α-galactosidase A deficiency promotes von Willebrand factor secretion in models of Fabry disease
Authors: Justin J. Kang, Nayiri M. Kaissarian, Karl C. Desch, Robert J. Kelly, Liming Shu, Peter F. Bodary, James A. Shayman
Journal: Kidney International (2018)

Fabry disease results from loss of activity of the lysosomal enzyme α-galactosidase A (GLA), leading to the accumulation of globoseries glycosphingolipids in vascular endothelial cells. Thrombosis and stroke are life-threatening complications of Fabry disease; however, the mechanism of the vasculopathy remains unclear. We explored the relationship between GLA deficiency and endothelial cell von Willebrand factor (VWF) secretion in in vivo and in vitro models of Fabry disease. Plasma VWF was significantly higher at two months and increased with age in Gla-null compared to wild-type mice. Disruption of GLA in a human endothelial cell line by siRNA and CRISPR/Cas9 resulted in a 3-fold and 5-fold increase in VWF secretion, respectively. The increase in VWF levels was associated with decreased endothelial nitric oxide synthase (eNOS) activity in both in vitro models. Pharmacological approaches that increase nitric oxide bioavailability or decrease reactive oxygen species completely normalized the elevated VWF secretion in GLA deficient cells. In contrast, the abnormality was not readily reversed by recombinant human GLA or by inhibition of glycosphingolipid synthesis with eliglustat. These results suggest that GLA deficiency promotes VWF secretion through eNOS dysregulation, which may contribute to the vasculopathy of Fabry disease. Fabry disease results from loss of activity of the lysosomal enzyme α-galactosidase A (GLA), leading to the accumulation of globoseries glycosphingolipids in vascular endothelial cells. Thrombosis and stroke are life-threatening complications of Fabry disease; however, the mechanism of the vasculopathy remains unclear. We explored the relationship between GLA deficiency and endothelial cell von Willebrand factor (VWF) secretion in in vivo and in vitro models of Fabry disease. Plasma VWF was significantly higher at two months and increased with age in Gla-null compared to wild-type mice. Disruption of GLA in a human endothelial cell line by siRNA and CRISPR/Cas9 resulted in a 3-fold and 5-fold increase in VWF secretion, respectively. The increase in VWF levels was associated with decreased endothelial nitric oxide synthase (eNOS) activity in both in vitro models. Pharmacological approaches that increase nitric oxide bioavailability or decrease reactive oxygen species completely normalized the elevated VWF secretion in GLA deficient cells. In contrast, the abnormality was not readily reversed by recombinant human GLA or by inhibition of glycosphingolipid synthesis with eliglustat. These results suggest that GLA deficiency promotes VWF secretion through eNOS dysregulation, which may contribute to the vasculopathy of Fabry disease. Fabry disease is a rare and often devastating lysosomal storage disorder caused by mutations in the α-galactosidase A (GLA) gene resulting in a partial or complete absence of GLA activity.1Brady R.O. Gal A.E. Bradley R.M. et al.Enzymatic defect in Fabry's disease. Ceramidetrihexosidase deficiency.N Engl J Med. 1967; 276: 1163-1167Crossref PubMed Scopus (844) Google Scholar Although X-linked, heterozygous females may also develop clinically significant cardiovascular disease.2MacDermot K.D. Holmes A. Miners A.H. Anderson-Fabry disease: clinical manifestations and impact of disease in a cohort of 60 obligate carrier females.J Med Genet. 