Publications by authors named "Soracha Ward"

14 Publications

  • Page 1 of 1

The Biological Significance of von Willebrand Factor O-Linked Glycosylation.

Semin Thromb Hemost 2021 Jun 15. Epub 2021 Jun 15.

Haemostasis Research Group, Irish Centre for Vascular Biology, School of Pharmacy and Bimolecular Sciences, Royal College of Surgeons in Ireland, Dublin, Ireland.

Glycosylation is a key posttranslational modification, known to occur on more than half of all secreted proteins in man. As such, the role of N- and O-linked glycan structures in modulating various aspects of protein biology is an area of much research. Given their prevalence, it is perhaps unsurprising that variations in glycan structures have been demonstrated to play critical roles in modulating protein function and have been implicated in the pathophysiology of human diseases. von Willebrand factor (VWF), a plasma glycoprotein that is essential for normal hemostasis, is heavily glycosylated, containing 13 N-linked and 10 O-linked glycans. Together, these carbohydrate chains account for 20% of VWF monomeric mass, and have been shown to modulate VWF structure, function, and half-life. In this review, we focus on the specific role played by O-linked glycans in modulating VWF biology. Specifically, VWF O-linked glycans have been shown to modulate tertiary protein structure, susceptibility to ADAMTS13 proteolysis, platelet tethering, and VWF circulatory half-life.
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http://dx.doi.org/10.1055/s-0041-1726373DOI Listing
June 2021

ADAMTS13 regulation of VWF multimer distribution in severe COVID-19.

J Thromb Haemost 2021 May 30. Epub 2021 May 30.

Irish Centre for Vascular Biology, School of Pharmacy and Biomolecular Sciences, Royal College of Surgeons in Ireland, Dublin, Ireland.

Background: Consistent with fulminant endothelial cell activation, elevated plasma von Willebrand factor (VWF) antigen levels have been reported in patients with COVID-19. The multimeric size and function of VWF are normally regulated through A Disintegrin And Metalloprotease with ThrombSpondin Motif type 1 motif, member 13 (ADAMTS-13)--mediated proteolysis.

Objectives: This study investigated the hypothesis that ADAMTS-13 regulation of VWF multimer distribution may be impaired in severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection contributing to the observed microvascular thrombosis.

Patients And Methods: Patients with COVID-19 (n = 23) were recruited from the Beaumont Hospital Intensive Care Unit (ICU) in Dublin. Plasma VWF antigen, multimer distribution, ADAMTS-13 activity, and known inhibitors thereof were assessed.

Results: We observed markedly increased VWF collagen-binding activity in patients with severe COVID-19 compared to controls (median 509.1 versus 94.3 IU/dl). Conversely, plasma ADAMTS-13 activity was significantly reduced (median 68.2 IU/dl). In keeping with an increase in VWF:ADAMTS-13 ratio, abnormalities in VWF multimer distribution were common in patients with COVID-19, with reductions in high molecular weight VWF multimers. Terminal sialylation regulates VWF susceptibility to proteolysis by ADAMTS-13 and other proteases. We observed that both N- and O-linked sialylation were altered in severe COVID-19. Furthermore, plasma levels of the ADAMTS-13 inhibitors interleukin-6, thrombospondin-1, and platelet factor 4 were significantly elevated.

Conclusions: These findings support the hypothesis that SARS-CoV-2 is associated with profound quantitative and qualitative increases in plasma VWF levels, and a multifactorial down-regulation in ADAMTS-13 function. Further studies will be required to determine whether therapeutic interventions to correct ADAMTS-13-VWF multimer dysfunction may be useful in COVID-microvascular thrombosis and angiopathy.
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http://dx.doi.org/10.1111/jth.15409DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8237059PMC
May 2021

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

Haematologica 2021 Mar 25. Epub 2021 Mar 25.

