The Physiological Function of von Willebrand's Factor Depends on Its Tubular Storage in Endothelial Weibel-Palade Bodies

SummaryWeibel-Palade bodies are the 1–5 μm long rod-shaped storage organelles of endothelial cells. We have investigated the determinants and functional significance of this shape. We find that the folding of the hemostatic protein von Willebrand's factor (VWF) into tubules underpins the rod-like shape of Weibel-Palade bodies. Further, while the propeptide and the N-terminal domains of mature VWF are sufficient to form tubules, their maintenance relies on a pH-dependent interaction between the two. We show that the tubular conformation of VWF is essential for a rapid unfurling of 100 μm long, platelet-catching VWF filaments when exposed to neutral pH after exocytosis in cell culture and in living blood vessels. If tubules are disassembled prior to exocytosis, then short or tangled filaments are released and platelet recruitment is reduced. Thus, a 100-fold compaction of VWF into tubules determines the unique shape of Weibel-Palade bodies and is critical to this protein's hemostatic function

Re-establishment of VWF-dependent Weibel-Palade bodies in VWD endothelial cells

Type 3 von Willebrand disease (VWD) is a severe hemorrhagic defect in humans. We now identify the homozygous mutation in the Chapel Hill strain of canine type 3 VWD that results in premature termination of von Willebrand factor (VWF) protein synthesis. We cultured endothelium from VWD and normal dogs to study intracellular VWF trafficking and Weibel-Palade body formation. Weibel-Palade bodies could not be identified in the canine VWD aortic endothelial cells (VWD-AECs) by P-selectin, VWFpp, or VWF immunostaining and confocal microscopy. We demonstrate the reestablishment of Weibel-Palade bodies that recruit endogenous P-selectin by expressing wild-type VWF in VWD-AECs. Expression of mutant VWF proteins confirmed that VWF multimerization is not necessary for Weibel-Palade body creation. Although the VWF propeptide is required for the formation of Weibel-Palade bodies, it cannot independently induce the formation of the granule. These VWF-null endothelial cells provide a unique opportunity to examine the biogenesis of Weibel-Palade bodies in endothelium from a canine model of type 3 VWD

Enhanced VWF clearance in low VWF pathogenesis: Limitations of the VWFpp/VWF:Ag ratio and clinical significance

Increased von Willebrand factor (VWF) clearance plays a key role in the pathogenesis of type 1 and type 2 von Willebrand disease (VWD). However, the pathological mechanisms involved in patients with mild to moderate reductions in plasma VWF:Ag (range, 30-50 IU/dL; low VWF) remain poorly understood. In this study, we investigated the hypothesis that enhanced VWF clearance may contribute to the pathobiology of low VWF. Patients with low VWF were recruited to the LoVIC study after ethics approval and receipt of informed consent. Desmopressin was administered IV in 75 patients, and blood samples were drawn at baseline and at the 1-hour and 4-hour time points. As defined by recent ASH/ISTH/NHF/WFH guidelines, 20% of our low-VWF cohort demonstrated significantly enhanced VWF clearance. Importantly, from a clinical perspective, this enhanced VWF clearance was seen after desmopressin infusion, but did not affect the steady-state VWF propeptide (VWFpp)-to-VWF antigen (VWF:Ag) ratio (VWFpp/VWF:Ag) in most cases. The discrepancy between the VWFpp/VWF:Ag ratio and desmopressin fall-off rates in patients with mild quantitative VWD may have reflected alteration in VWFpp clearance kinetics. Finally, bleeding scores were significantly lower in patients with low VWF with enhanced VWF clearance, compared with those in whom reduced VWF biosynthesis represented the principle pathogenic mechanism. This trial was registered at http://www.clinicaltrials.gov as #NCT03167320

Platelet-targeted gene therapy with human factor VIII establishes haemostasis in dogs with haemophilia A

It is essential to improve therapies for controlling excessive bleeding in patients with haemorrhagic disorders. As activated blood platelets mediate the primary response to vascular injury, we hypothesize that storage of coagulation Factor VIII within platelets may provide a locally inducible treatment to maintain haemostasis for haemophilia A. Here we show that haematopoietic stem cell gene therapy can prevent the occurrence of severe bleeding episodes in dogs with haemophilia A for at least 2.5 years after transplantation. We employ a clinically relevant strategy based on a lentiviral vector encoding the ITGA2B gene promoter, which drives platelet-specific expression of human FVIII permitting storage and release of FVIII from activated platelets. One animal receives a hybrid molecule of FVIII fused to the von Willebrand Factor propeptide-D2 domain that traffics FVIII more effectively into α-granules. The absence of inhibitory antibodies to platelet-derived FVIII indicates that this approach may have benefit in patients who reject FVIII replacement therapies. Thus, platelet FVIII may provide effective long-term control of bleeding in patients with haemophilia A. Haemophilia is a genetic bleeding disorder associated with a deficiency in the coagulation factor VIII. Here, the authors use gene therapy to achieve stable overexpression of factor VIII in platelets of dogs with haemophilia A, preventing the occurrence of severe bleeding episodes for over 2.5 years

VWF and FVIII: the origins of a great friendship

In this issue of Blood, van den Biggelaar and colleagues demonstrate the cotargeting of FVIII and VWF to secretory granules in the absence of a high-affinity interactions between VWF and FVIII, using recombinant VWF type 2N variants