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.
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.
Improved understanding of the plasmin-VWF interaction offers exciting opportunities to develop novel adjunctive therapies for the treatment of refractory thrombotic thrombocytopenic purpura.
Although most plasma FVIII (Factor VIII) circulates in complex with VWF (von Willebrand factor), a minority (3%-5%) circulates as free-FVIII, which is rapidly cleared. Consequently, 20% of total ...FVIII may be cleared as free-FVIII. Critically, the mechanisms of free-FVIII clearance remain poorly understood. However, recent studies have implicated the MGL (macrophage galactose lectin) in modulating VWF clearance.
Since VWF and FVIII share similar glycosylation, we investigated the role of MGL in FVIII clearance. FVIII binding to MGL was assessed in immunosorbent and cell-based assays. In vivo, FVIII clearance was assessed in
and
mice.
In vitro-binding studies identified MGL as a novel macrophage receptor that binds free-FVIII in a glycan-dependent manner.
and
mice who received an anti-MGL1/2 blocking antibody both showed significantly increased endogenous FVIII activity compared with wild-type mice (
=0.036 and
<0.0001, respectively). MGL inhibition also prolonged the half-life of infused FVIII in
mice. To assess whether MGL plays a role in the clearance of free FVIII in a VWF-independent manner, in vivo clearance experiments were repeated in dual
mice. Importantly, the rapid clearance of free FVIII in
mice was significantly (
=0.012) prolonged in the presence of anti-MGL1/2 antibodies. Finally, endogenous plasma FVIII levels in
mice were significantly increased following MGL inhibition (
=0.016).
Cumulatively, these findings demonstrate that MGL plays an important role in regulating macrophage-mediated clearance of both VWF-bound FVIII and free-FVIII in vivo. We propose that this novel FVIII clearance pathway may be of particular clinical importance in patients with type 2N or type 3 Von Willebrand disease.
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, A
A
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.
Severe COVID-19 is associated with marked endothelial cell (EC) activation that plays a key role in immunothrombosis and pulmonary microvascular occlusion. However, the biological mechanisms through ...which SARS-CoV-2 causes EC activation and damage remain poorly defined.
We investigated EC activation in patients with acute COVID-19, and specifically focused on how proteins stored within Weibel-Palade bodies may impact key aspects of disease pathogenesis.
Thirty-nine patients with confirmed COVID-19 were recruited. Weibel-Palade body biomarkers (von Willebrand factor VWF, angiopoietin-2 Angpt-2, and osteoprotegerin) and soluble thrombomodulin (sTM) levels were determined. In addition, EC activation and angiogenesis were assessed in the presence or absence of COVID-19 plasma incubation.
Markedly elevated plasma VWF antigen, Angpt-2, osteoprotegerin, and sTM levels were observed in patients with acute COVID-19. The increased levels of both sTM and Weibel-Palade body components (VWF, osteoprotegerin, and Angpt-2) correlated with COVID-19 severity. Incubation of COVID-19 plasma with ECs triggered enhanced VWF secretion and increased Angpt-2 expression, as well as significantly enhanced in vitro EC tube formation and angiogenesis.
We propose that acute SARS-CoV-2 infection leads to a complex and multifactorial EC activation, progressive loss of thrombomodulin, and increased Angpt-2 expression, which collectively serve to promote a local proangiogenic state.
•Severe COVID-19 is associated with endotheliopathy that plays a key role in immunothrombosis.•Endothelial cell activation correlates with COVID-19 severity and results in Weibel-Palade body exocytosis.•Plasma levels of von Willebrand factor, angiopoietin-2, osteoprotegerin, and soluble thrombomodulin are elevated in patients with severe COVID-19.•Plasma alterations result in endothelial cell activation and Weibel-Palade body secretion and may promote local angiogenesis.
Introduction
Although the biological mechanisms underpinning VWF clearance remain poorly understood, accumulating data has shown that increased clearance is a common pathophysiology in type 1 VWD ...patients. Moreover, enhanced clearance has been implicated in the etiology of types 2 and 3 VWD. Previous studies have shown that loss of terminal sialic acid from VWF glycans results in enhanced clearance. Furthermore, ST3Gal-IV knockout mice exhibit reduced plasma VWF levels due to rapid in vivo clearance. Initial studies suggested that clearance of hyposialylated VWF was mediated via the asialoglycoprotein receptor (ASGPR). However, we recently reported a novel role for the Macrophage Galactose Lectin (MGL) in regulating clearance of VWF (Ward et al, Blood 2018). In this study, we sought to elucidate the molecular mechanisms through which MGL interacts with human VWF.
