Geoffrey Kemball-Cook

The Factor VIII Structure and Mutation Resource Site: HAMSTeRS Version 4

Since 1996 the HAMSTeRS (Haemophilia A Mutation, Search, Test and Resource Site) WWW site has provided an online resource for access to data on the molecular pathology of haemophilia A, replacing previous text editions of the Haemophilia A Database published in Nucleic Acids Research . This report describes the continued development of the site (version 4), and in particular the expansion of factor VIII (FVIII) structure-related features. Access to the mutation database itself, both for searching the listings and for submission of new mutations, is via custom-designed forms: more powerful Boolean searches of the point mutations in the database are also available. During 1997 a total of 22 novel missense mutations were reported, increasing the total number of unique variants now described to 252 (238 in exonic sequences and 14 at intronic splice junctions). Currently, a total of 586 individual reports with associated phenotypic data are available for searching by any category including phenotype. The FVIII structure section now includes a download of a FVIII A domain homology model in Protein Data Bank format and a multiple alignment of the FVIII amino-acid sequencies from four species (human, murine, porcine and canine) in addition to the virtual reality simulations, secondary structural data and FVIII animation already available. Finally, to aid navigation across this site, a clickable roadmap of the main features provides easy access to the page desired. Our intention is that continued development and updating of the site shall provide workers in the fields of molecular and structural biology with a one-stop resource site to facilitate FVIII research and education. The HAMSTeRS URL is http://europium.mrc.rpms.ac.uk

The Factor VIII Mutation Database on the World Wide Web: The Haemophilia A Mutation, Search, Test and Resource Site HAMSTeRS Update (version 3.0)

The HAMSTeRS WWW site was set up in 1996 in order to facilitate easy access to, and aid understanding of, the causes of haemophilia A at the molecular level; previously, the first and second text editions of the database have been published in Nucleic Acids Research. This report describes the facilities originally available at the site and the recent additions which we have made to increase its usefulness to clinicians, the molecular genetics community and structural biologists interested in factor VIII. The database (version 3.0) has been completely updated with easy submission of point mutations, deletions and insertions via e-mail of custom-designed forms. The searching of point mutations in the database has been made simpler and more robust, with a concomitantly expanded real-time bioinformatic analysis of the database. A methods section devoted to mutation detection has been added, highlighting issues such as choice of technique and PCR primer sequences. Finally, a FVIII structure section gives access to 3D VRML (Virtual Reality Modelling Language) files for any user-definable residue in a FVIII A domain homology model based on the crystal structure of human caeruloplasmin, together with secondary structural data and a sound+video animation of the model. It is intended that the general availability of this model will assist both in interpretation of causative mutations and selection of candidate residues forin vitromutagenesis. The HAMSTeRS URL is http://europium.mrc.rpms.ac.uk.

The Haemophilia A Mutation Search Test and Resource Site, Home Page of the Factor VIII Mutation Database: HAMSTeRS

In order to facilitate easy access to and aid understanding of the causes of haemophilia A at the molecular level we have constructed HAMSTeRS, the third release of the factor VIII mutation database and the first release of this database that may be accessed and interrogated over the internet through a World Wide Web browser. The database also presents a review of the structure and function of factor VIII and the molecular genetics of haemophilia A, a real time update of the biostatistics of each parameter in the database, a molecular model of the A1, A2 and A3 domains of the factor VIII protein (based on the crystal structure of caeruloplasmin) and a bulletin board for discussion of issues in the molecular biology of factor VIII.

Factor VIIa and the extracellular domains of human tissue factor form a compact complex: A study by X-ray and neutron solution scattering

The four‐domain structure of human factor VIIa and the two‐domain structure of tissue factor form a tight complex to initiate blood coagulation. By solution scattering, the mean X‐ray and neutron radii of gyration R G (which determine macromolecular elongation) were found to be 3.25 nm, 2.13 nm and 3.14 nm (± 0.13 nm) for factor VIIa, the extracellular region of tissue factor and their complex in that order. The mean cross‐sectional radii of gyration R XS were 1.33 nm, 0.56 nm and 1.42 nm (± 0.13 nm) in that order. The mean lengths were 10.3 nm, 7.7 nm and 10.2 nm in that order. The data show that, in solution, the free proteins have extended domain structures, and the complex is formed by a compact side‐by‐side alignment of the two proteins along their long axes. The high binding affinity of tissue factor for factor VIIa may thus be accounted for by the occurrence of many intermolecular contacts in the complex.

Structural determination of lipid-bound human blood coagulation factor IX

Human coagulation factor IX (FIX) is a serine protease which binds to a negatively charged phospholipid surface in the presence of Ca ions (Ca2+). FIX two-dimensional (2-D) crystals were obtained by the lipid layer crystallisation technique under near physiological conditions. The 2-D projection map of the protein was calculated to a resolution of 3 nm using electron crystallographic analysis. The structural organisation of membrane-bound FIX is discussed and compared with the known X-ray crystallographic data.