Estudio del factor XII aberrante en diferentes patologíasnuevas perspectivas de esta molécula pleiotrópica

Resumo

Factor XII (FXII) remains an unknown and enigmatic element despite being a pleotropic molecule and being involved in various systems such as coagulation, inflammation, fibrinolysis and complement. To obtain information about this protein, we decided to study 3 patterns with the presence of aberrant FXII molecules. The characterization of this aberrant FXII could provide new molecular, biochemical, functional and pathological information. First, we identified a patient with acute leukemia who presented all of his FXII in plasma 5 KDa greater than the wild FXII. The congenital basis of this defect was demonstrated by observing the same FXII in his brother. However, the complete sequencing of the F12 gene did not identify any molecular alteration that would explain the aberrant FXII. In addition, under certain conditions (activation with silica or dextran sulfate (DXS), increase in SDS %, or purification of FXII by affinity chromatography), wild FXII was detected. This result suggests that aberrant FXII could be explained by an almost covalent interaction of wild FXII with a 5 KDa peptide. The purification and proteomic analysis of the aberrant FXII suggests that it could be a GRIP1 peptide. In fact, the complete sequencing of the exome identifies a mutation in this gene shared by the two brothers. Functionally, aberrant FXII seems to be less sensitive to activation with low doses of silica and DXS, which could contribute to explain the almost zero complications that the patient has presented throughout an extensive and aggressive oncological treatment that included two allogeneic transplants. Second, we studied 58 patients with congenital glycosylation disorders (CDGs). This defect does not cause FXII deficiency but the appearance in plasma of a hypoglycosylated form of FXII. The characteristics of the heavy chain of FXII after its activation showed that all the hypoglycosylated FXII lacks the N-glycan located in position Asn414. The recombinant expression of mutated variants in insect cells affecting the two N-glycosylation sites described in FXII confirms that the absence of N-glycan in Asn230 is key for the correct folding and/or secretion of the molecule. In addition, hypoglicosylated FXII seems more sensitive to its activation and to the generation of kallikrein. Finally, our project included 33 patients with hereditary angioedema type III (FXII-HAE) carrying the p.Thr309Lys mutation. Until now, this pathology is clearly associated with mutations in the F12 gene. All patients, except 3 who were carriers of this mutation, presented two forms of FXII in plasma. The size of the aberrant FXII could not be justified by a defect in O-glycosylation as proposed. In fact, the mutated recombinant form, also expressed in insect cells, had the same size as the wild type. The studies carried out suggest this aberrant form could be the result of an alternative processing of exon 9. The aberrant FXII, both plasma and recombinant, is more sensitive to activation in response to lower stimulation, generating kallikrein but without causing coagulation activation. In conclusion, the study of aberrant FXII in different pathologies has allowed us to know the complexity of this protein, its interactions and the repercussion that a post-translational modification, such as glycosylation, has on its secretion and functionality.