VWF-deficient Mice Research Articles

Utility of a high VWF: FVIII ratio in preventing FVIII accumulation: a study in VWF-deficient mice.

Treatment of von Willebrand disease typically requires multiple infusions of von Willebrand factor (VWF)/factor VIII (FVIII) concentrate. Accumulation of FVIII is a clinical concern due to potential risk for thromboembolism. This study sought to determine whether VWF/FVIII concentrate of high VWF:FVIII ratio can prevent FVIII accumulation. VWF-deficient knockout mice received four 150 IU/kg VWF:ristocetin cofactor (RCo) infusions at 3-h intervals, with VWF/FVIII concentrates of a high (Haemate P/Humate-P) or low (Wilate) VWF:FVIII ratio. After each infusion, trough FVIII and VWF levels in plasma were determined. Separately, pharmacokinetic analysis was performed after single 250-IU/kg VWF:RCo infusions of each concentrate. Over the course of the four infusions, trough FVIII increased significantly in the group receiving Wilate (P < 0.001), but not Haemate P/Humate P (P = 0.058). After the first infusion, mean trough FVIII level in the Wilate group (31.7 IU/dl) was greater by 82% (P = 0.017) than that in the Haemate P/Humate P group (17.4 IU/dl). After the final infusion, mean trough FVIII of animals receiving Wilate (55.1 IU/dl) continued to exceed that of Haemate P/Humate P recipients (30.2 IU/dl) significantly (P < 0.001). Trough VWF levels were similar in the two groups. The VWF pharmacokinetics of the two concentrates coincided closely; however, the FVIII peak concentration and area under the curve were approximately twice as great in the mice treated with Wilate. In a murine model of severe von Willebrand disease, a VWF/FVIII concentrate with a high VWF:FVIII ratio prevented persistent exposure to elevated trough FVIII levels.

Probing ADAMTS13 substrate specificity using phage display.

Von Willebrand factor (VWF) is a large, multimeric protein that regulates hemostasis by tethering platelets to the subendothelial matrix at sites of vascular damage. The procoagulant activity of plasma VWF correlates with the length of VWF multimers, which is proteolytically controlled by the metalloprotease ADAMTS13. To probe ADAMTS13 substrate specificity, we created phage display libraries containing randomly mutated residues of a minimal ADAMTS13 substrate fragment of VWF, termed VWF73. The libraries were screened for phage particles displaying VWF73 mutant peptides that were resistant to proteolysis by ADAMTS13. These peptides exhibited the greatest mutation frequency near the ADAMTS13 scissile residues. Kinetic assays using mutant and wild-type substrates demonstrated excellent agreement between rates of cleavage for mutant phage particles and the corresponding mutant peptides. Cleavage resistance of selected mutations was tested in vivo using hydrodynamic injection of corresponding full-length expression plasmids into VWF-deficient mice. These studies confirmed the resistance to cleavage resulting from select amino acid substitutions and uncovered evidence of alternate cleavage sites and recognition by other proteases in the circulation of ADAMTS13 deficient mice. Taken together, these studies demonstrate the key role of specific amino acids residues including P3-P2’ and P11’, for substrate specificity and emphasize the importance in flowing blood of other ADAMTS13–VWF exosite interactions outside of VWF73.

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 groupO have ~25% lower plasma levels. The exact mechanism behind this relationship remains unknown, although effects on clearance have been postulated. To determine whether clearance of VWF is directly dependent on the presence of ABH antigens on VWF. Three type3 von Willebrand disease (VWD) patients were infused with Haemate-P, and the relative loading of VWF with ABH antigens at different time points was measured. VWF-deficient mice were injected with purified plasma-derived human VWF obtained from donors with either blood groupA, blood groupB, or blood groupO. In mice, we found no difference in clearance rate between plasma-derived blood groupA, blood groupB and blood groupO VWF. Faster clearance of the blood groupO VWF present in Haemate-P infused in type3 VWD patients would have resulted in a relative increase in the loading of VWF with A and B antigens over time. However, we observed a two-fold decrease in the loading with A and B antigens in two out of three patients, and stable loading in the third patient. There is no direct effect of ABH antigens on VWF in VWF clearance. We demonstrate that, in a direct comparison within one individual, blood groupO VWF is not cleared faster than blood groupA or blood groupB VWF. Clearance differences between blood groupO and non-blood groupO individuals may therefore be related to the blood group status of the individual rather than the ABH antigen loading on VWF itself.

