A Modified Model of Type 2B von Willebrand Disease: Taking ADAMTS13-Mediated Cleavage out of the Equation

Type 2B von Willebrand disease (2B VWD), a qualitative variant of VWD, is characterized by thrombocytopenia and loss of high molecular weight multimers. 2B VWF has both an increased affinity for platelet glycoprotein Ibα (GPIbα) and increased susceptibility to ADAMTS13-mediated cleavage. In order to determine whether the loss of high molecular weight (HMW) multimers associated with 2B VWD occurs due to the increased ADAMTS13 susceptibility or due to the increased binding to platelet GPIbα, we created a modified type 2B mouse model, in which the mice expressed ADAMTS13-insensitive 2B VWF.

Three common type 2B VWD mutations (R1306W, V1316M, and R1341Q) were independently introduced into the mouse Vwf (mVWF) cDNA sequence containing the ADAMTS13 cleavage site knockout, Y1605A/M1606A (CSKO). Recombinant mutant mVWF was produced using transient transfection of HEK293T cells. ADAMTS13 digestion of the recombinant mVWF was performed using a Tris-Urea system. Platelet binding affinity was assessed using a botrocetin-induced platelet-binding ELISA assay. 8–9 week old C57Bl6 VWF KO mice were hydrodynamically injected with 100 μ g of naked plasmid DNA containing the liver specific ET promoter and wildtype (WT) or mutant mouse Vwf DNA. The mice were sampled weekly; complete blood counts, VWF:Ag levels, VWFpp levels, and VWF multimer structure were examined.

Following transient transfections, mutant and wildtype mVWF were secreted at similar levels, with a full range of VWF multimer sizes. Recombinant 2B/CSKO mVWF is markedly ADAMTS13 cleavage insensitive, with >80 U mADAMTS13 required for 50% cleavage (WT mVWF requires 1 U mADAMTS13). Similar to 2B VWF, 2B/CSKO mVWF showed enhanced platelet binding affinity in the presence of botrocetin. Hydrodynamic injections of the naked expression plasmids did not result in adverse events in the mice. Mean 2B/CSKO platelet counts at day 7 were significantly reduced compared to WT platelet counts (149 and 370, respectively). At day 14, only the mice expressing V1316M/CSKO remained thrombocytopenic, with platelet counts similar to those seen with the V1316M mutation alone (170 and 171, respectively). By day 14, the mice expressing 2B/CSKO VWF had significantly lower mean VWF:Ag levels than WT mice or mice expressing the CSKO alone (see table below). The mean VWFpp/VWF:Ag ratios were significantly increased in the 2B/CSKO mice when compared to the WT mice (1.53 and 1.00, respectively). There was no loss of HMW multimers in the mice expressing R1306W/CSKO and R1341Q/CSKO and, indeed, these mice showed significantly more multimer bands than WT mice (days 7 to 21; 14.5±0.6 and 13.9±1.2 multimer bands versus 10.2±0.5 bands for WT). In marked contrast, the mice expressing V1316M/CSKO VWF showed complete loss of high molecular weight multimers, similar to mice expressing the same 2B VWF mutation alone (8.3±1.8 and 7.7±0.9 multimer bands, respectively).

Mice expressing R1306W/CSKO and R1341Q/CSKO VWF have an absence of thrombocytopenia and show the presence of supranormal high molecular weight VWF multimer bands. These results suggest that enhanced ADAMTS13-mediated cleavage plays an important role in the type 2B phenotype associated with these mutations. In contrast, low V1316M/CSKO VWF:Ag was associated with both thrombocytopenia and loss of HMW multimers, demonstrating that enhanced GPIbα binding affinity is the predominant mechanism associated with this mutation. Thus, the importance of ADAMTS13 susceptibility in 2B VWD is mutation-dependent, and ADAMTS13-mediated cleavage plays an important role in enhancing the severity of the type 2B VWD phenotype associated with the R1306W and R1341Q mutations.

No relevant conflicts of interest to declare.