Irish Centre for Vascular Biology, School of Pharmacy and Biomolecular Sciences, Royal College of Surgeons in Ireland; National Children's Research Centre, Our Lady's Children's Hospital, Dublin, Ireland; National Coagulation Centre, St James's Hospital, Dublin.

Terminal sialylation determines plasma VWF half-life. A role for macrophage galactose lectin (MGL) in regulating hyposialylated VWF clearance has recently been proposed. In this study, we show 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:Ag levels (p.
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http://dx.doi.org/10.3324/haematol.2020.274720DOI Listing
March 2021

Prolonged elevation of D-dimer levels in convalescent COVID-19 patients is independent of the acute phase response.

J Thromb Haemost 2021 04 8;19(4):1064-1070. Epub 2021 Mar 8.

Irish Centre for Vascular Biology, School of Pharmacy and Biomolecular Sciences, Royal College of Surgeons in Ireland, Dublin, Ireland.

Background: Persistent fatigue, breathlessness, and reduced exercise tolerance have been reported following acute COVID-19 infection. Although immuno-thrombosis has been implicated in acute COVID-19 pathogenesis, the biological mechanisms underpinning long COVID remain unknown. We hypothesized that pulmonary microvascular immuno-thrombosis may be important in this context.

Methods: One hundred fifty COVID-19 patients were reviewed at St James's Hospital Dublin between May and September 2020 at a median of 80.5 (range 44-155) days after initial diagnosis. These included patients hospitalized during initial illness (n = 69) and others managed entirely as out-patients (n = 81). Clinical examination, chest x-ray, and 6-min walk tests were performed. In addition, a range of coagulation and inflammatory markers were assessed.

Results: Increased D-dimer levels (>500 ng/ml) were observed in 25.3% patients up to 4 months post-SARS-CoV-2 infection. On univariate analysis, elevated convalescent D-dimers were more common in COVID-19 patients who had required hospital admission and in patients aged more than 50 years (p < .001). Interestingly, we observed that 29% (n = 11) of patients with elevated convalescent D-dimers had been managed exclusively as out-patients during their illness. In contrast, other coagulation (prothrombin time, activated partial thromboplastin time, fibrinogen, platelet count) and inflammation (C-reactive protein, interleukin-6, and sCD25) markers had returned to normal in >90% of convalescent patients.

Conclusions: Elucidating the biological mechanisms responsible for sustained D-dimer increases may be of relevance in long COVID pathogenesis and has implications for clinical management of these patients.
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http://dx.doi.org/10.1111/jth.15267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8013297PMC
April 2021

Von Willebrand factor propeptide in severe coronavirus disease 2019 (COVID-19): evidence of acute and sustained endothelial cell activation.

Br J Haematol 2021 02 16;192(4):714-719. Epub 2020 Dec 16.

Irish Centre for Vascular Biology, School of Pharmacy and Biomolecular Sciences, Royal College of Surgeons in Ireland (RCSI), Dublin, Ireland.

Endothelial cell (EC) activation plays a key role in the pathogenesis of pulmonary microvascular occlusion, which is a hallmark of severe coronavirus disease 2019 (COVID-19). Consistent with EC activation, increased plasma von Willebrand factor antigen (VWF:Ag) levels have been reported in COVID-19. Importantly however, studies in other microangiopathies have shown that plasma VWF propeptide (VWFpp) is a more sensitive and specific measure of acute EC activation. In the present study, we further investigated the nature of EC activation in severe COVID-19. Markedly increased plasma VWF:Ag [median (interquatile range, IQR) 608·8 (531-830)iu/dl] and pro-coagulant factor VIII (FVIII) levels [median (IQR) 261·9 (170-315) iu/dl] were seen in patients with severe severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. Sequential testing showed that these elevated VWF-FVIII complex levels remained high for up to 3 weeks. Similarly, plasma VWFpp levels were also markedly elevated [median (IQR) 324·6 (267-524) iu/dl]. Interestingly however, the VWFpp/VWF:Ag ratio was reduced, demonstrating that decreased VWF clearance contributes to the elevated plasma VWF:Ag levels in severe COVID-19. Importantly, plasma VWFpp levels also correlated with clinical severity indices including the Sequential Organ Failure Assessment (SOFA) score, Sepsis-Induced Coagulopathy (SIC) score and the ratio of arterial oxygen partial pressure to fractional inspired oxygen (P/F ratio). Collectively, these findings support the hypothesis that sustained fulminant EC activation is occurring in severe COVID-19, and further suggest that VWFpp may have a role as a biomarker in this setting.
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http://dx.doi.org/10.1111/bjh.17273DOI Listing
February 2021