Adhesion of Staphylococcus aureus to the vessel wall under flow is mediated by von Willebrand factor–binding protein

AbstractAdhesion of Staphylococcus aureus to blood vessels under shear stress requires von Willebrand factor (VWF). Several bacterial factors have been proposed to interact with VWF, including VWF-binding protein (vWbp), a secreted coagulase that activates the host's prothrombin to generate fibrin. We measured the adhesion of S aureus Newman and a vWbp-deficient mutant (vwb) to VWF, collagen, and activated endothelial cells in a microparallel flow chamber. In vivo adhesion of S aureus was evaluated in the mesenteric circulation of wild-type (WT) and VWF-deficient mice. We found a shear-dependent increase in adhesion of S aureus to the (sub)endothelium that was dependent on interactions between vWbp and the A1-domain of VWF. Adhesion was further enhanced by coagulase-mediated fibrin formation that clustered bacteria and recruited platelets into bacterial microthrombi. In vivo, deficiency of vWbp or VWF as well as inhibition of coagulase activity reduced S aureus adhesion. We conclude that vWbp contributes to vascular adhesion of S aureus through 2 independent mechanisms: shear-mediated binding to VWF and activation of prothrombin to form S aureus–fibrin–platelet aggregates.

Neutrophils mediate edema formation but not mechanical allodynia during zymosan-induced inflammation

Inflammatory pain is based on stimulation and sensitization of peripheral endings of sensory neurons (nociceptors) by pronociceptive mediators. These mediators can be released by resident cells, as well as invading immune cells. Although neutrophils are known to release various mediators, which can stimulate or sensitize nociceptors, the extent of their contribution to nociceptive responses is unclear. Here, we studied the contribution of neutrophils to zymosan-induced inflammatory pain, which is characterized by an early recruitment of high numbers of neutrophils. Surprisingly, antibody-mediated neutrophil depletion caused a complete loss of edema formation but had no effect on mechanical pain thresholds. Blockage of the interaction between neutrophils and platelets or endothelial cells using antibodies directed against CD11b and CD162 reduced neutrophil recruitment to the site of inflammation. Again, the treatment decreased zymosan-induced edemas without altering mechanical pain thresholds. Also, HLB-219 mice, which have five to 10 times less platelets than WT mice, showed reduced neutrophil recruitment to the site of inflammation and decreased edema sizes, whereas, again, mechanical thresholds were unaltered. The effects observed in HLB-219 mice were relatively small and not reproduced in vWF-deficient mice or after antibody-mediated blockage of GPIbα. Flow chamber and transmigration assays showed that platelets were not necessary for neutrophil adhesion to endothelial cells but increased their transmigration. Taken together, zymosan-induced mechanical allodynia is, in contrast to edema formation, independent of neutrophils, and recruitment of neutrophils is only partly influenced by interactions with platelets.

Anfibatide, a novel GPIb complex antagonist, inhibits platelet adhesion and thrombus formation in vitro and in vivo in murine models of thrombosis

Platelet adhesion and aggregation at the sites of vascular injury are key events for thrombosis and haemostasis. It has been well demonstrated that interaction between glycoprotein (GP) Ibα and von Willebrand factor (VWF) initiates platelet adhesion and contributes to platelet aggregation, particularly at high shear. GPIb has long been suggested as a desirable antithrombotic target, but anti-GPIb therapy has never been successfully developed. Here, we evaluated the antithrombotic potential of Anfibatide, a novel snake venom-derived GPIb antagonist.We found Anfibatide inhibited washed murine platelet aggregation induced by ristocetin and recombinant murine VWF. It also blocked botrocetin-induced binding of murine plasma VWF to recombinant human GPIbα. Interestingly, Anfibatide did not inhibit botrocetin-induced aggregation of platelet-rich plasma, indicating that its binding site may differ from other snake venom-derived GPIb antagonists. Anfibatide strongly inhibited platelet adhesion, aggregation, and thrombus formation in perfusion chambers at high shear conditions and efficiently dissolved preformed thrombi. Anfibatide also inhibited thrombus growth at low shear conditions, though less than at high shear. Using intravital microscopy, we found that Anfibatide markedly inhibited thrombosis in laser-injured cremaster vessels and prevented vessel occlusion in FeCl3-injured mesenteric vessels. Importantly, Anfibatide further inhibited residual thrombosis in VWF-deficient mice, suggesting that Anfibatide has additional antithrombotic effect beyond its inhibitory role in GPIb-VWF interaction. Anfibatide did not significantly cause platelet activation, prolong tail bleeding time, or cause bleeding diathesis in mice. Thus, consistent with the data from an ongoing clinical trial, the data from this study suggests that Anfibatide is a potent and safe antithrombotic agent.