2001; 38: 769-775Crossref PubMed Google Scholar, 3Whybra C. Kampmann C. Willers I. et al.Anderson-Fabry disease: clinical manifestations of disease in female heterozygotes.J Inherit Metab Dis. 2001; 24: 715-724Crossref PubMed Scopus (245) Google Scholar α-Gal A deficiency causes a progressive deposition of globotriaosylceramide (Gb3) in vascular endothelial and smooth muscle cells.4Eng C.M. Desnick R.J. Molecular basis of Fabry disease: mutations and polymorphisms in the human alpha-galactosidase A gene.Hum Mutat. 1994; 3: 103-111Crossref PubMed Scopus (135) Google Scholar, 5Shen J.S. Meng X.L. Moore D.F. et al.Globotriaosylceramide induces oxidative stress and up-regulates cell adhesion molecule expression in Fabry disease endothelial cells.Mol Genet Metab. 2008; 95: 163-168Crossref PubMed Scopus (169) Google Scholar Increased levels of deacylated Gb3, lyso-Gb3, also occur.6Aerts J.M. Groener J.E. Kuiper S. et al.Elevated globotriaosylsphingosine is a hallmark of Fabry disease.Proc Natl Acad Sci U S A. 2008; 105: 2812-2817Crossref PubMed Scopus (524) Google Scholar Fabry disease is manifest in childhood with angiokeratomas, neuropathic pain, hypohidrosis, and gastrointestinal pain.7Sestito S. Ceravolo F. Concolino D. Anderson-Fabry disease in children.Curr Pharm Des. 2013; 19: 6037-6045Crossref PubMed Scopus (27) Google Scholar Fabry disease patients may develop life-threatening complications as adults, notably stroke, cardiomyopathy, and renal failure.8Germain D.P. Fabry disease.Orphanet J Rare Dis. 2010; 5: 30Crossref PubMed Scopus (739) Google Scholar Thrombotic events occur in Fabry disease patients.9Utsumi K. Yamamoto N. Kase R. et al.High incidence of thrombosis in Fabry's disease.Intern Med. 1997; 36: 327-329Crossref PubMed Scopus (67) Google Scholar, 10Sims K. Politei J. Banikazemi M. et al.Stroke in Fabry disease frequently occurs before diagnosis and in the absence of other clinical events: natural history data from the Fabry Registry.Stroke. 2009; 40: 788-794Crossref PubMed Scopus (265) Google Scholar, 11Lenders M. Karabul N. Duning T. et al.Thromboembolic events in Fabry disease and the impact of factor V Leiden.Neurology. 2015; 84: 1009-1016Crossref PubMed Scopus (29) Google Scholar In addition, accelerated arterial thrombosis is observed following oxidant injury in Gla-knockout mice.12Eitzman D.T. Bodary P.F. Shen Y. et al.Fabry disease in mice is associated with age-dependent susceptibility to vascular thrombosis.J Am Soc Nephrol. 2003; 14: 298-302Crossref PubMed Scopus (64) Google Scholar, 13Shen Y. Bodary P.F. Vargas F.B. et al.Alpha-galactosidase A deficiency leads to increased tissue fibrin deposition and thrombosis in mice homozygous for the factor V Leiden mutation.Stroke. 2006; 37: 1106-1108Crossref PubMed Scopus (25) Google Scholar More generally, the Gla-knockout mouse has provided an important model in elucidating potential mechanisms of vasculopathy in Fabry disease as well as the basis for proof of principle studies for substrate reduction agents such as eliglustat.14Shayman J.A. Eliglustat tartrate: glucosylceramide synthase inhibitor treatment of type 1 Gaucher disease.Drugs Future. 2010; 35: 613-620Crossref PubMed Scopus (82) Google Scholar Prior work has demonstrated that α-Gal A deficiency in this model is also associated with atherogenesis,15Bodary P.F. Shen Y. Vargas F.B. et al.Alpha-galactosidase A deficiency accelerates atherosclerosis in mice with apolipoprotein E deficiency.Circulation. 2005; 111: 629-632Crossref PubMed Scopus (74) Google Scholar oxidant formation,16Shu L. Vivekanandan-Giri A. Pennathur S. et al.Establishing 3-nitrotyrosine as a biomarker for the vasculopathy of Fabry disease.Kidney Int. 2014; 86: 58-66Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar and impaired vaso-relaxation.17Park J.L. Whitesall S.E. D'Alecy L.G. et al.Vascular dysfunction in the alpha-galactosidase A-knockout mouse is an endothelial cell-, plasma membrane-based defect.Clin Exp Pharmacol Physiol. 