Dafna Groeneveld

Biogenesis of Weibel-Palade bodies in von Willebrand's disease variants with impaired von Willebrand factor intrachain or interchain disulfide bond formation

Background Mutations of cysteine residues in von Willebrand factor are known to reduce the storage and secretion of this factor, thus leading to reduced antigen levels. However, one cysteine mutation, p.Cys2773Ser, has been found in patients with type 2A(IID) von Willebrand’s disease who have normal plasma levels of von Willebrand factor. We hypothesize that disruption of either intra- or interchain disulfide bonds by cysteine mutations in von Willebrand factor has different effects on the biogenesis of Weibel-Palade bodies. Design and Methods The effect of specific cysteine mutations that either disrupt intrachain (p.Cys1130Phe and p.Cys2671Tyr) or interchain (p.Cys2773Ser) disulfide bonds on storage and secretion of von Willebrand factor was studied by transient transfection of human embryonic kidney cell line 293. Upon expression of von Willebrand factor these cells formed endothelial Weibel-Palade body-like organelles called pseudo-Weibel-Palade bodies. Storage of von Willebrand factor was analyzed with both confocal immunofluorescence and electron microscopy. Regulated secretion of von Willebrand factor was induced by phorbol 12-myristate 13-acetate. Results p.Cys1130Phe and p.Cys2671Tyr reduced the storage of von Willebrand factor into pseudo-Weibel-Palade bodies with notable retention of von Willebrand factor in the endoplasmic reticulum, whereas p.Cys2773Ser-von Willebrand factor was stored normally. As expected, wild-type von Willebrand factor formed proteinaceous tubules that were seen under electron microscopy as longitudinal striations in pseudo-Weibel-Palade bodies. p.Cys2773Ser caused severe defects in von Willebrand factor multimerization but the factor formed normal tubules. Furthermore, the basal and regulated secretion of von Willebrand factor was drastically impaired by p.Cys1130Phe and p.Cys2671Tyr, but not by p.Cys2773Ser. Conclusions We postulate that natural mutations of cysteines involved in the formation of interchain disulfide bonds do not affect either the storage in Weibel-Palade bodies or secretion of von Willebrand factor, whereas mutations of cysteines forming intrachain disulfide bonds lead to reduced von Willebrand factor storage and secretion because the von Willebrand factor is retained in the endoplasmic reticulum.

von Willebrand factor propeptide and the phenotypic classification of von Willebrand disease

UNLABELLED The ratios between von Willebrand factor propeptide (VWFpp) or factor VIII activity ( FVIII C) and VWF antigen (VWF:Ag) reflect synthesis, secretion, and clearance of VWF. We aimed to define the pathophysiology of 658 patients with type 1, 2, or 3 von Willebrand disease (VWD) with VWF levels ≤30 U/dL from the Willebrand in The Netherlands (WiN) study using the VWFpp/VWF:Ag and FVIII C/VWF:Ag ratios. We evaluated the use of VWFpp in the classification and diagnosis of VWD. On the basis of the ratios, reduced VWF synthesis was observed in 18% of type 1 and only 2% of type 2 patients. A significant proportion of type 3 patients had detectable VWFpp (41%). These patients had a lower bleeding score than type 3 patients who had a complete absence of VWF:Ag and VWFpp (14.0 vs 19.5; P = .025). The majority of these patients had missense mutations with rapid VWF clearance, whereas type 3 patients with no VWFpp were homozygous for null alleles. In conclusion, VWFpp identified severe type 1 VWD with very low VWF levels in patients who had previously been classified as type 3 VWD. This study underlines the clinical significance of the VWFpp assay in the diagnosis and classification of VWD.

Thrombomodulin-modified thrombin generation testing detects a hypercoagulable state in patients with cirrhosis regardless of the exact experimental conditions