Regulation Of Factor VIII Clearance By Mannose-Binding Lectins

The coagulation factors von Willebrand factor (FVIII) and factor VIII (FVIII) circulate in the plasma in a non-covalent complex. VWF acts as a carrier for FVIII and protects it from proteolysis and accelerated clearance. However, in the absence of VWF, the molecular mechanisms that regulate FVIII clearance are largely uncharacterized.The glycosylation of FVIII may regulate its interaction with lectin clearance receptors such as the asialoglycoprotein receptor and siglec-5. The FVIII polypeptide is modified by the addition of 24 N-linked glycans and 7 O-linked glycans. The majority of N-linked glycans on FVIII are complex type and predominantly localized to the B-domain. However, two high mannose glycans are found on the A1 and C1 domains. In these studies, we characterized the ability of mannose binding lectins to interact with FVIII and mediate its clearance from the plasma.We have previously reported that the endothelial mannose binding lectin CLEC4M is a novel clearance receptor for FVIII. Removal of N-linked glycans, or pre-incubation of recombinant CLEC4M with the mannose polymer mannan attenuated binding of CLEC4M to FVIII. Additionally, the pre-incubation of CLEC4M-expressing cells with mannan significantly decreased binding and internalization of FVIII.In these studies, high mannose glycans on recombinant human FVIII were removed using the endoglycosidase endoH. Effective removal of FVIII high mannose glycans by endoH was confirmed via a lectin binding assay to Galanthus Nivalis lectin (50%, p=0.006). The binding of recombinant CLEC4M-Fc to endoH-treated FVIII was measured using a solid phase binding assay. Removal of high mannose glycans attenuated the interaction between FVIII and CLEC4M-Fc by 41% (p=0.005). A 49% decrease in the internalization of endoH-treated FVIII was observed on CLEC4M-expressing cells relative to control (p=0.046).We next sought to confirm the contribution of mannose binding lectins to the clearance of FVIII in a murine model. Although there is no murine ortholog to CLEC4M, mice express a series of CLEC4M homologs termed SIGNRs. Similar to CLEC4M, SIGNR1 is expressed on liver sinusoidal endothelial cells. While significant structural differences exist between CLEC4M and SIGNR1, they retain 59% amino acid identity between their carbohydrate recognition domains, and SIGNR1 has been shown to bind mannosylated as well as fucosylated glycoproteins.We assessed the contribution of SIGNR1 to VWF and FVIII clearance. Plasma levels of VWF:Ag, and FVIII:C were quantified in SIGNR1 deficient mice by ELISA and chromogenic assay respectively. Endogenous VWF (88% of wild type, p=0.233, n=35) and FVIII (110% of wild type, p=0.435, n=35) in SIGNR1 deficient mice were not different compared to wild type mice. SIGNR1/VWF deficient mice were bred on a C57Bl/6 background; mice received tail vein infusions of recombinant human FVIII (200 U/kg) or plasma derived VWF-FVIII complex (200 U/kg). Blood was sampled via retro-orbital puncture. VWF:Ag and FVIII:Ag levels were measured with ELISA and elimination half life was calculated using one phase exponential decay. Half-lives of infused recombinant human FVIII (VWF-/- t½=8.74 minutes, SIGNR1-/-/VWF-/- t½=10.03 minutes, p=0.898) and human plasma-derived VWF-FVIII complex (VWF-/- t½=46.09 minutes, SIGNR1-/-/VWF-/- t½=49.83 minutes, p=0.934) were not significantly different.We finally sought to characterize the contribution of other mannose-binding lectins to FVIII clearance in mice. VWF deficient mice received tail vein infusions of mannan (6 mg/kg) or saline followed after 3 minutes by tail vein infusions of recombinant human FVIII (200 U/kg). Pre-infusion of mannan into VWF deficient mice did not modify the half-life of FVIII relative to the control (control t½= 10.56 minutes, mannan t½= 9.38 minutes. p=0.33). We also assessed the clearance of endoH-treated FVIII in VWF deficient mice, and did not observe a change relative to the control (control t½=12.28 minutes, endoH FVIII t½=11.23 minutes, p=0.812).These studies suggest that CLEC4M interacts with FVIII in part through its high mannose glycans in vitro. However, our in vivo studies were unable to reveal a significant role for mannose-binding lectins in FVIII clearance in mouse models. Disclosures:James:CSL Behring: Honoraria, Research Funding; Octapharma: Honoraria, Research Funding; Baxter: Honoraria; Bayer: Honoraria.