2008; 35: 1156-1163Crossref PubMed Scopus (45) Google Scholar Clinical studies suggest that cerebral vascular events continue to occur in Fabry disease patients treated despite reduction in Gb3 by long-term enzyme replacement therapy (ERT).18Wilcox W.R. Banikazemi M. Guffon N. et al.Long-term safety and efficacy of enzyme replacement therapy for Fabry disease.Am J Hum Genet. 2004; 75: 65-74Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar, 19Rombach S.M. Smid B.E. Bouwman M.G. et al.Long term enzyme replacement therapy for Fabry disease: effectiveness on kidney, heart and brain.Orphanet J Rare Dis. 2013; 8: 47Crossref PubMed Scopus (121) Google Scholar, 20Weidemann F. Niemann M. Stork S. et al.Long-term outcome of enzyme-replacement therapy in advanced Fabry disease: evidence for disease progression towards serious complications.J Intern Med. 2013; 274: 331-341Crossref PubMed Scopus (150) Google Scholar, 21Beck M. Ricci R. Widmer U. et al.Fabry disease: overall effects of agalsidase alfa treatment.Eur J Clin Invest. 2004; 34: 838-844Crossref PubMed Scopus (202) Google Scholar Endothelial cells are a critically affected cell type in Fabry disease,22Desnick R.J. Brady R. Barranger J. et al.Fabry disease, an under-recognized multisystemic disorder: expert recommendations for diagnosis, management, and enzyme replacement therapy.Ann Intern Med. 2003; 138: 338-346Crossref PubMed Scopus (605) Google Scholar as evidenced by decreased nitric oxide (NO) bioavailability and endothelial nitric oxide synthase (eNOS) uncoupling. Von Willebrand factor (VWF), stored in Weibel-Palade bodies (WPB) of endothelial cells, is a large adhesive glycoprotein that plays a pivotal role in platelet adhesion and subsequent thrombus formation.23Xiang Y. Hwa J. Regulation of VWF expression, and secretion in health and disease.Curr Opin Hematol. 2016; 23: 288-293Crossref PubMed Scopus (31) Google Scholar High levels of VWF are predictive of an increased risk of thrombotic cardiovascular diseases, including stroke and coronary heart disease.24Vischer U.M. von Willebrand factor, endothelial dysfunction, and cardiovascular disease.J Thromb Haemost. 2006; 4: 1186-1193Crossref PubMed Scopus (278) Google Scholar, 25van Schie M.C. de Maat M.P. Isaacs A. et al.Variation in the von Willebrand factor gene is associated with von Willebrand factor levels and with the risk for cardiovascular disease.Blood. 2011; 117: 1393-1399Crossref PubMed Scopus (53) Google Scholar, 26Morange P.E. Simon C. Alessi M.C. et al.Endothelial cell markers and the risk of coronary heart disease: the Prospective Epidemiological Study of Myocardial Infarction (PRIME) study.Circulation. 2004; 109: 1343-1348Crossref PubMed Scopus (194) Google Scholar Although a prothrombotic profile and increased production of reactive oxygen species (ROS) have been documented in Fabry disease,5Shen J.S. Meng X.L. Moore D.F. et al.Globotriaosylceramide induces oxidative stress and up-regulates cell adhesion molecule expression in Fabry disease endothelial cells.Mol Genet Metab. 2008; 95: 163-168Crossref PubMed Scopus (169) Google Scholar, 27DeGraba T. Azhar S. Dignat-George F. et al.Profile of endothelial and leukocyte activation in Fabry patients.Ann Neurol. 2000; 47: 229-233Crossref PubMed Scopus (171) Google Scholar, 28Gelderman M.P. Schiffmann R. Simak J. Elevated endothelial microparticles in Fabry children decreased after enzyme replacement therapy.Arterioscler Thromb Vasc Biol. 2007; 27: e138-e139Crossref PubMed Scopus (20) Google Scholar, 29Vedder A.C. Biro E. Aerts J.M. et al.Plasma markers of coagulation and endothelial activation in Fabry disease: impact of renal impairment.Nephrol Dial Transplant. 