Expresser phenotype determines ABO(H) blood group antigen loading on platelets and von Willebrand factor.

Sci Rep 2020 10 27;10(1):18366. Epub 2020 Oct 27.

Department of Haematology, University Hospital of Wales, Cardiff, Wales, UK.

ABO blood group is associated with cardiovascular disease, with significantly lower risk in blood group O individuals. ABO(H) blood group determinants are expressed on different glycoproteins on platelet surfaces. In addition, ABO(H) structures are also present on VWF glycans. These ABO(H) carbohydrates influence both platelet and VWF function. Previous studies have reported that approximately 5-10% of normal blood donors express abnormally high or low levels of A or B blood group antigens on their platelet surfaces (high expresser phenotype, HXP or low expresser phenotype, LXP respectively). In this study, the biological effects of the ABO Expresser phenotype were investigated. ABO(H) expression on platelets and plasma VWF was studied in a series of 541 healthy blood donors. Overall, 5.6% of our study cohort were classified as HXP, whilst 4.4% satisfied criteria for LXP. We demonstrate that genotype at the ABO blood group locus plays a critical role in modulating the platelet HXP phenotype. In particular, AA genotype is a major determinant of ABO high-expresser trait. Our data further show that ABH loading on VWF is also affected by ABO expresser phenotype. Consequently, A antigen expression on VWF was significantly elevated in HXP individuals and moderately reduced in LXP subjects (P < 0.05). Collectively, these findings suggest that ABO expresser phenotype influences primary hemostasis though several different pathways. Further studies will be required to define whether inter-individual variations in ABO(H) expression on platelets and/or VWF (particularly HXP and LXP) impact upon risk for cardiovascular disease.
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http://dx.doi.org/10.1038/s41598-020-75462-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7591562PMC
October 2020

The relationship between ABO blood group, von Willebrand factor, and primary hemostasis.

Blood 2020 12;136(25):2864-2874

Irish Centre for Vascular Biology, School of Pharmacy and Biomolecular Sciences, Royal College of Surgeons in Ireland, Dublin, Ireland.

Numerous studies have reported significant associations between ABO blood group and risk of cardiovascular disease. These studies have consistently demonstrated that thrombotic risk is significantly reduced in individuals in blood group O. Nevertheless, the biological mechanisms through which ABO influences hemostasis have remained poorly understood. Exciting recent data have provided novel insights into how these ABO effects are modulated and have highlighted that ABO group significantly influences platelet plug formation at sites of vascular injury (primary hemostasis). In particular, ABO affects multiple aspects of von Willebrand factor (VWF) biology. In keeping with their reduced thrombotic risk, plasma VWF levels are ∼25% lower in healthy group O compared with healthy group non-O individuals. In addition, blood group O VWF demonstrates enhanced susceptibility to ADAMTS13 proteolysis. Finally, preliminary findings suggest that the interaction of group O VWF with platelets may also be reduced. Although the molecular mechanisms underlying these ABO effects on VWF have not been fully elucidated, it seems likely that they are mediated in large part by the ABO(H) carbohydrate structures that are carried on both the N- and O-linked glycans of VWF. Interestingly, ABO(H) determinants are also expressed on several different platelet surface glycoprotein receptors. Recent studies support the hypothesis that ABO group not only exerts major quantitative and qualitative effects on VWF, but also affect specific aspects of platelet function. Given the severe morbidity and the mortality associated with thrombotic disorders, defining the mechanisms underlying these ABO effects is not only of scientific interest, but also of direct clinical importance.
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http://dx.doi.org/10.1182/blood.2020005843DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7751360PMC
December 2020

Endothelial cells orchestrate COVID-19 coagulopathy.