VON WILLEBRAND FACTOR ABNORMALITIES STUDIED IN THE MOUSE MODEL: WHAT WE LEARNED ABOUT VWF FUNCTIONS

Up until recently, von Willebrand Factor (VWF) structure-function relationships have only been studied through in vitro approaches. A powerful technique known as hydrodynamic gene transfer, which allows transient expression of a transgene by mouse hepatocytes, has led to an important shift in VWF research. Indeed this approach has now enabled us to transiently express a number of VWF mutants in VWF-deficient mice in order to test the relative importance of specific residues in different aspects of VWF biology and functions in an in vivo setting. As a result, mice reproducing various types of von Willebrand disease have been generated, models that will be useful to test new therapies. This approach also allowed a more precise identification of the importance of VWF interaction with subendothelial collagens and with platelets receptors in hemostasis and thrombosis. The recent advances gathered from these studies as well as the pros and cons of the technique will be reviewed here.

A novel B-domain O-glycoPEGylated FVIII (N8-GP) demonstrates full efficacy and prolonged effect in hemophilic mice models

AbstractFrequent infusions of intravenous factor VIII (FVIII) are required to prevent bleeding associated with hemophilia A. To reduce the treatment burden, recombinant FVIII with a longer half-life was developed without changing the protein structure. FVIII–polyethylene glycol (PEG) conjugates were prepared using an enzymatic process coupling PEG (ranging from 10 to 80 kDa) selectively to a unique O-linked glycan in the FVIII B-domain. Binding to von Willebrand factor (VWF) was maintained for all conjugates. Upon cleavage by thrombin, the B-domain and the associated PEG were released, generating activated FVIII (FVIIIa) with the same primary structure and specific activity as native FVIIIa. In both FVIII- and VWF-deficient mice, the half-life was found to increase with the size of PEG. In vivo potency and efficacy of FVIII conjugated with a 40-kDa PEG (N8-GP) and unmodified FVIII were not different. N8-GP had a longer duration of effect in FVIII-deficient mouse models, approximately a twofold prolonged half-life in mice, rabbits, and cynomolgus monkeys; however, the prolongation was less pronounced in rats. Binding capacity of N8-GP on human monocyte-derived dendritic cells was reduced compared with unmodified FVIII, resulting in several-fold reduced cellular uptake. In conclusion, N8-GP has the potential to offer efficacious prevention and treatment of bleeds in hemophilia A at reduced dosing frequency.

Mutations in the A3 domain of Von Willebrand factor inducing combined qualitative and quantitative defects in the protein

AbstractTwo 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 Ib and IIbIIIa. Co-transfections 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.

ADAMTS13 Deficiency Exacerbates VWF-Dependent Acute Myocardial Ischemia/Reperfusion Injury in Mice