2009; 24: 3074-3081Crossref PubMed Scopus (17) Google Scholar less attention has been paid to the direct effect of α-Gal A deficiency on VWF, the focus of this study. In this study we further explore the basis for the prothrombotic phenotype. We observe that α-Gal A deficiency is associated with increased VWF secretion in the Gla-knockout mouse and in 2 endothelial cell models of α-Gal A deficiency. Previously, α-Gal A deficiency was demonstrated to accelerate vascular complications in mouse models of atherosclerosis and oxidant-induced arterial thrombosis.12Eitzman D.T. Bodary P.F. Shen Y. et al.Fabry disease in mice is associated with age-dependent susceptibility to vascular thrombosis.J Am Soc Nephrol. 2003; 14: 298-302Crossref PubMed Scopus (64) Google Scholar, 15Bodary P.F. Shen Y. Vargas F.B. et al.Alpha-galactosidase A deficiency accelerates atherosclerosis in mice with apolipoprotein E deficiency.Circulation. 2005; 111: 629-632Crossref PubMed Scopus (74) Google Scholar Soluble vascular cell adhesion molecule-1 (sVCAM-1) and VWF were measured in plasma to assess whether endothelial inflammation was evident in young Fabry disease mice without experimental injury or other alteration. The sVCAM-1 levels in Gla-null mice were similar to those in wild-type (WT) mice at 2 months, but were significantly elevated at 12 months compared with levels in younger Gla-deficient mice at 2 months and age-matched WT mice (Figure 1a). Intriguingly, plasma VWF levels were significantly higher in Gla-null compared with WT mice even at 2 months of age (Figure 1b). VWF levels from Gla-null mice at 5 and 17 months, but not WT mice, were significantly elevated in comparison with the same genotype at 2 months. No difference in VWF mRNA levels in lung homogenates was observed between Gla-deficient and WT mice by quantitative reverse-transcription polymerase chain reaction analysis (Supplementary Figure S1A; WT vs. Gla-null; 1.02 ± 0.12 vs. 0.97 ± 0.15 arbitrary units), indicating that the observed VWF levels in Gla-deficient mice is not due to increased VWF production from the endothelium. These data suggest that α-Gal A deficiency resulted in an age-dependent endothelial dysfunction and elevated VWF secretion. An established in vitro model of Fabry disease was employed to explore whether α-Gal-A deficiency directly promotes VWF secretion from the endothelial cells.16Shu L. Vivekanandan-Giri A. Pennathur S. et al.Establishing 3-nitrotyrosine as a biomarker for the vasculopathy of Fabry disease.Kidney Int. 2014; 86: 58-66Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar A positive linear relationship was confirmed between VWF expression and the number of cells (r2 = 0.9242) in the EA.hy926 human endothelial cell line. After GLA disruption by small, interfering RNA (siRNA), GLA expression was not detectable by immunoblotting (Figure 2a). Cells were then incubated in 2% serum media under control siRNA (siSCR) or GLA siRNA (siGLA) conditions for 3 days to measure the amount of VWF released into the media. A 3-fold higher VWF level was observed by enzyme-linked immunosorbent assay in the supernatants from siGLA compared with siSCR (Figure 2b). Thus the loss of GLA results in the release of VWF from endothelial cells consistent with the increased plasma VWF levels observed in knockout mice. Because α-Gal A deficiency results in decreased eNOS activity,16Shu L. Vivekanandan-Giri A. Pennathur S. et al.Establishing 3-nitrotyrosine as a biomarker for the vasculopathy of Fabry disease.Kidney Int. 2014; 86: 58-66Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 30Shu L. Park J.L. Byun J. et al.Decreased nitric oxide bioavailability in a mouse model of Fabry disease.J Am Soc Nephrol. 