von Willebrand factor binds to angiopoietin-2 within endothelial cells and after release from Weibel-Palade bodies

BACKGROUND: The von Willebrand factor (VWF) is a multimeric plasma glycoprotein essential for hemostasis, inflammation, and angiogenesis. The majority of VWF is synthesized by endothelial cells (ECs) and stored in Weibel-Palade bodies (WPB). Among the range of proteins shown to co-localize to WPB is angiopoietin-2 (Angpt-2), a ligand of the receptor tyrosine kinase Tie-2. We have previously shown that VWF itself regulates angiogenesis, raising the hypothesis that some of the angiogenic activity of VWF may be mediated by its interaction with Angpt-2. METHODS: Static-binding assays were used to probe the interaction between Angpt-2 and VWF. Binding in media from cultured human umbilical vein ECs s and in plasma was determined by immunoprecipitation experiments. Immunofluorescence was used to detect the presence of Angpt-2 on VWF strings, and flow assays were used to investigate the effect on VWF function. RESULTS: Static-binding assays revealed that Angpt-2 bound to VWF with high affinity (KD,app ∼3 nM) in a pH and calcium-dependent manner. The interaction was localized to the VWF A1 domain. Co-immunoprecipitation experiments demonstrated that the complex persisted following stimulated secretion from ECs and was present in plasma. Angpt-2 was also visible on VWF strings on stimulated ECs. The VWF-Angpt-2 complex did not inhibit the binding of Angpt-2 to Tie-2 and did not significantly interfere with VWF-platelet capture. CONCLUSIONS: Together, these data demonstrate a direct binding interaction between Angpt-2 and VWF that persists after secretion. VWF may act to localize Angpt-2; further work is required to establish the functional consequences of this interaction

Sialylation on O-linked glycans protects von Willebrand factor from macrophage galactose lectin mediated clearance

Terminal sialylation determines the plasma half-life of von Willebrand factor (VWF). A role for macrophage galactose lectin (MGL) in regulating hyposialylated VWF clearance has recently been proposed. In this study, we showed that MGL influences physiological plasma VWF clearance. MGL inhibition was associated with a significantly extended mean residence time and 3-fold increase in endogenous plasma VWF antigen levels (P<0.05). Using a series of VWF truncations, we further demonstrated that the A1 domain of VWF is predominantly responsible for enabling the MGL interaction. Binding of both full-length and VWF-A1-A2-A3 to MGL was significantly enhanced in the presence of ristocetin (P<0.05), suggesting that the MGL-binding site in A1 is not fully accessible in globular VWF. Additional studies using different VWF glycoforms demonstrated that VWF O-linked glycans, clustered at either end of the A1 domain, play a key role in protecting VWF against MGLmediated clearance. Reduced sialylation has been associated with pathological, increased clearance of VWF in patients with von Willebrand disease. Herein, we demonstrate that specific loss of α2-3 linked sialylation from O-glycans results in markedly increased MGL-binding in vitro, and markedly enhanced MGL-mediated clearance of VWF in vivo. Our data further show that the asialoglycoprotein receptor (ASGPR) does not have a significant role in mediating the increased clearance of VWF following loss of O-sialylation. Conversely however, we observed that loss of N-linked sialylation from VWF drives enhanced circulatory clearance predominantly via the ASGPR. Collectively, our data support the hypothesis that in addition to regulating physiological VWF clearance, the MGL receptor works in tandem with ASGPR to modulate enhanced clearance of aberrantly sialylated VWF in the pathogenesis of von Willebrand disease

The heparin binding domain of von Willebrand factor binds to growth factors and promotes angiogenesis in wound healing

During wound healing, the distribution, availability, and signaling of growth factors (GFs) are orchestrated by their binding to extracellular matrix components in the wound microenvironment. Extracellular matrix proteins have been shown to modulate angiogenesis and promote wound healing through GF binding. The hemostatic protein von Willebrand factor (VWF) released by endothelial cells (ECs) in plasma and in the subendothelial matrix has been shown to regulate angiogenesis; this function is relevant to patients in whom VWF deficiency or dysfunction is associated with vascular malformations. Here, we show that VWF deficiency in mice causes delayed wound healing accompanied by decreased angiogenesis and decreased amounts of angiogenic GFs in the wound. We show that in vitro VWF binds to several GFs, including vascular endothelial growth factor-A (VEGF-A) isoforms and platelet-derived growth factor-BB (PDGF-BB), mainly through the heparin-binding domain (HBD) within the VWF A1 domain. VWF also binds to VEGF-A and fibroblast growth factor-2 (FGF-2) in human plasma and colocalizes with VEGF-A in ECs. Incorporation of the VWF A1 HBD into fibrin matrices enables sequestration and slow release of incorporated GFs. In vivo, VWF A1 HBD-functionalized fibrin matrices increased angiogenesis and GF retention in VWF-deficient mice. Treatment of chronic skin wounds in diabetic mice with VEGF-A165 and PDGF-BB incorporated within VWF A1 HBD-functionalized fibrin matrices accelerated wound healing, with increased angiogenesis and smooth muscle cell proliferation. Therefore, the VWF A1 HBD can function as a GF reservoir, leading to effective angiogenesis and tissue regeneration