Patients with cirrhosis have substantial alterations in their hemostatic systemdue to impaired synthesis and/or catabolismof hemostatic proteins [1]. Despite this, coagulation potential appears intact due to a concomitant decline of proand anticoagulant proteins [2]. Evidence for a rebalanced status has been provided by several studies in which thrombin generation (TG) tests were performed in presence of thrombomodulin (TM) or Protac®, a thrombin-dependent or direct activator of the anticoagulant protein C system, respectively. Initially it was reported that thrombin generation with such a modified thrombin generation test was equal to that of healthy volunteers [3]. However, subsequent studies reported enhanced thrombin generation in patients [4–7]. Better insight in whether patients with cirrhosis are normoor hypercoagulable is essential in the discussions on prophylactic antithrombotic therapy for these patients [8,9]. The discrepancies between the studies might reflect differences in patient selection or differences in test conditions. Different concentrations and sources of TM have been used in TM-modified TG assays. For example Potze et al. [10] used a commercially available reagent with an unknown concentration of TM, Youngwon et al. [11] used both 5nM and 2,5nM of TM,while Tripodi et al. [3] and Chaireti et al. [7] used 4nMTM. Tripodi et al. [3] and Potze et al. [10] triggered the coagulation with 1pM of tissue factor (TF). In the study of Youngwon et al. [11] both 1pM and 5pM of TF were used to trigger coagulation. We measured thrombin generation at varying concentrations of thrombomodulin and TF, to investigate whether the normalor hypercoagulable state observed in liver disease patients is dependent on the test conditions used in the TG assay. Twenty three patients that were previously diagnosed with cirrhosis, who were under routine control by or admitted at the department of Hepatology of the UniversityMedical Center Groningen, were included in this study. Patients were classified according to theModel for EndStage Liver Disease (MELD) score. The control group consisted of eighteen healthy individuals from our institution. Thrombin generation wasmeasured using calibrated automated thrombography as described [12]. Coagulationwas triggered byusing commercially available reagents containing a final concentration of 1 and 5 pM recombinant TF and 4 μM phospholipids (Thrombinoscope BV. Maastricht, The Netherlands). Tests were performed after the addition of 0, 1, 2, 4 or 8 nM rabbit-lung thrombomodulin (Sekisui Diagnostics, Stamford, CT, USA) to the plasma. Fluorescence was monitored using the Fluoroscan Ascent fluorometer (ThermoLabsystems, Helsinki, Finland) and Endogenous Thrombin Potential (ETP) was calculated using the Thrombinoscope® software (Thrombinoscope BV). Data are expressed as numbers (with

Angiogenic characteristics of blood outgrowth endothelial cells from patients with von Willebrand disease

Endothelial von Willebrand factor (VWF) inhibits angiogenesis. Accordingly, blood outgrowth endothelial cells (BOECs) isolated from von Willebrand disease (VWD) patients showed enhanced in vitro angiogenesis when compared with healthy control BOECs. Characterization of the angiogenic response of VWD BOECs is limited and differences between the different types of VWD have not been investigated in detail.

Ex vivo addition of fibrinogen concentrate improves the fibrin network structure in plasma samples taken during liver transplantation

Optimal hemostatic management during orthotopic liver transplantation (OLT) remains a challenge. The cause of bleeding during OLT is multifactorial, and may include hemostatic imbalance. Fibrinogen concentrates are increasingly being used to control perioperative bleeding during OLT. However, administration is based on arbitrary thresholds of fibrinogen levels. Importantly, studies on fibrin clot structure during OLT are lacking.

Balance between von Willebrand factor and ADAMTS13 following major partial hepatectomy

Conventional coagulation tests are frequently prolonged after liver surgery, suggesting a postoperative bleeding tendency. At the same time, thrombotic complications following partial hepatectomy (PH) are not uncommon. Little is known about changes in the platelet adhesive protein von Willebrand factor (VWF) and its cleaving protease a disintegrin and metalloprotease with a thrombospondin type 1 motif, member 13 (ADAMTS13) following a PH.

No evidence for a direct effect of von Willebrand factor's ABH blood group antigens on von Willebrand factor clearance

One of the major determinants of von Willebrand factor (VWF) plasma levels is ABO blood group status, and individuals with blood group O have ~ 25% lower plasma levels. The exact mechanism behind this relationship remains unknown, although effects on clearance have been postulated.

