availability of whole-genome sequences for a variety of vertebrates is making it possible to reconstruct the step-by-step evolution of complex phenomena such as blood coagulation, an event that in mammals involves the interplay of more than two dozen genetically encoded factors. Gene inventories for different organisms are revealing when during vertebrate evolution certain factors first made their appearance in or, on occasion, disappeared from some lineages. The whole-genome sequence databases of two protochordates and seven nonmammalian vertebrates were examined in search of approximately 20 genes known to be associated with blood clotting in mammals. No genuine orthologs were found in the protochordate genomes (sea squirt and amphioxus). As for vertebrates, although the jawless fish have genes for generating the thrombin-catalyzed conversion of fibrinogen to fibrin, they lack several clotting factors, including two thought to be essential for the activation of thrombin in mammals. Fish in general lack genes for the "contact factor" proteases, the predecessor forms of which make their first appearance in tetrapods. The full complement of factors known to be operating in humans does not occur until pouched marsupials (opossum), at least one key factor still absent in egg-laying mammals such as platypus.

Evolution of the contact phase of vertebrate blood coagulation.

BACKGROUND: Previous reports have noted that factor (F) XI and FXII and prekallikrein (the contact phase proteases) are absent in fish. OBJECTIVES: A broad survey of recently completed genomes was undertaken to find where during the course of vertebrate evolution these coagulation factors appeared. METHODS: BLAST searches were conducted for the various factors on genomes of lamprey, puffer fish, zebra fish, frog, chicken, platypus, and opossum. RESULTS: It was confirmed that FXII is absent from fish; it is present in frog, platypus, and opossum, but is absent in chicken, an apparent example of gene loss. A single gene corresponding to the evolutionary predecessor of FXI and prekallikrein occurs in frog, chicken, and platypus. The opossum (a marsupial) has both prekallikrein and FXI, completing the full complement of these genes that occurs in eutherian mammals. CONCLUSIONS: The step-by-step accrual of genes for these factors by a series of timely gene duplications has been confirmed by phylogenetic analysis and other considerations.

Determining the crystal structure of fibrinogen.

Summary. I have enjoyed reading previous historical sketches that have appeared in Journal of Thrombosis and Haemostasis, and especially those by Ted Tuddenham on factor VIII and Bjorn Dahlback on activated protein C resistance. Like those authors, I have tried to capture some of the excitement-as well as the disappointments-that occurred along the way to a long-term goal.

X-ray crystallographic studies on fibrinogen and fibrin.

More than a dozen crystal structures of fibrinogen and fibrin fragments have been reported, including a structure of a native fibrinogen. The majority of the other structures are fragments D and d-dimer crystallized in the presence or absence of synthetic peptide ligands patterned on the A and B 'knobs'. Overall, fibrinogens or their fragments from four different species--human, bovine, chicken and lamprey--have been studied so far, with only minor differences in the structures being observed. Although these studies have thrown much light on the details of the fibrinogen to fibrin conversion, much remains to be found out.

Crystal structure of native chicken fibrinogen at 2.7 A resolution.

The crystal structure of native chicken fibrinogen (320 kDa) complexed with two synthetic peptides has been determined at a resolution of 2.7 A. The structure provides the first atomic-resolution view of the polypeptide chain arrangement in the central domain where the two halves of the molecule are joined, as well as of a putative thrombin-binding site. The amino-terminal segments of the alpha and beta chains, including fibrinopeptides A and B, are not visible in electron density maps, however, and must be highly disordered. The alphaC domain is also very disordered. A residue by residue analysis of the coiled coils with regard to temperature factor shows a strong correlation between mobility and plasmin attack sites. It is concluded that structural flexibility is an inherent feature of fibrinogen that plays a key role in both its conversion to fibrin and its subsequent destruction by plasmin.

Crystal structure studies on fibrinogen and fibrin.

X-ray crystallography studies on fragments D and double-D from human fibrinogen and fibrin have revealed the details of knob-hole interactions between fibrin units, as well as the nature of the association at their ends. More recently, a lower-resolution structure of native chicken fibrinogen has provided details about the structure of the central domain, and particularly the arrangement of disulfide bonds. Parts of the fibrinogen molecule are so flexible that they have not been visualized in electron density maps. The elusive regions include the alpha C domain, the amino-terminal segments of the alpha and beta chains, and the carboxyl-terminal segments of the gamma chains. Nonetheless, when all the structural data are considered together, it is possible to construct a realistic model not only of a fibrinogen molecule but also of a fibrin protofibril.

A model of fibrin formation based on crystal structures of fibrinogen and fibrin fragments complexed with synthetic peptides.

A blood clot is a meshwork of fibrin fibers built up by the systematic assembly of fibrinogen molecules proteolyzed by thrombin. Here, we describe a model of how the assembly process occurs. Five kinds of interaction are explicitly defined, including two different knob-hole interactions, an end-to-end association between gamma-chains, a lateral association between gamma-chains, and a hypothetical lateral interaction between beta-chains. The last two of these interactions are responsible for protofibril association and are predicated on intermolecular packing arrangements observed in crystal structures of fibrin double-D fragments cocrystallized with synthetic peptides corresponding to the knobs exposed by the release of the fibrinopeptides A and B.

