Molecular mechanisms impairing biosynthesis and function of hemostatic proteins

The gene mutations leading to hemorrhagic disorders provide peculiar models to elucidate molecular mechanisms underlying protein biosynthesis, and the relationship between the structure and function. The research activity has been focused on the molecular defects leading to severe deficiency of Factor IX (FIX), a serine protease with a key role in the intrinsic pathway of blood coagulation, and of von Willebrand factor (VWF), a large multimeric protein essential for the primary hemostasis. In particular, I investigated mechanisms due to three different molecular defects such as nonsense and missense mutations in F9 gene, associated with type I hemophilia B, and an in-frame deletion in the VWF gene, displaying a dominant-negative effect. Results from investigations in patients’ plasma and the expression of nonsense FIX variants in eukaryotic cells led to the demonstration of trace levels of full-length FIX molecules even in the presence of nonsense mutations through a mechanism of ribosome read-through. The efficiency was dependent of the specific nonsense mutation and on it sequence context. Moreover, I investigated the susceptibility of a panel of nonsense FIX mutations to the induction of readthrough by aminoglycosides. The data suggested a direct relationship between the spontaneous and the drug-induced readthrough. Overall data indicated that not all nonsense mutations can be considered truly “null-mutations”, a finding that have pathophysiological implications. The severe p.Tyr450Cys mutation in the carboxyl-terminal region of coagulation FIX was chosen as model to study the interplay between impaired protein biosynthesis and/or function caused by missense mutations in relation to specific protein regions, which has been poorly investigated. Results from the expression of a panel of recombinant variants demonstrated the key role of the tyrosine phenyl group for both FIX secretion and coagulant activity. Comparison among highly homologous coagulation serine proteases indicate that additive or compensatory pleiotropic effects on secretion and function by carboxyl-terminus mutations produce life-threatening or mild phenotypes in the presence of similarly reduced protein amounts. Finally I contributed to the characterization of the dominant inheritance in VWD, due to two essential process in VWF dimerization and multimerization VWD can express dominant-negative features. Previous study characterized and demonstrate d the modulation of this dominant effect. In our study we reproduced in vivo the effect of dominance and through a creation of an artificial mutation, we demonstrated in vitro an in vivo the key role of interaction between wild type and mutant protein monomers during dimerization and or multimerization, We believe that our finding have general implication for the dominant forms of VWD, the most frequent inherited bleeding disorders in humans.