AbstractAbstract 264 Background and objective:ADAMTS13 (A Disintegrin And Metalloprotease with Thrombospondin type I repeats-13) cleaves von Willebrand factor (VWF), a large multimeric protein that plays an important role in thrombus formation by binding to platelets following vascular injury. Epidemiological studies suggest that elevated VWF levels and reduced ADAMTS13 activity in the plasma are risk factors for myocardial infarction. It remains unknown, however, whether the ADAMTS13-VWF axis plays a causal role in the pathophysiology of myocardial infarction. We tested the hypothesis that ADAMTS13 reduces VWF-mediated acute myocardial ischemia/reperfusion (I/R) injury in mice. Methods:Myocardial infarction was induced in male mice (8–10 weeks of age) by ligating the left anterior descending coronary artery for 30 minutes followed by 23.5 hours of reperfusion. The extent of myocardium damage was evaluated by measuring infarct size (%) in 2 mm serial sections stained with 2% triphenyl-2, 3, 4-tetrazolium-chloride. Neutrophil infiltration and myocyte apoptosis in the left ventricular area was quantified by immunohistochemistry and TUNEL staining respectively. Results:Adamts13-/- mice exhibited significantly increased infarct size (22.2 % ± 1.1 %, P <.01) compared with WT mice (16.9 % ± 1.2 %, P<0.05). Plasma levels of cardiac troponin T (cTnT), an index of myocyte injury, were significantly higher in Adamts13−/− mice compared with WT mice (P <0.01). Adamts13+/− mice, which have a 50% reduction in ADAMTS13 activity, had similar sized infarcts (16.6 ± 1.3%) and cTnT levels compared to those in WT mice. Larger infarcts in the Adamts13−/− mice were concordant with increased neutrophil infiltration and myocyte apoptosis compared with WT mice. Because VWF remains the only known substrate of ADAMTS13 in multiple experimental models, we hypothesized that ADAMTS13 reduces myocardial injury through its proteolytic effect on hyper adhesive ULVWF and /or VWF. Vwf−/− mice exhibited significantly reduced infarct size, neutrophil infiltration, and myocyte apoptosis compared with WT mice, suggesting a detrimental role for VWF in myocardial I/R injury. VWF-deficient mice have a defect in regulation of endothelial P-selectin due to the loss of Weibel-Palade body formation. To confirm that exacerbated myocardial I/R injury in the setting of ADAMTS13 deficiency is dependent on VWF rather than P-selectin, we compared WT and Adamts13−/− mice treated with anti-VWF inhibitory antibodies. Treating WT or Adamts13−/− mice with neutralizing antibodies to VWF prior to myocardial I/R injury significantly reduced infarct size compared with control Ig-treated mice, suggesting that exacerbated myocardial I/R injury observed in Adamts13−/− mice is entirely VWF-dependent. Finally, myocardial I/R injury in Adamts13−/−/Vwf−/− mice was similar to that in Vwf−/− mice, suggesting that the exacerbated myocardial I/R injury observed in the setting of ADAMTS13 deficiency is VWF-dependent. Conclusion:These findings reveal a new role for anti-thrombotic enzyme ADAMTS13 in reducing VWF-mediated myocardial ischemia/reperfusion injury. Disclosures:Lentz:Novo Nordisk A/S: Consultancy, Investigator Other.

ADAMTS13 reduces VWF‐mediated acute inflammation following focal cerebral ischemia in mice

ADAMTS13 cleaves hyperactive ultra-large von Willebrand factor (ULVWF) multimers into smaller and less active forms. It remains unknown whether VWF-mediated inflammatory processes play a role in the enhanced brain injury due to ADAMTS13 deficiency. We tested the hypothesis that the deleterious effect of ADAMTS13 deficiency on ischemic brain injury is mediated through VWF-dependent enhanced vascular inflammation. Transient focal cerebral ischemia was induced by 60 min of occlusion of the right middle cerebral artery. Myeloperoxidase (MPO) activity and inflammatory cytokines in the infarcted region were evaluated 23 h after reperfusion injury. Neutrophil infiltration within the infarct and surrounding areas was quantitated by immunohistochemistry. We report that ADAMTS13-deficient mice exhibited significantly enlarged infarct size, concordant with increased myeloperoxidase (MPO) activity, neutrophil infiltration and expression of the pro-inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). In contrast, VWF-deficient mice exhibited significantly reduced MPO activity, neutrophil infiltration and inflammatory cytokine induction, demonstrating a role of VWF in these inflammatory processes. Mice deficient for both ADAMTS13 and VWF exhibited an identical reduction of the same inflammatory parameters, demonstrating that the increased inflammation observed in ADAMTS13-deficient mice is VWF dependent. Finally, the increased infarct size observed in ADAMTS13-deficient mice was completely abrogated by prior immunodepletion of neutrophils, demonstrating a causal role for acute inflammation in the enhanced brain injury that occurs in the setting of ADAMTS13 deficiency. These findings provide new evidence for ADAMTS13 in reducing VWF-mediated acute cerebral inflammation following ischemic stroke.

