Mechanisms and Therapeutic Modulation of the Bleeding Tendency in Genetically-Engineered Von Willebrand Disease Type 2B Mice

Von Willebrand disease type 2B (VWD2B) is characterized by an abnormal increased affinity of von Willebrand factor (VWF) for platelets and related to mutations in the A1 domain of VWF. In patients, the VWD2B-mutant p.V1316M is specifically associated with a severe phenotype including hemorrhagic tendency, loss of high molecular weight VWF multimers, thrombocytopenia and platelet dysfunction related to defective αIIbβ3 integrin activation (Casari et al. JCI 2013, 123:5071). Genetically-engineered mice carrying the p.V1316M mutation (V1316M+/+ mice) perfectly mimic the human phenotype. We used this murine model to investigate the relative contribution of platelet count and platelet dysfunction to the occurrence of bleeding tendency and explore new therapeutic options.

Bleeding tendency was evaluated using a novel tail-vein transection bleeding model involving the specific transection of the lateral vein at standardized depth and diameter (0.7 and 2.3mm respectively). Time (TVT) and blood loss were measured in wild-type (WT) and V1316M+/+ mice at baseline and after platelet count alteration.

Compared to WT mice (platelet count: 1019±154G/L, n=58), V1316M+/+ mice (platelet count: 455±139G/L, n=52) have a significant increase (p<0.01) in TVT (25±9min, n=12 vs 2±1min, n=14) and blood loss (448±273µl vs 34±19µl). Induced thrombocytopenia (after infusion of anti-GPIb antibody) in WT mice (platelet count: 347±93G/L, n=6), to a similar level of V1316M+/+ mice, did not induce a hemorrhagic tendency (TVT: 2±1min, blood loss: 14±13µl). An increased bleeding phenotype in WT mice was exclusively observed in case of severe thrombocytopenia (platelet count: 70±25G/L, TVT>30min, blood loss: 532±168ml, n=6). Conversely, romiplostim-treated V1316M+/+ mice with normalized platelet count (platelet count: 866±100G/L, n=8) did not improve their bleeding tendency (TVT: 21±8min, blood loss: 448±282ml). These results suggest the bleeding phenotype in V1316M+/+ mice is independent of their thrombocytopenia, and could be related to the p.V1316M-associated defect in platelet function in general, and αIIbβ3 integrin activation in particular.

Based on the hemostatic efficacy of recombinant activated factor VII (rFVIIa) in Glanzmann thrombasthenia, we tested rFVIIa in thrombocytopenic V1316M+/+ mice.

A single infusion of rFVIIa (3 mg/kg) induced a complete and immediate correction of the bleeding phenotype (TVT: 4±2min, blood loss: 69±25ml, n=6). Thrombin generation measured in platelet-rich plasma before and after treatment showed a significant reduction of the lag time (1.13min, n=12 vs 1.8min, n=8 respectively, p<0.01).

In conclusion, genetically-engineered mice carrying the VWD2B p.V1316M mutation display a bleeding phenotype that is not explained by their reduced platelet count, but is possibly related to the impaired platelet function induced by this VWF mutant. The bleeding tendency was rapidly and completely controlled by using rFVIIa. Based on these promising data, it would be of interest to consider rFVIIa as a therapeutic option in VWD-2B patients characterized by a severe hemorrhagic phenotype.