2009; 20: 1975-1985Crossref PubMed Scopus (50) Google Scholar we assessed whether the elevated VWF secretion was associated with decreased NO bioavailability. The activity of eNOS was significantly lower in siGLA than siSCR (Figure 3a). Because NO inhibits VWF release,31Lowenstein C.J. Nitric oxide regulation of protein trafficking in the cardiovascular system.Cardiovasc Res. 2007; 75: 240-246Crossref PubMed Scopus (49) Google Scholar siRNA-treated cells were incubated in the presence or absence of an exogenous NO donor, DETA-NONOate. In contrast to siSCR, the supplemental provision of NO (50 μM) resulted in a 35% reduction in VWF secretion in siGLA cells (Figure 3b). A previous study reported that VWF secretion is dependent on cell density as well as the duration of cell culture32LoMonaco M.B. Lowenstein C.J. Enhanced assay of endothelial exocytosis using extracellular matrix components.Anal Biochem. 2014; 452: 19-24Crossref PubMed Scopus (2) Google Scholar Because siRNA treatments do not persist for more than 3 days, and the efficiency of the transfection is dependent on cell seeding density (data not shown), a permanent GLA-deficient cell line was generated to avoid these confounding factors. GLA activity level was first determined in CRISPR-GLA (CR-GLA) and CRISPR-WT cells (CR-WT). As expected, the enzyme activity was not detectable in CR-GLA (Figure 4a). Next, VWF secretion into the media was measured in confluent CR-WT and CR-GLA over a more extended period of 96 hours. VWF levels in CR-GLA were significantly elevated at the 24-hour time point (Figure 4b). In additional sets of experiments, an approximately 4.7-fold increase in the level of VWF was observed in the media from CR-GLA compared with CR-WT at 48- and 72-hour time points (Figure 4c). VWF mRNA levels were slightly but significantly increased in CR-GLA compared with CR-WT (Supplementary Figure S1B; CR-WT vs. CR-GLA; 1.00 ± 0.02 vs. 1.23 ± 0.14 arbitrary unit, P < 0.01). The levels of interleukin-8, another component of WPBs, were also measured to confirm that elevated VWF secretion in α-Gal A deficiency was due to enhanced WPB exocytosis. The interleukin-8 concentration was also significantly higher in the media of CR-GLA than CR-WT (Supplementary Figure S1C; CR-WT vs. CR-GLA; 544.8 ± 15.2 vs. 1086.7 ± 23.5 pg/ml). Flow cytometry was used to compare CR-GLA and CR-WT cells for VWF content (Supplementary Table S1). A 25% increase in VWF staining was observed in CR-GLA cells in fixed and permeabilized cells stained with anti-VWF antibody as measured by fluorescence-activated cell sorting analysis. We next determined whether exogenous NO inhibits VWF secretion in CRISPR cells. Compared with the siRNA-treated cells, CRISPR cells were viable at higher concentrations of exogenous NO without affecting endothelial morphology (data not shown). Provision of NO donor (100 μM) for 3 days significantly decreased VWF secretion from both CR-WT and CR-GLA (Figure 4d). Surprisingly, NO completely normalized VWF level from CR-GLA to the level seen in vehicle-treated CR-WT. These data reinforced the prior observations that α-Gal A deficiency promotes VWF secretion and exogenous NO inhibits the release. Because CRISPR cells were more stable for pharmacologic studies than the siRNA-treated cells, this cell line was used in subsequent studies. Decreased eNOS expression and eNOS uncoupling have been documented in in vitro models of Fabry disease.16Shu L. Vivekanandan-Giri A. Pennathur S. et al.Establishing 3-nitrotyrosine as a biomarker for the vasculopathy of Fabry disease.Kidney Int. 2014; 86: 58-66Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 30Shu L. Park J.L. Byun J. et al.Decreased nitric oxide bioavailability in a mouse model of Fabry disease.J Am Soc Nephrol. 