bioRxiv Science⌕ Search

Biology subjects

Ferretti, A.

Publications and source records attributed to Ferretti, A..

2 recordsLinked to original sources

Natural variants of von Willebrand factor R1205 causing von Willebrand disease with accelerated von Willebrand factor clearance: in silico docking models and energetics of the interaction with both LRP1 and GpIb A1 domain

Type 1 von Willebrand disease (VWD) is often caused by variants in von Willebrand factor (VWF), including p.R1205H ("Vicenza mutation"), which accelerate VWF clearance via macrophage receptor LRP1 and impair platelet adhesion. However, the structural mechanisms underlying these phenotypes remain unclear. Here, we use integrative computational modeling (I-TASSER, HADDOCK2.4, and PRODIGY) to predict how p.R1205H/C/L/S variants alter VWF interaction with LRP1 and GPIb. Our models reveal that R1205 acts as a structural hinge: its variants disrupt polar networks in VWFs D3 domain, exposing neo-epitopes that enhance LRP1 binding ({Delta}{Delta}G up to -5.3 kcal/mol) while destabilizing the A1 domains 1-{beta}2 loop, reducing GPIb affinity (33-fold for R1205L/C). These findings explain clinical observations, p.R1205H rapid clearance yet retained platelet adhesion, and establish R1205 as a dual-functional switch regulating VWF circulatory lifetime and hemostatic activity. This analytical procedute provides a template for predicting pathogenicity of VWF variants and designing targeted therapies for VWD. Author SummaryIn our study, we explored how specific genetic changes in von Willebrand factor (VWF), a protein crucial for haemostsis, lead to a bleeding disorder called von Willebrand disease (VWD). We focused on mutations at position R1205 in VWF, known to cause rapid clearance of the protein from the bloodstream, resulting in low levels and impaired clotting. Using advanced computer modeling, we investigated how these mutations alter VWFs interactions with two key partners: LRP1, a receptor that removes VWF from circulation, and GpIb, a platelet receptor essential for clotting. Our models revealed that R1205 acts like a structural hinge. Mutations disrupt its stabilizing role, exposing regions that bind more tightly to LRP1, thus explaining the accelerated clearance seen in patients. Simultaneously, these changes can also weaken VWFs ability to interact with platelets, further impairing platelet haemostatic function. This work provides a blueprint for understanding how some VWF variants cause disease and could guide the design of targeted therapies to correct or compensate for these defects, improving treatment for VWD patients.

biochemistry↗

EVs analysis in the COVID-19 era: insights on serum inactivation protocols towards downstream isolation and analysis

Since the outbreak of COVID-19 crisis, the handling of biological samples from confirmed or suspected SARS-CoV-2 positive individuals demanded the use of inactivation protocols to ensure laboratory operators safety. While not standardized, these practices can be roughly divided in two categories, namely heat inactivation and solvent-detergent treatments. As such, these routine procedures should also apply to samples intended for Extracellular Vesicles (EVs) analysis. Assessing the impact of virus inactivating pre-treatments is therefore of pivotal importance, given the well-known variability introduced by different pre-analytical steps on downstream EVs isolation and analysis. Arguably, shared guidelines on inactivation protocols tailored to best address EVs-specific requirements will be needed among the EVs community, yet deep investigations in this direction havent been reported so far. In the attempt of sparking interest on this highly relevant topic, we here provide preliminary insights on SARS-CoV-2 inactivation practices to be adopted prior serum EVs analysis by comparing solvent/detergent treatment vs. heat inactivation. Our analysis entailed the evaluation of EVs recovery and purity along with biochemical, biophysical and biomolecular profiling by means of Nanoparticle Tracking Analysis, Western Blotting, Atomic Force Microscopy, miRNA content (digital droplet PCR) and tetraspanin assessment by microarrays. Our data suggest an increase in ultracentrifugation (UC) recovery following heat-treatment, however accompanied by a marked enrichment in EVs-associated contaminants. On the contrary, solvent/detergent treatment is promising for small EVs (< 150 nm range), yet a depletion of larger vesicular entities was detected. This work represents a first step towards the identification of optimal serum inactivation protocols targeted to EVs analysis.

biochemistry↗