MOLECULAR BASES OF THE MODULATION OF COAGULATION FACTORS LEVELS: IN VITRO AND IN VIVO STUDY.

Hemostasis is a nely tuned equilibrium status between procoagulant and anticoagulant activities. Despite the presence of several regulatory factors, hemostatic balance is nevertheless sensitive to perturbation. In this thesis, we have analyzed the effects of some acquired and inherited components that could modulate specific coagulation factors. The study of coagulation factor level variations could be instrumental to discover new parameters for the evaluation of the risk of thrombotic and hemorrhagic events. In the first study we evaluated the effects of a whole-diet therapy on a wide panel of hemostatic and in ammatory parameters in a population of overweighted premenopausal women with moderate CVD risk. We observed specific changes on proteins important in coagulation initiation (factor VII, [FVII]) and amplication phases (factor VIII, [FVIII]). Levels of Tumor Necrosis Factor-alfa, a cytokine over-produced by adipocytes and macrophages of adipose tissue in the obese state, were correlated with those of FVIII, thus suggesting new relations between coagulation and cellular components of in ammation. Also temporal parameters of thrombin generation (lag time and time to peak) were significantly prolonged, perhaps re ecting changes related with initial stages of coagulation cascade. In the second study, we evaluated the effect of exercise training on end-stage renal disease (ESRD) patients on hemodialysis (HD), a population characterized by high CVD risk and a seriously impaired physical function. The results we have obtained suggest the presence of factors capable of modulating the coagulation phenotype in both the two groups of patients, albeit independently of physical activity. The lack of association between physical exercise and the coagulation phenotype, cannot exclude that exercise alone is not able to modulate blood coagulation in the ESRD population. We have found an extremely significant reduction in the levels of FVIII, FVII and FX levels, that are of potential clinical importance, even though they proved to be independent from lifestyle change. For a better understanding of these results, we will need the expertise of clinicians, and particularly of nephrologists. Finally, we have investigated the molecular mechanisms induced by a new mutation (R1698W) of coagulation factor V (FV), in the modulation of its protein levels. Our results indicate that the R1698W mutation has a pleiotropic effect on FV by reducing its cellular secretion, impairing its binding affinity to FXa, reducing the prothrombinase catalytic efficiency and deeply increasing its instability after activation. These data also provide an interpretation of the cross-reacting material negative (CRM -) phenotype shown in R1698W carriers. This study gives new structural information at the A3-A2 domains interface, a region presenting several differences between the two available FV models. The replacement of the arginine with a relatively large and hydrophobic residue (tryptophan or alanine) at 1698 is not tolerated since it probably abolishes an important inter-domains interaction. The next step of our research will be focused at understanding the potentially unique role of 1698R in FVa stability by the creation of new FV mutants.