Lancet Haematol 2020 08 30;7(8):e553-e555. Epub 2020 Jun 30.

Irish Centre for Vascular Biology, School of Pharmacy and Biomolecular Sciences, Royal College of Surgeons in Ireland, Dublin 2, Ireland. Electronic address:

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http://dx.doi.org/10.1016/S2352-3026(20)30215-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7326442PMC
August 2020

More on 'Association between ABO blood groups and risk of SARS-CoV-2 pneumonia'.

Br J Haematol 2020 07 1;190(1):27-28. Epub 2020 Jun 1.

Irish Centre for Vascular Biology, School of Pharmacy and Biomolecular Sciences, Royal College of Surgeons in Ireland, Dublin, Ireland.

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http://dx.doi.org/10.1111/bjh.16845DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7276715PMC
July 2020

von Willebrand factor sialylation-A critical regulator of biological function.

J Thromb Haemost 2019 07 30;17(7):1018-1029. Epub 2019 May 30.

Haemostasis Research Group, Irish Centre for Vascular Biology, Royal College of Surgeons in Ireland, Dublin, Ireland.

Essentials Von Willebrand Factor (VWF) is extensively glycosylated with serial studies demonstrating that these carbohydrate determinants play critical roles in regulating multiple aspects of VWF biology. Terminal sialic acid residues, expressed on both the N- and O-linked glycans of VWF, regulate VWF functional activity, susceptibility to proteolysis and plasma clearance in vivo. Quantitative and qualitative variations in VWF sialylation have been reported in patients with von Willebrand Disease, as well as in a number of other physiological and pathological states. Further studies are warranted to define the molecular mechanisms through which N- and O-linked sialylation impacts upon the multiple biological activities of VWF. von Willebrand factor (VWF) undergoes complex post-translational modification prior to its secretion into the plasma. Consequently, VWF monomers contain complex N-glycan and O-glycan structures that, together, account for approximately 20% of the final monomeric mass. An increasing body of evidence has confirmed that these carbohydrate determinants play critical roles in regulating multiple aspects of VWF biology. In particular, studies have demonstrated that terminal ABO blood group has an important effect on plasma VWF levels. This effect is interesting, given that only 15% of the N-glycans and 1% of the O-glycans of VWF actually express terminal ABO(H) determinants. In contrast, the vast majority of the N-glycans and O-glycans on human VWF are capped by terminal negatively charged sialic acid residues. Recent data suggest that sialylation significantly regulates VWF functional activity, susceptibility to proteolysis, and clearance, through a number of independent pathways. These findings are of direct clinical relevence, in that quantitative and qualitative variations in VWF sialylation have been described in patients with VWD, as well as in patients with a number of other physiologic and pathologic conditions. Moreover, platelet-derived VWF is significantly hyposialylated as compared with plasma-derived VWF, whereas the recently licensed recombinant VWF therapeutic is hypersialylated. In this review, we examine the evidence supporting the hypothesis that VWF sialylation plays multiple biological roles. In addition, we consider data suggesting that quantitative and qualitative variations in VWF sialylation may play specific roles in the pathogenesis of VWD, and that sialic acid expression on VWF may also differ across a number of other physiologic and pathologic conditions.
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http://dx.doi.org/10.1111/jth.14471DOI Listing
July 2019

von Willebrand factor clearance - biological mechanisms and clinical significance.

