We characterized a symptomatic CRM^red factor X (FX) deficiency produced by the Glu19Ala mutation in the γ-carboxyglutamic-rich domain. FX activity levels in plasma were markedly reduced in prothrombin time assays (< 1-5%), whereas in activated partial thromboplastin assays (16%) and in RVV assays (17%) the reduction in activity mirrored that in antigen levels (17%). Activation of recombinant 19Ala-FX by factor IXa/factor VIIIa or RVV, and the activity in thrombin generation assays, were comparable to those of wild-type FX. Differently, complete activation of recombinant 19AlaFX required a factor VIIa/TF concentration 30-fold higher than that of wild-type FX. The recombinant FVIIa significantly reduced PT values in 19Ala-FX reconstituted plasma, thus suggesting an alternative approach for treatment of FX deficiencies characterized by defective FX activation. The study of this FX deficiency provides an “in vivo” and “in vitro” model for the investigation of Gla domain interactions.

Congenital coagulation factor (F) VII deficiency is a rare bleeding disorder caused by mutations in the F7 gene. The missense factor FVII variant p.Q160R is the disease-causing mutation in all Norwegian FVII deficient patients and results in reduced biological activity and antigen levels of FVII in patient plasma. Previous in vitro studies on this variant demonstrated impaired intracellular trafficking and reduced secretion, possibly due to protein misfolding. The aim of the study was therefore to assess the impact of chemical chaperones on cellular processing and secretion of this variant using a cell model based on overexpression of the recombinant protein. Through screening of compounds, we identified 4-phenylbutyrate (4-PBA) to increase the secretion of recombinant (r) FVII-160R by ~ 2.5-fold. Additionally, treatment with 4-PBA resulted in a modest increase in specific biological activity. Intracellular localization studies revealed that upon treatment with 4-PBA, rFVII-160R was secreted through Golgi and Golgi reassembly-stacking protein (GRASP)-structures. The present study demonstrates that the chemical chaperone 4-PBA, restores intracellular trafficking and increases the secretion of a missense FVII variant with functional properties in the extrinsic coagulation pathway.

Aiming at exploring vascular components in multiple sclerosis (MS) with brain outflow disturbance, we combined transcriptome analysis in MS internal jugular vein (IJV) wall with WES in MS families with vertical transmission of disease. Main results were the differential expression in IJV wall of 16 MS-GWAS genes and of seven genes (GRIN2A, GRIN2B, IL20RB, IL26, PER3, PITX2, and PPARGC1A) not previously indicated by GWAS but encoding for proteins functionally interacting with MS candidate gene products. Strikingly, 22/23 genes have been previously associated with vascular or neuronal traits/diseases, nine encoded for transcriptional factors/regulators and six (CAMK2G, GRIN2A, GRIN2B, N1RD1, PER3, PPARGC1A) for circadian entrainment/rhythm components. Among the WES low-frequency (MAF ≤ 0.04) SNPs (n = 7) filtered in the 16 genes, the NR1D1 rs17616365 showed significantly different MAF in the Network for Italian Genomes affected cohort than in the 1000 Genome Project Tuscany samples. This pattern was also detected in five nonintronic variants (GRIN2B rs1805482, PER3 rs2640909, PPARGC1A rs2970847, rs8192678, and rs3755863) in genes coding for functional partners. Overall, the study proposes specific markers and low-frequency variants that might help (i) to understand perturbed biological processes in vascular tissues contributing to MS disease, and (ii) to characterize MS susceptibility genes for functional association with disease-pathways.

Recent observations highlight a notable surge in West Nile Virus (WNV) infections in Europe that can lead to neuroinvasive consequences associated with neurodegeneration, mainly triggered by WNV Non-Structural protein 1 (NS1). During viral replication, various protein-protein interactions take place, allowing viral proteins to interact with host factors. NS1 is actively secreted in the bloodstream by infected cells and is known to affect endothelial permeability and host immune response. Focusing on the recently discovered antimicrobial roles of Amyloid-Beta (Aβ) in the context Central Nervous System (CNS), we connected WNV late pathology to overlapping features encountered in neurodegenerative diseases. In fact, CNS viral infections, or presence of specific viral components, activate glial cells, which in turn increase Aβ expression as an antiviral mechanism, leading to Aβ accumulation and neuronal damage. Considering West Nile neuroinvasive disease (WNND) as a possible complication of WNV infection, we investigated the impact of soluble WNV (s)NS1 on glial and neuronal cells, in 2D and 3D in vitro models. We reported an increased Aβ deposition after WNV sNS1 treatment, particularly of Aβ-142 isoform, and increased glial activation with a subsequent neurotoxicity. These findings underscore the crucial role of sNS1 in CNS-related effects during WNV infection, suggesting a novel pathogenetic role.