Methods
Recombinant VWF variants, truncated A1A2A3 and isolated A1, A2 and A3 domains were expressed in HEK293T cells. Human plasma derived (pdVWF) was purified from commercial VWF concentrates as before. VWF glycoforms were generated by digestion with exoglycosidases. VWF-MGL interaction was assessed using plate binding ELISA assays. In vivo clearance of VWF variants was assessed in both Asgr1+/+VWF-/- and Asgr1-/-VWF-/-mice in the presence/absence of anti-MGL blocking antibodies.
Results
Previous studies have shown 80% of total sialic acid on VWF is expressed on N glycans in α2-6 linkage. In contrast, the other 20% of VWF sialylation is α2-3 linked on O linked glycans. Removal O linked sialylation (with α2-3 neuraminidase) was sufficient to increase VWF clearance in VWF-/- and Asgr1-/-VWF-/-mice (half life = 9.0 ± 1 mins and 8.3 ± 2mins). Interestingly, in vivo clearance of α2-3 Neu VWF in VWF-/- andAsgr1-/-VWF-/- mice was almost as rapid as that of asialo-VWF (digested with α2-3,6,8,9 neuraminidase). Critically, the increased clearance of α2-3 Neu VWF in VWF-/- is attenuated in the presence of MGL blocking antibodies (120mins residual α2-3 Neu VWF is 10.6% ±2% , compared to 10.2% ±1% for that of pdVWF). Plate-binding studies confirmed that α2-3 Neu VWF and α2-3,6,8,9 Neu VWF demonstrated enhanced binding to MGL compared to pdVWF (155% and 134% versus 100%; p =0.017 and 0.006). In keeping with in vivo clearance data, removal of α2-6 linked sialic acid from N glycans did not further promote binding to MGL. Finally, although PNGase removal of VWF N glycans did not affect MGL binding, treatment with O glycosidase significantly attenuated binding (p<0.0001). All together, these data demonstrate that O-linked sialylation plays a critical role in protecting VWF from MGL mediated clearance.
The mature VWF monomer contains 10 O linked glycans, with eight clustered around the A1 domain. To further investigate the role of these glycans in regulating MGL interaction, site directed mutagenesis was used to generate VWF molecules lacking either O-linked cluster 1 (T1248, T1255, T1256, T1263) or O-linked cluster 2 (T1468, T1477, S1486, T1487) respectively. Binding of Δcluster 1 VWF to MGL was similar to that of wild-type VWF. In contrast, binding of the Δcluster 2 VWF variant to MGL was markedly reduced (84 % vs 40%; p=0.0027). O linked glycan structures are known to play important roles in maintaining glycoprotein conformation. To further define the mechanism through which these O glycan structures influence MGL mediated clearance, binding of a truncated A1A2A3 fragment and isolated recombinant A1, A2 and A3 domains were examined. Interestingly, MGL-binding to full length rVWF and A1A2A3 were both significantly increased in the presence of ristocetin. Interestingly, no binding of isolated A2 or A3 domains to MGL was observed, while A1 domain binding to MGL was seen both in the presence and absence of ristocetin.
Conclusions
In conclusion, these findings define a novel role for O linked sialylation in protecting VWF from MGL mediated clearance. Our data further demonstrate that the cluster of O linked glycans located at the C-terminal end of the A1 domain play a specific role in regulating VWF clearance via MGL, and that conformation of A1A2A3 may be important in determining accessibility of these glycans for the clearance receptor. Further studies will be required to determine whether abnormalities in these O-glycan determinants may be important in the pathophysiology of VWD, particularly in patients with type 1C VWD who lack VWF gene coding mutations.
O'Donnell:Bayer: Research Funding, Speakers Bureau; Novo Nordisk: Research Funding, Speakers Bureau; Leo Pharma: Speakers Bureau; Octapharma: Speakers Bureau; CSL Behring: Consultancy; Daiichi Sankyo: Consultancy; Pfizer: Consultancy, Research Funding; Baxter: Research Funding, Speakers Bureau; Shire: Research Funding, Speakers Bureau.
The plasma multimeric glycoprotein von Willebrand factor (VWF) plays a critical role in primary hemostasis by tethering platelets to exposed collagen at sites of vascular injury. Recent studies have ...identified additional biological roles for VWF, and in particular suggest that VWF may play an important role in regulating inflammatory responses. However, the molecular mechanisms through which VWF exerts its immuno-modulatory effects remain poorly understood. In this study, we report that VWF binding to macrophages triggers downstream MAP kinase signaling, NF-κB activation and production of pro-inflammatory cytokines and chemokines. In addition, VWF binding also drives macrophage M1 polarization and shifts macrophage metabolism towards glycolysis in a p38-dependent manner. Cumulatively, our findings define an important biological role for VWF in modulating macrophage function, and thereby establish a novel link between primary hemostasis and innate immunity.
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