2009; 20: 1975-1985Crossref PubMed Scopus (50) Google Scholar Consistent with these previous findings, a greater than 50% reduction in the eNOS expression of CR-GLA was observed compared with CR-WT (Figure 5a). An attempt was made to “recouple” eNOS activity with sepiapterin, a stable precursor of tetrahydrobiopterin, in CR-GLA cells. The activity of eNOS from CR-WT and CR-GLA was increased with sepiapterin in a dose-dependent manner (Figure 5b). However, eNOS activity of CR-GLA was approximately 50% lower than CR-WT at any given doses of sepiapterin. Nevertheless, eNOS activity of CR-GLA was 2.5-fold higher with 300 μM sepiapterin than CR-WT vehicle conditions (Figure 5b). Three days of sepiapterin (300 μM) treatment reduced VWF secretion from CR-GLA to the level of CR-WT control conditions (Figure 5c), similar to the effect of the exogenous NO donor (Figure 4d). Nevertheless, the level of VWF with sepiapterin treatment (300 μM) in CR-GLA remained significantly higher than CR-WT, presumably due to the lower level of eNOS activity compared with CR-WT (Figure 5d). Endothelial-derived NO regulates vascular function, in part, through a soluble guanylate cyclase (sGC) and cyclic guanosine monophosphate (cGMP) signaling pathway.33Qian J. Zhang Q. Church J.E. et al.Role of local production of endothelium-derived nitric oxide on cGMP signaling and S-nitrosylation.Am J Physiol Heart Circ Physiol. 2010; 298: H112-H118Crossref PubMed Scopus (30) Google Scholar However, the inhibition of sGC by a specific irreversible inhibitor, ODQ, did not modify the effects of the NO donor and sepiapterin on VWF release in CR-WT and CR-GLA (Supplementary Figure S2). Together, these data indicate that both exogenous and endogenous NO repletion inhibit increased VWF release via a sGC/cGMP-independent signaling pathway in the setting of α-Gal A deficiency. Matsushita and colleagues demonstrated that NO inhibits WPB exocytosis through NSF S-nitrosylation (SNO-NSF).34Matsushita K. Morrell C.N. Cambien B. et al.Nitric oxide regulates exocytosis by S-nitrosylation of N-ethylmaleimide-sensitive factor.Cell. 2003; 115: 139-150Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar In addition, thioredoxin-1 is known to regulate VWF secretion by denitrosylating SNO-NSF in endothelial cells.35Ito T. Yamakuchi M. Lowenstein C.J. Thioredoxin increases exocytosis by denitrosylating N-ethylmaleimide-sensitive factor.J Biol Chem. 2011; 286: 11179-11184Crossref PubMed Scopus (22) Google Scholar Having characterized that α-Gal A deficiency resulted in decreased NO production and increased VWF secretion (Figure 3, Figure 4, Figure 5), it was next determined whether elevated VWF secretion is associated with decreased SNO-NSF in the setting of α-Gal A deficiency. The level of SNO-NSF in siGLA was approximately 75% higher than siSCR confirmed by a biotin switch assay (Figure 6a). In addition, a 12% lower level of TRX-1 was associated with increased SNO-NSF in the siGLA compared to siSCR (Figure 6b). Similarly, CR-GLA also had approximately 50% higher and 24% lower levels of SNO-NSF and TRX-1, respectively, than those in CR-WT (Figure 6c and d). Together, these data show that α-Gal A deficiency resulted in increased SNO-NSF and decreased TRX-1 level in the endothelial cells. Decreased eNOS expression, reduced enzymatic activity, and deactivation of NO by ROS can decrease NO bioavailability. Based on the previous finding that α-Gal A deficiency elevated ROS,5Shen J.S. Meng X.L. Moore D.F. et al.Globotriaosylceramide induces oxidative stress and up-regulates cell adhesion molecule expression in Fabry disease endothelial cells.Mol Genet Metab. 