Storage and secretion of naturally occurring von Willebrand factor A domain variants

Von Willebrand disease (VWD) is a bleeding disorder characterized by reduced plasma von Willebrand factor (VWF) levels or functionally abnormal VWF. Low VWF plasma levels in VWD patients are the result of mutations in the VWF gene that lead to decreased synthesis, impaired secretion, increased clearance or a combination thereof. However, expression studies of variants located in the A domains of VWF are limited. We therefore characterized the biosynthesis of VWF mutations, located in the VWF A1–A3 domains, that were found in families diagnosed with VWD. Human Embryonic Kidney 293 (HEK293) cells were transiently transfected with plasmids encoding full‐length wild‐type VWF or mutant VWF. Six mutations in the A1–A3 domains were expressed. We found that all mutants, except one, showed impaired formation of elongated pseudo‐Weibel‐Palade bodies (WPB). In addition, two mutations also showed reduced numbers of pseudo‐WPB, even in the heterozygous state, and increased endoplasmic reticulum retention, which is in accordance with the impaired regulated secretion seen in patients. Regulated secretion upon stimulation of transfected cells reproduced the in vivo situation, indicating that HEK293 cells expressing VWF variants found in patients with VWD can be used to properly assess defects in regulated secretion.

Circulating Angiogenic Mediators in Patients with Moderate and Severe von Willebrand Disease: A Multicentre Cross-Sectional Study

Inhibition of von Willebrand factor (VWF) expression in endothelial cells results in enhanced, possible dysfunctional angiogenesis, consistent with observations of severe gastrointestinal bleedings caused by vascular malformations in patients with von Willebrand disease (VWD). VWF is stored in endothelial Weibel-Palade bodies (WPB) with several other mediators of angiogenesis, like angiopoietin-2, osteoprotegerin and galectin-3. Increased release of angiopoietin-2 has been observed in medium of endothelial cells lacking VWF, but data on circulating levels of angiogenic factors in patients with VWD are lacking. The aim of this study was therefore to investigate plasma levels of angiogenic factors in patients with various types of VWD to obtain more insight into the pathogenesis of vascular malformations in these patients. We hypothesized that VWF deficiency leads to increased circulating levels of other WPB components. We therefore measured plasma levels of the WPB components angiopoietin-2, osteoprotegerin and galectin-3 as well as two other angiogenic factors (angiopoietin-1 and vascular endothelial growth factor [VEGF]) that are not stored within WPB. We observed that various angiogenic mediators are significantly different between types of VWD patients. Type 2A VWD patients had higher angiopoietin-1 levels compared with type 2B patients. Patients who have increased VWF clearance had higher angiopoietin-2 levels, whereas patients who have impaired VWF synthesis had higher galectin-3 levels. VEGF levels were negatively associated with VWF levels as type 3 VWD patients had the highest VEGF levels. However, complete VWF deficiency did not lead to increased circulating levels of other WPB components.

Von Willebrand factor deficiency reduces liver fibrosis in mice

ABSTRACT Liver diseases are associated with complex changes in the hemostatic system and elevated levels of the platelet‐adhesive protein Von Willebrand factor (VWF) are reported in patients with acute and chronic liver damage. Although elevated levels of VWF are associated with fibrosis in the general population, the role of VWF in acute and chronic liver injury has not been examined in depth in experimental settings. We tested the hypothesis that VWF deficiency inhibits experimental liver injury and fibrosis. Wild‐type (WT) and VWF‐deficient mice were challenged with carbon tetrachloride (CCl4) and the impact of VWF deficiency on acute liver injury and chronic liver fibrosis was determined. VWF deficiency did not significantly affect acute CCl4‐induced hepatocellular necrosis in mice. Chronic CCl4 challenge, twice weekly for 6 weeks, significantly increased hepatic stellate cell activation and collagen deposition in livers of WT mice. Interestingly, hepatic induction of several profibrogenic and stellate cell activation genes was attenuated in VWF‐deficient mice. Moreover, birefringent sirius red staining (indicating type I and III collagens) and type I collagen immunofluorescence indicated a reduction in hepatic collagen deposition in CCl4‐exposed VWF‐deficient mice compared to CCl4‐exposed WT mice. The results indicate that VWF deficiency attenuates chronic CCl4‐induced liver fibrosis without affecting acute hepatocellular necrosis. The results are the first to demonstrate that VWF deficiency reduces the progression of liver fibrosis, suggesting a mechanistic role of elevated plasma VWF levels in cirrhosis. HIGHLIGHTSPlasma von Willebrand Factor (VWF) levels increase in experimental liver damage.VWF contributes to liver myofibroblast activation in experimental fibrosis.VWF deficiency reduces experimental fibrosis without affecting acute liver injury.