Abstract

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 (>KdCl) and an increased (>kcat/Km) susceptibility to proteolysis by ADAMTS13 (De Cristofaro, R. et al Molecular mapping of the chloride-binding site in VWF.

J Biol Chem
2007
;
281
:
30400
–11
).

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 KdCl 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 (KdCl) 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.

Table (patients) WT --- R1341Q (4) R1341W (3) H1268D (1) V1316M (4) R1306W (15) 
KdCl (mM) 156 191 359 481 526 540 
kcat/Km (M−1s−17.25x104 8.8x104 1.2x105 1.39x105 1.6x105 1.95x105 
VWF:RCo/Ag ratio (range) 0.7 – 1.2 0.8 (0.77–0.84) 0.7 (0.6–0,8) 0.5 0.3 (0.2–0.7) 0.5 (0.25–1.0) 
Table (patients) WT --- R1341Q (4) R1341W (3) H1268D (1) V1316M (4) R1306W (15) 
KdCl (mM) 156 191 359 481 526 540 
kcat/Km (M−1s−17.25x104 8.8x104 1.2x105 1.39x105 1.6x105 1.95x105 
VWF:RCo/Ag ratio (range) 0.7 – 1.2 0.8 (0.77–0.84) 0.7 (0.6–0,8) 0.5 0.3 (0.2–0.7) 0.5 (0.25–1.0) 

Disclosures: No relevant conflicts of interest to declare.

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