2008; 95: 163-168Crossref PubMed Scopus (169) Google Scholar it was evaluated whether decreasing ROS could lower VWF levels. CR-WT and CR-GLA cells were grown to confluence in the presence of ebselen (10 μM) or TEMPOL (1mM), glutathione peroxidase-1 and superoxide dismutase mimetics, respectively. After confluency was attained, fresh media was provided with one of the treatments or vehicle. Ebselen treatment for 3 days significantly reduced VWF secretion in both CR-WT and CR-GLA compared with vehicle controls (Figure 7a). In addition, the treatment in CR-GLA decreased VWF levels below that of CR-WT vehicle. TEMPOL incubation for 3 days resulted in decreased VWF in CR-GLA to levels of CR-WT vehicle condition (Figure 7b). However, VWF in CR-GLA after treatment remained significantly higher than CR-WT with TEMPOL. These data are consistent with α-Gal A deficiency causing VWF secretion, in part, by increased production of ROS. Previously it was shown that 2 days of recombinant human GLA (α-Gal A) treatment lowered Gb3 in Gla-null mouse aortic endothelial cells.36Shu L. Murphy H.S. Cooling L. et al.An in vitro model of Fabry disease.J Am Soc Nephrol. 2005; 16: 2636-2645Crossref PubMed Scopus (29) Google Scholar When eNOS activity was examined in a subsequent study, the reduction of Gb3 by the potent glucosylceramide synthase inhibitor, eliglustat, failed to restore the decreased eNOS activity significantly in these cells.30Shu L. Park J.L. Byun J. et al.Decreased nitric oxide bioavailability in a mouse model of Fabry disease.J Am Soc Nephrol. 2009; 20: 1975-1985Crossref PubMed Scopus (50) Google Scholar The effects of recombinant α-Gal A and eliglustat were evaluated to examine whether α-Gal A deficiency per se or elevated globo-series glycosphingolipids was the primary cause for the elevated secretion of VWF. CRISPR cells were pre-treated with α-Gal A (10 μg/ml) or eliglustat (200 nM) for 2 days prior to becoming confluent. VWF accumulation in the presence of α-Gal A or eliglustat was measured over 3 days. VWF was no different with or without the enzyme treatment in CR-WT cells (Figure 8a). α-Gal A reduced VWF secretion in CR-GLA by 18% compared with vehicle controls, but this was not statistically significant (P = 0.13). Eliglustat treatment slightly but significantly increased VWF secretion from both CR-WT and CR-GLA cells (Figure 8b). We report that α-Gal A deficiency decreases eNOS activity and promotes VWF secretion in knockout mice and in vitro models of Fabry disease, identifying a potential mechanism for the vasculopathy. Furthermore, pharmacological approaches that increase NO bioavailability or decreasing ROS normalize VWF levels in an in vitro model of α-Gal A deficiency. However, recombinant human GLA only partially decreases VWF secretion, while the inhibition of glycosphingolipid synthesis results in an increase in VWF secretion in vitro. Together, these results suggest that α-Gal A deficiency may promote VWF secretion through decreased NO bioavailability and elevated ROS, but this abnormality is not readily reversed by normalization of glycosphingolipid levels. Clinical observations have revealed a high incidence of thrombosis and stroke in Fabry disease patients9Utsumi K. Yamamoto N. Kase R. et al.High incidence of thrombosis in Fabry's disease.Intern Med. 1997; 36: 327-329Crossref PubMed Scopus (67) Google Scholar, 10Sims K. Politei J. Banikazemi M. et al.Stroke in Fabry disease frequently occurs before diagnosis and in the absence of other clinical events: natural history data from the Fabry Registry.Stroke. 2009; 40: 788-794Crossref PubMed Scopus (265) Google Scholar, 11Lenders M. Karabul N. Duning T. et al.Thromboembolic events in Fabry disease and the impact of factor V Leiden.Neurology. 