Von Willebrand disease biology

*Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden; Department of Thrombosis and Hemostasis, C2-R Einthoven Laboratory for Experimental Vascular Medicine, LeidenUniversity Medical Center, Leiden, The Netherlands; Centre for Haematology, Imperial College London, HammersmithHospital Campus, London;§INSERM U770, Cedex, France; and –Department of Pathology and Molecular Medicine, QueensUniversity, Kingston, ON, Canada

Platelets, glycoprotein Ib-IX, and von Willebrand factor are required for FeCl3-induced occlusive thrombus formation in the inferior vena cava of mice

Venous thromboembolism is a leading cause of death from cardiovascular disease. Despite the importance of the glycoprotein (GP) Ib-IX/von Willebrand factor (vWF) axis in arterial thrombosis, its requirement in venous, not venule thrombosis in response to endothelial injury (not stenosis or stasis) is uncharacterized. GPIbα-vWF participation in FeCl3-induced thrombus formation was evaluated in the inferior vena cava (IVC). Stable, occlusive thrombus formation in response to FeCl3-induced injury of the IVC was studied. FeCl3 (20% FeCl3, 10 minutes)-induced occlusive thrombosis required platelets as confirmed by a lack of occlusion in thrombocytopenic mice, and stable occlusion in control animals. No IVC occlusion was observed using GPIbα-deficient animals, a model of the human Bernard-Soulier syndrome (BSS). Transgenic IL-4 R/GPIbα mice (lack murine GPIbα, but express the extracellular domain of the human interleukin (IL-4 receptor fused to the transmembrane and cytoplasmic domains of human GPIbα) were studied to determine if the absence of IVC occlusion in the BSS mouse was caused by GPIbα extracellular domain deficiency rather than platelet BSS phenotype associated abnormalities. As with GPIbα knock-out mice, no occlusion was observed in the IVC of IL-4 R/GPIbα mice. The IVC of vWF-deficient mice also failed to occlude in response to FeCl3 treatment. The chimeric protein GPIbα(2V)-Fc prevented occlusion, demonstrating that GPIbα-vWF A1 domain interaction is required for FeCl3-induced stable thrombus formation in the IVC. Therefore, FeCl3-induced stable, occlusive thrombus formation in the IVC is platelet, and apparently GPIbα-vWF interaction dependent, despite the large diameter and low venous flow rate in the IVC.

In VivoGene Transfer Strategies to Achieve Partial Correction of von Willebrand Disease

von Willebrand disease (VWD), the most common hereditary coagulation disorder, results from mutations in the 52-exon gene for von Willebrand factor (VWF), which encodes an 8.4-kB cDNA. Studies with VWF cDNA plasmids have demonstrated that in vivo gene transfer to the liver will correct the coagulation dysfunction in VWF(-/-) mice, but the correction is transient. To develop gene therapy for VWF that would mediate long-term expression of the VWF cDNA in liver, we first evaluated segmental pre-mRNA trans-splicing (SPTS) with two adeno-associated virus (AAV) serotype 8 vectors, each delivering one-half of the VWF cDNA. However, although the two vectors functioned well to generate VWF multimers after infection of cells in vitro, the efficiency of SPTS was insufficient to correct the VWF(-/-) mouse in vivo. As an alternative, we assessed the ability of a lentiviral vector to transfer the intact murine VWF cDNA in vivo directly to the neonatal liver of VWF(-/-) mice, using generation of VWF multimers, bleeding time, and bleeding volume as efficacy parameters. The VWF lentivirus generated VWF multimers and partially or completely corrected the coagulation defect on a persistent basis in 33% of the treated VWF-deficient mice. On the basis of the concept that partial persistent correction with gene transfer could be beneficial in VWD patients, these observations suggest that lentiviral delivery of VWF cDNA should be explored as a candidate for gene therapy in patients with a severe form of VWD.

In vivo analysis of the role of O-glycosylations of von Willebrand factor.

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.

Identification of Galectin-1 and Galectin-3 as Novel Partners for Von Willebrand Factor

Although von Willebrand factor (VWF) is a heavily glycosylated protein, its potential to associate with glycan-binding proteins is poorly investigated. Here, we explored its interaction with the glycan-binding proteins galectin-1 and galectin-3. Immunofluorescence analysis using Duolink proximity ligation assays revealed that VWF colocalizes with galectin-1 and galectin-3 in endothelial cells, both before and after stimulation of endothelial cells. Moreover, galectin-1 was found along the typical VWF bundles that are released by endothelial cells. Galectin-1 and galectin-3 could be coprecipitated with VWF from plasma in immunoprecipitation assays, whereas plasma levels of galectin-1 and galectin-3 were significantly reduced in VWF-deficient mice. Binding studies using purified proteins confirmed that VWF could directly interact with both galectins, predominantly via its N-linked glycans. In search of the physiological relevance of the VWF-galectin interaction, we found that inhibition of galectins in in vitro perfusion assays was associated with increased VWF-platelet string formation, a phenomenon that was reproduced in galectin-1/galectin-3 double-deficient mice. These mice were also characterized by a more rapid formation of initial thrombi following ferric chloride-induced injury. We have identified galectin-1 and galectin-3 as novel partners for VWF, and these proteins may modulate VWF-mediated thrombus formation.

