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This report describes the first case of splenic injury in a patient with p.V1316M-associated von Willebrand disease type 2B (VWD2B) with chronic thrombocytopenia, successfully treated with nonoperative management including von Willebrand factor (VWF) replacement therapy, and platelet transfusions relayed by a thrombopoietin receptor agonist (TPO-RA, Eltrombopag). Eltrombopag was initially introduced to rescue an unusual post-platelet-transfusion reaction exacerbating the thrombocytopenia. In-depth analysis of the dramatic platelet count drop and VWF measurements timeline ruled out an allo-immune reaction and supported an alternative hypothesis of a sudden platelet clearance as a consequence of stress-induced release of abnormal VWF. One year later, a second life-threatening bleeding episode required urgent surgery successfully managed with VWF replacement therapy and platelet transfusions. Eltrombopag was further introduced in the post-surgery period to allow bleeding-free and platelet-transfusion-free successful recovery. Treatment decisions are particularly challenging in patients with VWD2B, and this case highlights how such decisions can benefit from understanding the molecular origin of platelet count fluctuations observed in these patients. Here, we successfully used a new therapeutic approach combining VWF-replacement therapy and initial platelet-transfusion relayed by TPO-RA to optimize patient management. Plain language summary A combination of von Willebrand factor replacement and thrombopoietin receptor agonist in thrombocytopenic patients with von Willebrand disease type 2B: a new therapy approach to optimize patient management? Therapeutic management of patients with von Willebrand disease type 2B are particularly challenging in case of severe thrombocytopenia. Treatment includes von Willebrands factor replacement therapy and iterative platelet transfusions. We describe the first case of splenic injury in a patient with p.V1316M-associated von Willebrand disease type 2B successfully treated with nonoperative management including von Willebrand factor replacement therapy and platelet transfusions relayed by a thrombopoietin receptor agonist. We showed that the unusual post-platelet-transfusion reaction associated with a dramatic platelet count drop was a consequence of stress-induced release of abnormal von Willebrand factor. The combination of von Willebrand factor replacement therapy and thrombopoietin receptor agonist may offer a new therapeutic approach to optimize patient management.

Patients with type 2B von Willebrand disease (vWD) (caused by gain-of-function mutations in the gene coding for von Willebrand factor) display bleeding to a variable extent and, in some cases, thrombocytopenia. There are several underlying causes of thrombocytopenia in type 2B vWD. It was recently suggested that desialylation-mediated platelet clearance leads to thrombocytopenia in this disease. However, this hypothesis has not been tested in vivo. The relationship between platelet desialylation and the platelet count was probed in 36 patients with type 2B von Willebrand disease (p.R1306Q, p.R1341Q, and p.V1316M mutations) and in a mouse model carrying the severe p.V1316M mutation (the 2B mouse). We observed abnormally high elevated levels of platelet desialylation in both patients with the p.V1316M mutation and the 2B mice. In vitro, we demonstrated that 2B p.V1316M/von Willebrand factor induced more desialylation of normal platelets than wild-type von Willebrand factor did. Furthermore, we found that N-glycans were desialylated and we identified αIIb and β3 as desialylation targets. Treatment of 2B mice with sialidase inhibitors (which correct platelet desialylation) was not associated with the recovery of a normal platelet count. Lastly, we demonstrated that a critical platelet desialylation threshold (not achieved in either 2B patients or 2B mice) was required to induce thrombocytopenia in vivo. In conclusion, in type 2B vWD, platelet desialylation has a minor role and is not sufficient to mediate thrombocytopenia.

Previously, we found that LDL-receptor related protein-1 on macrophages mediated shear stress-dependent clearance of von Willebrand factor. In control experiments, however, we observed that von Willebrand factor also binds to macrophages independently of this receptor under static conditions, suggesting the existence of additional clearance-receptors. In search for such receptors, we focused on the macrophage-specific scavenger-receptor SR-AI. von Willebrand factor displays efficient binding to SR-AI (half-maximum binding 14±5 nM). Binding is calcium-dependent and is inhibited by 72±4% in the combined presence of antibodies against the A1- and D4-domains. Association with SR-AI was confirmed in cell-binding experiments. In addition, binding to bone marrow-derived murine SR-AI-deficient macrophages was strongly reduced compared to binding to wild-type murine macrophages. Following expression via hydrodynamic gene transfer, we determined ratios for von Willebrand factor-propeptide over von Willebrand factor-antigen, a marker of von Willebrand factor clearance. Propeptide/antigen ratios were significantly reduced in SR-AI-deficient mice compared to wild-type mice (0.6±0.2 versus 1.3±0.3; P