Determining the relative rates of change for prokaryotic and eukaryotic proteins with anciently duplicated paralogs.

The relative rates of change for eight sets of ubiquitous proteins were determined by a test in which anciently duplicated paralogs are used to root the universal tree and distances are calculated between each taxonomic group and the last common ancestor. The sets included ATPase subunits, elongation factors, signal recognition particle and its receptor, three sets of tRNA synthetases, transcarbamoylases, and an internal duplication in carbamoyl phosphate synthase. In each case phylogenetic trees were constructed and the distances determined for all pairs. Taken over the period of time since their last common ancestor, average evolutionary rates are remarkably similar for Bacteria and Eukarya, but Archaea exhibit a significantly slower average rate.

Searching for the common ancestor.

In this brief mini-review I address the current controversy surrounding the nature of the last common ancestor of all life on Earth. The pros and cons of the various positions have been hotly debated of late with no sign of the tumult subsiding. As such, I could not possibly do justice to all the varied and opposing views, nor even cite them. Let me just say at the outset that my own views on the subject at hand have been greatly influenced by the writings of T. Cavalier-Smith, W.F. Doolittle, P. Gogarten, R. Gupta, H. Hartman, O. Kandler, E. Koonin, J. Lake, W. Martin, M. Sogin, and C. Woese, inter alia. I hope they will forgive me if my depiction of events does not do full justice to their contributions.

Crystal structure of native chicken fibrinogen at 5.5-A resolution.

The crystal structure of native chicken fibrinogen has been determined at a resolution of 5.5 A. The full-length molecule is 460 A in length and sigmoidally shaped. The structure includes the full sweep of the coiled coils that connect the central and terminal domains; the chain paths of the central domain confirm a predicted scheme of planar disulfide rings in apposition with each other. Electron density maps have revealed the outlines of disordered alphaC domains nestled within the confines of the sinuous coiled coils. The amino-terminal segments of the alpha- and beta-chains, including the fibrinopeptides A and B, are also disordered.

An attempt to pinpoint the phylogenetic introduction of glutaminyl-tRNA synthetase among bacteria.

Until recently it was believed that most Bacteria form Gln-tRNA(GLN) by the amidation of Glu-tRNA(GLN), only a few members of the gamma subdivision of Proteobacteria being able to charge tRNA(GLN) directly. We undertook a phylogenetic study in an attempt to determine at what point the changeover to the direct system may have occurred. To this end, we selected a number of representative Proteobacteria to see if we could find a division point. We constructed degenerate primers and conducted PCR analysis to identify which Bacteria had Gln-tRNA synthetase, on the one hand, and which had the amidotransferase system, on the other. At the same time, we surveyed data banks of completely sequenced microbial genomes, as well as those for genomes in the process of being sequenced. These combined efforts revealed four Proteobacteria in a phylogenetically intermediate position which have the genetic potential for both mechanisms. Perplexingly, however, three distantly related bacteria were also found to have both enzymes.

Conformational changes in fragments D and double-D from human fibrin(ogen) upon binding the peptide ligand Gly-His-Arg-Pro-amide.

The structure of fragment double-D from human fibrin has been solved in the presence and absence of the peptide ligands that simulate the two knobs exposed by the removal of fibrinopeptides A and B, respectively. All told, six crystal structures have been determined, three of which are reported here for the first time: namely, fragments D and double-D with the peptide GHRPam alone and double-D in the absence of any peptide ligand. Comparison of the structures has revealed a series of conformational changes that are brought about by the various knob-hole interactions. Of greatest interest is a moveable "flap" of two negatively charged amino acids (Glubeta397 and Aspbeta398) whose side chains are pinned back to the coiled coil with a calcium atom bridge until GHRPam occupies the beta-chain pocket. Additionally, in the absence of the peptide ligand GPRPam, GHRPam binds to the gamma-chain pocket, a new calcium-binding site being formed concomitantly.

Crystal structure of a recombinant alphaEC domain from human fibrinogen-420.

The crystal structure of a recombinant alphaEC domain from human fibrinogen-420 has been determined at a resolution of 2.1 A. The protein, which corresponds to the carboxyl domain of the alphaE chain, was expressed in and purified from Pichia pastoris cells. Felicitously, during crystallization an amino-terminal segment was removed, apparently by a contaminating protease, allowing the 201-residue remaining parent body to crystallize. An x-ray structure was determined by molecular replacement. The electron density was clearly defined, partly as a result of averaging made possible by there being eight molecules in the asymmetric unit related by noncrystallographic symmetry (P1 space group). Virtually all of an asparagine-linked sugar cluster is present. Comparison with structures of the beta- and gamma-chain carboxyl domains of human fibrinogen revealed that the binding cleft is essentially neutral and should not bind Gly-Pro-Arg or Gly-His-Arg peptides of the sort bound by those other domains. Nonetheless, the cleft is clearly evident, and the possibility of binding a carbohydrate ligand like sialic acid has been considered.