Br J Haematol 2018 10;183(2):185-195

Haemostasis Research Group, Irish Centre for Vascular Biology, Royal College of Surgeons in Ireland, Dublin, Ireland.

The mechanisms involved in regulating von Willebrand factor (VWF) clearance remain poorly understood. However recent studies have shown that macrophages play a critical role in regulating the half-life of VWF, and have identified specific lectin (including asialoglycoprotein, macrophage galactose-type lectin, Sigec-5 and C-type lectin domain family 4 member M) and scavenger receptors (including low-density lipoprotein receptor-related protein-1, scavenger receptor A1 and stabilin-2) that are involved in VWF clearance. Further studies will be required to determine the relative importance of these individual receptors with respect to physiological and pathological VWF clearance. Nevertheless, recent clinical data have highlighted the importance of enhanced VWF clearance in the pathogenesis of type 1 von Willebrand disease (VWD). Moreover, increased clearance also contributes to reduced VWF levels in many patients with type 2 and type 3 VWD. Improved understanding regarding VWF clearance is not only of direct biological relevance, but may also have important implications for the development of novel therapeutic agents with extended plasma half-lives for the treatment of both VWD and haemophilia A.
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http://dx.doi.org/10.1111/bjh.15565DOI Listing
October 2018

A novel role for the macrophage galactose-type lectin receptor in mediating von Willebrand factor clearance.

Blood 2018 02 27;131(8):911-916. Epub 2017 Dec 27.

Irish Centre for Vascular Biology, Molecular and Cellular Therapeutics, Royal College of Surgeons in Ireland, Dublin, Ireland.

Previous studies have shown that loss of terminal sialic acid causes enhanced von Willebrand factor (VWF) clearance through the Ashwell-Morrell receptor (AMR). In this study, we investigated (1) the specific importance of - vs -linked sialic acid in protecting against VWF clearance and (2) whether additional receptors contribute to the reduced half-life of hyposialylated VWF. α2-3-linked sialic acid accounts for <20% of total sialic acid and is predominantly expressed on VWF -glycans. Nevertheless, specific digestion with α2-3 neuraminidase (α2-3Neu-VWF) was sufficient to cause markedly enhanced VWF clearance. Interestingly, in vivo clearance experiments in dual mice demonstrated enhanced clearance of α2-3Neu-VWF even in the absence of the AMR. The macrophage galactose-type lectin (MGL) is a C-type lectin that binds to glycoproteins expressing terminal -acetylgalactosamine or galactose residues. Importantly, the markedly enhanced clearance of hyposialylated VWF in mice was significantly attenuated in the presence of an anti-MGL inhibitory antibody. Furthermore, dose-dependent binding of human VWF to purified recombinant human MGL was confirmed using surface plasmon resonance. Additionally, plasma VWF:Ag levels were significantly elevated in mice compared with controls. Collectively, these findings identify MGL as a novel macrophage receptor for VWF that significantly contributes to the clearance of both wild-type and hyposialylated VWF.
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http://dx.doi.org/10.1182/blood-2017-06-787853DOI Listing
February 2018

Plasmin Cleaves Von Willebrand Factor at K1491-R1492 in the A1-A2 Linker Region in a Shear- and Glycan-Dependent Manner In Vitro.

Arterioscler Thromb Vasc Biol 2017 05 9;37(5):845-855. Epub 2017 Mar 9.

From the Haemostasis Research Group, Institute of Molecular Medicine, Trinity Centre for Health Sciences, St. James's Hospital, Trinity College Dublin, Ireland (T.M.B., S.E.W., T.R.M.G., C.D., J.M.O., A.C., J.S.O.); Medilys Laborgesellschaft mbH, Department of Hämostaseology, Hamburg, Germany (S.S., U.B.); National Centre for Hereditary Coagulation Disorders, St. James's Hospital, Dublin, Ireland (J.S.O.); and Irish Centre for Vascular Biology, Royal College of Surgeons in Ireland, Dublin (J.S.O.).

