Molecular and structural analysis of human Factor X deficiency

Factor X (FX) is one of the vitamin K-dependent serine proteases, and is g of a catalytic serine protease domain, two EGF domains and a Gla domain. It is crucial for coagulation, however its role in disease has not been fully characterised. This thesis describes the phenotypic and genotypic analysis of kindred with FX deficiency. Because FX deficiency is rare in the general population, patient samples were drawn from across the world. Laboratory assays for FX and direct sequencing of the gene were performed on samples from ten kindred. A total of ten mutations were identified, nine of which were novel and summarised as follows. One mutation was identified in two unrelated families (Asp373Asn). Compound heterozygous mutations were identified in two kindred (Glul9Val and IVS5+3 A-G in one kindred; Cysl32Stop and Arg273Trp in the other). Two other mutations were identified in the catalytic domain (Gly222Asp and Pro382Leu), one in the EGF-1 domain (Glu51Lys), and one in the signal peptide (Ala-26Asp). No mutation was identified in one kindred. Molecular modelling of mutations in terms of the available crystal structure of factor Xa was performed in order to correlate genotype with phenotype. Explanations were proposed in terms of the perturbation of the structure itself or its biochemical function. For Glu51Lys and Arg273Trp, it was predicted that ligand binding would be disrupted. In the case of Asp373Asn, it was proposed that perturbation of the FX sodium-binding site would impair its enzymatic function. It was predicted that the Gly222Asp and Pro382Leu mutations would disrupt protein folding. In order to show that selected known mutations are causative for FX deficiency, recombinant FX with the Arg-1Thr, Glu51Lys, Arg273Trp and Asp373Asn substitutions was expressed. Their expression was successful. In preliminary work with these, it was not possible to recapitulate the in vivo data for these expressed proteins