Von Willebrand Factor (VWF) Regulates Angiogenesis: Studies in Blood-Derived Endothelial Progenitor Cells From Von Willebrand's Disease Patients and VWF-Deficient Mice

Abstract 5311Dysregulation of angiogenesis is implicated in many diseases. Von Willebrand factor (VWF), a large plasma glycoprotein essential for normal haemostasis is synthesized by endothelial cells (EC) and megakaryocytes. Raised VWF plasma levels are a risk factor for arterial thrombosis, whilst deficiency of VWF causes Von Willebrand disease (VWD), the most common congenital bleeding disorder in man. VWD can be associated with angiodysplasia, vascular malformations linked to defective angiogenesis which are responsible for intractable bleeding. We recently showed that VWF is involved in angiogenesis. Inhibition of VWF expression in human umbilical vein EC (HUVEC) with specific siRNA resulted in increased in vitro angiogenesis on Matrigel, proliferation and migration. Mechanism studies implicated the endothelial VWF receptor, integrin αvβ3 and the angiogenesis regulator angiopoietin-2. The findings were confirmed in EC from VWD patients and in VWF-deficient mice. Blood outgrowth endothelial cells (BOEC) isolated from peripheral blood of patients with VWD showed decreased VWF release, compared to control BOEC, and increased in vitro angiogenesis, migration and proliferation, similar to what observed with VWF siRNA-treated HUVEC. In vivo studies using the matrigel model and imaging of blood vessels in the ear showed increased angiogenesis and vascular network in VWF-deficient mice compared to controls. Recent studies in patients BOEC and in VWF deficient mice provide new insight into the complexity of this phenotype. We have used the mouse model of post-natal angiogenesis in the retina to carry out detailed analysis of angiogenic networks in the VWF-deficient mouse, and found increased vascular density and defective vascular network. Moreover, using BOEC from patients with type I and type II VWD, we have studied VWF intracellular distribution by immunofluorescence confocal analysis and found patterns of expression that point to a variety of defects in synthesis, storage and secretion. These studies define a new function for VWF, which may have clinical implications for VWD and for patients at risk of CV disease. Moreover, studies with BOEC from VWF patients provide a novel understanding of the physiopathology of this disease. Disclosures:No relevant conflicts of interest to declare.

39 Blood-derived endothelial progenitor cells from Von Willebrand's disease patients demonstrate that Von Willebrand factor regulates angiogenesis

Dysregulation of angiogenesis is implicated in many diseases. Von Willebrand factor (VWF), a large plasma glycoprotein essential for normal haemostasis is synthesised by endothelial cells (EC) and megakaryocytes. Raised VWF...

Endothelial von Willebrand factor regulates angiogenesis

AbstractThe regulation of blood vessel formation is of fundamental importance to many physiological processes, and angiogenesis is a major area for novel therapeutic approaches to diseases from ischemia to cancer. A poorly understood clinical manifestation of pathological angiogenesis is angiodysplasia, vascular malformations that cause severe gastrointestinal bleeding. Angiodysplasia can be associated with von Willebrand disease (VWD), the most common bleeding disorder in man. VWD is caused by a defect or deficiency in von Willebrand factor (VWF), a glycoprotein essential for normal hemostasis that is involved in inflammation. We hypothesized that VWF regulates angiogenesis. Inhibition of VWF expression by short interfering RNA (siRNA) in endothelial cells (ECs) caused increased in vitro angiogenesis and increased vascular endothelial growth factor (VEGF) receptor-2 (VEGFR-2)–dependent proliferation and migration, coupled to decreased integrin αvβ3 levels and increased angiopoietin (Ang)–2 release. ECs expanded from blood-derived endothelial progenitor cells of VWD patients confirmed these results. Finally, 2 different approaches, in situ and in vivo, showed increased vascularization in VWF-deficient mice. We therefore identify a new function of VWF in ECs, which confirms VWF as a protein with multiple vascular roles and defines a novel link between hemostasis and angiogenesis. These results may have important consequences for the management of VWD, with potential therapeutic implications for vascular diseases.