2015; 84: 1009-1016Crossref PubMed Scopus (29) Google Scholar and a robust sensitivity to oxidant-induced thrombosis in murine models of Fabry disease.12Eitzman D.T. Bodary P.F. Shen Y. et al.Fabry disease in mice is associated with age-dependent susceptibility to vascular thrombosis.J Am Soc Nephrol. 2003; 14: 298-302Crossref PubMed Scopus (64) Google Scholar, 13Shen Y. Bodary P.F. Vargas F.B. et al.Alpha-galactosidase A deficiency leads to increased tissue fibrin deposition and thrombosis in mice homozygous for the factor V Leiden mutation.Stroke. 2006; 37: 1106-1108Crossref PubMed Scopus (25) Google Scholar VWF may contribute to the pathogenesis of thrombosis and atherosclerosis in Fabry disease due to its role in the formation of platelet thrombi.37Sadler J.E. von Willebrand factor assembly and secretion.J Thromb Haemost. 2009; 7: 24-27Crossref PubMed Scopus (104) Google Scholar For example, inactivation of VWF or platelet glycoprotein Ib, a receptor for VWF, inhibits platelet adhesion to plaque-prone sites in murine models of atherosclerosis.38Massberg S. Brand K. Gruner S. et al.A critical role of platelet adhesion in the initiation of atherosclerotic lesion formation.J Exp Med. 2002; 196: 887-896Crossref PubMed Scopus (706) Google Scholar, 39Theilmeier G. Michiels C. Spaepen E. et al.Endothelial von Willebrand factor recruits platelets to atherosclerosis-prone sites in response to hypercholesterolemia.Blood. 2002; 99: 4486-4493Crossref PubMed Scopus (142) Google Scholar When VWF production is restricted to either from endothelial cells or platelets, endothelial-derived VWF was shown to be a major determinant for thrombus formation.40Kanaji S. Fahs S.A. Shi Q. et al.Contribution of platelet vs. endothelial VWF to platelet adhesion and hemostasis.J Thromb Haemost. 2012; 10: 1646-1652Crossref PubMed Scopus (90) Google Scholar, 41Nichols T.C. Samama C.M. Bellinger D.A. et al.Function of von Willebrand factor after crossed bone marrow transplantation between normal and von Willebrand disease pigs: effect on arterial thrombosis in chimeras.Proc Natl Acad Sci U S A. 1995; 92: 2455-2459Crossref PubMed Scopus (79) Google Scholar Numerous studies have documented abnormal procoagulant and proinflammatory profiles in Fabry disease patients.27DeGraba T. Azhar S. Dignat-George F. et al.Profile of endothelial and leukocyte activation in Fabry patients.Ann Neurol. 2000; 47: 229-233Crossref PubMed Scopus (171) Google Scholar, 29Vedder A.C. Biro E. Aerts J.M. et al.Plasma markers of coagulation and endothelial activation in Fabry disease: impact of renal impairment.Nephrol Dial Transplant. 2009; 24: 3074-3081Crossref PubMed Scopus (17) Google Scholar, 42Biancini G.B. Vanzin C.S. Rodrigues D.B. et al.Globotriaosylceramide is correlated with oxidative stress and inflammation in Fabry patients treated with enzyme replacement therapy.Biochim Biophys Acta. 2012; 1822: 226-232Crossref PubMed Scopus (91) Google Scholar, 43Biancini G.B. Jacques C.E. Hammerschmidt T. et al.Biomolecules damage and redox status abnormalities in Fabry patients before and during enzyme replacement therapy.Clinica Chimica Acta. 2016; 461: 41-46Crossref PubMed Scopus (20) Google Scholar Only 3 studies have reported measurements of VWF levels in this group. In 2 reports VWF levels were not different or only slightly elevated in Fabry disease patients when compared with control groups.27DeGraba T. Azhar S. Dignat-George F. et al.Profile of endothelial and leukocyte activation in Fabry patients.Ann Neurol. 2000; 47: 229-233Crossref PubMed Scopus (171) Google Scholar, 29Vedder A.C. Biro E. Aerts J.M. et al.Plasma markers of coagulation and endothelial activation in Fabry disease: impact of renal impairment.Nephrol Dial Transplant. 2009; 24: 3074-3081Crossref PubMed Scopus (17) Google Scholar In a third study, a significant elevation of VWF was observed in both hemizygous males an