Objective: Previous studies have demonstrated a role for plasmin in regulating plasma von Willebrand factor (VWF) multimer composition. Moreover, emerging data have shown that plasmin-induced cleavage of VWF is of particular importance in specific pathological states. Interestingly, plasmin has been successfully used as an alternative to ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motif) in a mouse model of thrombotic thrombocytopenic purpura. Consequently, elucidating the molecular mechanisms through which plasmin binds and cleaves VWF is not only of basic scientific interest but also of direct clinical importance. Our aim was to investigate factors that modulate the susceptibility of human VWF to proteolysis by plasmin.

Approach And Results: We have adapted the VWF vortex proteolysis assay to allow for time-dependent shear exposure studies. We show that globular VWF is resistant to plasmin cleavage under static conditions, but is readily cleaved by plasmin under shear. Although both plasmin and ADAMTS13 cleave VWF in a shear-dependent manner, plasmin does not cleave at the Tyr1605-Met1606 ADAMTS13 proteolytic site in the A2 domain. Rather under shear stress conditions, or in the presence of denaturants, such as urea or ristocetin, plasmin cleaves the K1491-R1492 peptide bond within the VWF A1-A2 linker region. Finally, we demonstrate that VWF susceptibility to plasmin proteolysis at K1491-R1492 is modulated by local N-linked glycan expression within A1A2A3, and specifically inhibited by heparin binding to the A1 domain.

Conclusions: Improved understanding of the plasmin-VWF interaction offers exciting opportunities to develop novel adjunctive therapies for the treatment of refractory thrombotic thrombocytopenic purpura.
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http://dx.doi.org/10.1161/ATVBAHA.116.308524DOI Listing
May 2017

N-linked glycans within the A2 domain of von Willebrand factor modulate macrophage-mediated clearance.

Blood 2016 10 23;128(15):1959-1968. Epub 2016 Aug 23.

Haemostasis Research Group, Institute of Molecular Medicine, Trinity Centre for Health Sciences, St. James's Hospital, National Centre for Hereditary Coagulation Disorders, St. James's Hospital, Dublin, Ireland; and Irish Centre for Vascular Biology, Royal College of Surgeons in Ireland, Dublin, Ireland.

Enhanced von Willebrand factor (VWF) clearance is important in the etiology of von Willebrand disease. However, the molecular mechanisms underlying VWF clearance remain poorly understood. In this study, we investigated the role of VWF domains and specific glycan moieties in regulating in vivo clearance. Our findings demonstrate that the A1 domain of VWF contains a receptor-recognition site that plays a key role in regulating the interaction of VWF with macrophages. In A1-A2-A3 and full-length VWF, this macrophage-binding site is cryptic but becomes exposed following exposure to shear or ristocetin. Previous studies have demonstrated that the N-linked glycans within the A2 domain play an important role in modulating susceptibility to ADAMTS13 proteolysis. We further demonstrate that these glycans presented at N1515 and N1574 also play a critical role in protecting VWF against macrophage binding and clearance. Indeed, loss of the N-glycan at N1515 resulted in markedly enhanced VWF clearance that was significantly faster than that observed with any previously described VWF mutations. In addition, A1-A2-A3 fragments containing the N1515Q or N1574Q substitutions also demonstrated significantly enhanced clearance. Importantly, clodronate-induced macrophage depletion significantly attenuated the increased clearance observed with N1515Q and N1574Q in both full-length VWF and A1-A2-A3. Finally, we further demonstrate that loss of these N-linked glycans does not enhance clearance in VWF in the presence of a structurally constrained A2 domain. Collectively, these novel findings support the hypothesis that conformation of the VWF A domains plays a critical role in modulating macrophage-mediated clearance of VWF in vivo.
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http://dx.doi.org/10.1182/blood-2016-04-709436DOI Listing
October 2016
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