Gene therapy for thrombotic thrombocytopaenic purpura

Thrombotic Thrombocytopaenic Purpura (TTP) is a rare (∼1/200,000 people) but life‐threatening disease caused by inherited or acquired deficiencies in ADAMTS13; a metalloprotease responsible for cleavage of large von Willebrand factor (VWF) multimers in the plasma. Reduced cleavage of thrombogenic VWF multimers through deficient ADAMTS13 can lead to spontaneous, wide‐spread accumulation of platelet‐rich thrombi. Without treatment, thrombi accumulation within the microvasculature causes organ failure and death in 90% of acute events. Individuals with TTP receive regular plasma infusions to restore ADAMTS13 levels. Despite current treatments reducing mortality rates, high treatment burden and morbidity associated with donor‐derived plasma warrants the development of a novel therapy for TTP. Gene therapy offers an alternative treatment which could prevent the onset of life‐threatening acute TTP episodes. The UK Cystic Fibrosis Gene Therapy Consortium, has developed a lentivirus pseudotyped with the Sendai virus envelope proteins F and HN for efficient lung gene transfer. Here, we assess whether lungs can be used as ‘factories’ for efficient and persistent ADAMTS13 production. We first cloned ADAMTS13 cDNA into a lentivirus producer plasmid and demonstrated proteolytic activity against VWF following co‐expression in HEK293T cells and subsequent detection of cleaved VWF by SDS‐PAGE. Vector is currently being manufactured using GMP‐compliant production methods. Next, ADAMTS13 knockout mice were characterised to determine suitable biomarkers (e.g. ADAMTS13 plasma levels and VWF cleavage activity) for assessing efficacy of pulmonary gene transfer. Future work will assess the restoration of plasma ADAMTS13 function in knockout mice and protection against TTP‐like symptoms

ADAMTS-13 glycans and conformation-dependent activity.

Background: ADAMTS-13 activity can be regulated by its conformation, whereby interactions between the C-terminal CUB domains and the spacer domain maintain ADAMTS-13 in a closed conformation. ADAMTS-13 contains 10 N-linked glycans, with four sites present in theTSP2 through to CUB domains that may contribute to its conformation. Objectives/Methods: We hypothesized that glycosylation contributes to ADAMTS-13 conformation and function. The proteolytic activity of glycan-modified ADAMTS-13 was assessed under static and shear stress conditions. Results: Enzymatic removal of terminal silaic acid or entire N-linked glycan chains decreased activity against FRETS-VWF73 at pH 7.4 and against full-length von Willebrand factor (VWF) under shear stress. Using truncated ADAMTS-13, we demonstrated that this was attributable to loss of sialic acid from the glycans in the metalloprotease domain and an effect of N-linked glycosylation in the TSP2 through to CUB domains. Mutation of the N-linked glycan sites in the MDTCS domains reduced or abolished protein expression. However, the N707Q, N828Q, N1235Q and N1354Q (TSP2, TSP4, CUB1, and CUB2 domains, respectively) variants were expressed normally. Interestingly, the N707Q and N828Q variants showed reduced activity against FRETS-VWF73, but normal activity under flow conditions. In contrast, the N1235Q and N1354Q variants had enhanced activity against FRETS-VWF73 and VWF under shear stress. Immunoprecipitation experiments confirmed that loss of N-linked glycans in the CUB domains significantly reduced the interaction with the spacer domain and enhanced binding to the 6A6 anti-ADAMTS-13 antibody, which recognizes a cryptic epitope in the metalloprotease domain. Conclusions: Together, these data demonstrate that the N-linked glycans of ADAMTS-13 play a crucial role in regulating ADAMTS-13 activity

Blocking von Willebrand factor free thiols inhibits binding to collagen under high and pathological shear stress

Background Von Willebrand factor (VWF) contains a number of free thiols, the majority of which are located in its C‐domains, and these have been shown to alter VWF function, However, the impact of free thiols on function following acute exposure of VWF to collagen under high and pathological shear stress has not been determined. Methods VWF free thiols were blocked with N‐ethylmaleimide and flow assays performed under high and pathological shear rates to determine the impact on platelet capture and collagen binding function. Atomic force microscopy (AFM) was used to probe the interaction of VWF with collagen and molecular simulations conducted to determine the effect of free thiols on the flexibility of the VWF‐C4 domain. Results Blockade of VWF free thiols reduced VWF‐mediated platelet capture to collagen in a shear‐dependent manner, with platelet capture virtually abolished above 5000 s−1 and in regions of stenosis in microfluidic channels. Direct visualization of VWF fibers formed under extreme pathological shear rates and analysis of collagen‐bound VWF attributed the effect to altered binding of VWF to collagen. AFM measurements showed that thiol‐blockade reduced the lifetime and strength of the VWF‐collagen bond. Pulling simulations of the VWF‐C4 domain demonstrated that with one or two reduced disulphide bonds the C4 domain has increased flexibility and the propensity to undergo free‐thiol exchange. Conclusions We conclude that free thiols in the C‐domains of VWF enhance the flexibility of the molecule and enable it to withstand high shear forces following collagen binding, demonstrating a previously unrecognized role for VWF free thiols