Relevance of Chloride Binding to Von Willebrand Factor in Type 2B Von Willebrand Disease Patients.

Background: In von Willebrand disease type 2B (VWD2B) the abnormal VWF spontaneously binds to circulating platelets, usually resulting into loss of high molecular weight multimers (HMWM) and thrombocytopenia. In these patients VWF:RCo/Ag ratio is indicative of reduced HMWM and roughly correlated with the severity of VWD2B defect. Physiological concentrations of Chloride ions (Cl⁻) inhibit the hydrolysis of VWF by ADAMTS-13. Cl⁻ acts as an allosteric effector and its specific binding to VWF A1 domain results into a reduced availability of A2 to be proteolised by ADAMTS-13. Type 2B VWF R1306W showed a reduced affinity to Cl⁻ (>K_dCl⁻) and an increased (>kcat/Km) susceptibility to proteolysis by ADAMTS13 (De Cristofaro, R. et al Molecular mapping of the chloride-binding site in VWF.

Aim: To determine if other VWD2B mutations affect Cl^– binding by changing the proteolysis of mutant VWF by ADAMTS-13.

Methods: Five distinct mutant recombinant (r)A1-A2-A3 proteins (Table), have been prepared along with the WT. Determination of K_dCl⁻ of each rA1- A2-A3 was achieved by intrinsic protein fluorescence, performed as a function of both Cl⁻ concentrations and temperature. Study on the kinetic rates of hydrolysis of each rA1- A2-A3 by ADAMTS-13 was evaluated using HPLC methods.

Results and Conclusions: The results (Table) confirmed that affinity of Cl⁻ (K_dCl⁻) was decreased in all mutant rA1-A2-A3s compared to the WT at different extent, with an inverse correlation to the catalytic specificity (kcat/Km) of ADAMTS-13 interaction. Higher VWF:RCo/Ag ratios were found associated with lower values of kcat/Km in our studied patients. This finding strongly support that the reduced affinity of type 2B VWF to Cl⁻ might contribute to the depletion of HMWM.