Thrombocytopenia and increased platelet clearance observed in von Willebrand disease-type 2B (VWD-2B) may be explained by platelet apoptosis triggered by the constitutive binding of VWF to its receptor, glycoprotein Ib (GPIb). Apoptosis was assessed in platelets from two patients with a severe VWD-2B mutation VWF/p.V1316M and from mice transiently expressing VWF/p.V1316M. We now report that the VWD-2B mutation VWF/p.V1316M which binds spontaneously to its receptor GPIbα does not induce apoptosis. In 2 unrelated patients (P1 and P2) exhibiting different VWF plasma levels (70% and 36%, respectively, compared with normal pooled human plasma given as 100%), inner transmembrane depolarization of mitochondria, characteristic of apoptotic events was undetectable in platelets, whether washed or in whole blood. No or a moderate phosphatidyl serine (PS) exposure as measured by annexin-V staining was observed for P1 and P2, respectively. Expression of pro-apoptotic proteins Bak and Bax, and caspase-3 activity were similar to control platelets. In the VWD-2B mouse model expressing high levels of mVWF/p.V1316M (423%), similar to what is found in inflammatory pathologies, no significant difference was observed between mice expressing mVWF/WT and mVWF/p.V1316M. These results strongly argue against apoptosis as a mechanism for the thrombocytopenia of severe VWD-2B exhibiting the VWF/p.V1316M mutation.

The objective of this project was to study the function of O-glycosylations in von Willebrand factor (VWF) life cycle. In total, 14 different murine Vwf cDNAs mutated on one or several O-glycosylations sites were generated: 9 individual mutants, 2 doublets, 2 clusters and 1 mutant with all 9 murine glycosylation sites mutated (Del-O-Gly). We expressed each mutated cDNA in VWF deficient-mice by hydrodynamic injection. An immunosorbent assay with Peanut Agglutinin (PNA) was used to verify the O-glycosylation status. Wild-type (WT) VWF expressed by hepatocytes after hydrodynamic injection was able to bind PNA with slightly higher affinity than endothelial-derived VWF. In contrast, the Del-O-Gly VWF mutant did not bind PNA, demonstrating removal of O-linked glycans. All mutants displayed a normal multimeric pattern. Two mutants, Del-O-Gly and T1255A/T1256A, led to expression levels 50% lower than those induced by WT VWF and their half-life in vivo was significantly reduced. When testing the capacity of each mutant to correct the bleeding time of VWF-deficient mice, we found that S1486A, T1255A, T1256A and the doublet T1255A/T1256A were unable to do so. In conclusion we have shown that O-glycosylations are dispensable for normal VWF multimerization and biosynthesis. It also appears that some O-glycosylation sites, particularly the T1255 and T1256 residues, are involved in the maintenance of VWF plasma levels and are essential for normal haemostasis. As for the S1486 residue, it seems to be important for platelet binding as demonstrated in vitro using perfusion experiments.

Two unrelated families were recruited in the French Reference Center for von Willebrand Disease with moderate bleeding symptoms associated with low von Willebrand factor (VWF) antigen levels, decreased collagen binding assay, and no or partial response to desmopressin. Genetic analysis showed the presence of heterozygous mutations in the A3 domain away from the collagen-binding surface: 1 never reported previously (p.L1696R) and another (p.P1824H) described in a Spanish family. The mutations were reproduced by site-directed mutagenesis and mutant VWF was expressed in different expression systems, COS-7 cells, baby hamster kidney cells, and in VWF-deficient mice through hydrodynamic injection. p.L1696R and p.P1824H were associated with very low expression levels both in vitro and in vivo, with intracellular retention for p.P1824H. Both homozygous mutants displayed decreased binding to collagen types I and III but also decreased binding to platelet glycoproteins lb and IIbilla. Co-transtections with wild-type VWF partially corrected these defects, except that collagen binding remained abnormal. The in vivo thrombosis response was severely reduced for both heterozygous mutants. In conclusion, we report 2 VWF A3 domain mutations that induce a combined qualitative and quantitative defect. [ABSTRACT FROM AUTHOR]