[Show abstract][Hide abstract] ABSTRACT: Von Willebrand disease (VWD) is a heterogeneous bleeding disorder caused by decrease or dysfunction of von Willebrand factor (VWF). A wide range of mutations in the VWF gene have been characterised; however their cellular consequences are still poorly understood. Here we have used a recently developed approach to study the molecular and cellular basis of VWD. We isolated blood outgrowth endothelial cells (BOEC) from peripheral blood of four type 1 VWD, four type 2 VWD patients and nine healthy controls. We confirmed the endothelial lineage of BOEC, then measured VWF mRNA and protein levels, both before and after stimulation, and VWF multimers. Decreased mRNA levels were predictive of plasma VWF levels in type 1 VWD, confirming a defect in VWF synthesis. However BOEC from this group of patients also showed defects in processing, storage and/or secretion of VWF. Levels of VWF mRNA and protein were normal in BOEC from three type 2 VWD patients, supporting the dysfunctional VWF model. However, one type 2M patient showed decreased VWF synthesis and storage, indicating a complex cellular defect. These results demonstrate for the first time that isolation of endothelial cells from VWD patients provides novel insight into the cellular mechanisms of the disease.
[Show abstract][Hide abstract] ABSTRACT: Protein S is a cofactor for tissue factor pathway inhibitor (TFPI) that, critically, reduces the inhibition constant for FXa to below the plasma concentration of TFPI. TFPI Kunitz domain 3 is required for this enhancement to occur. To delineate the molecular mechanism underlying enhancement of TFPI function, we produced a panel of Kunitz domain 3 variants of TFPI encompassing all 12 surface-exposed charged residues. Thrombin generation assays in TFPI-depleted plasma identified a novel variant, TFPI E226Q, which exhibited minimal enhancement by protein S. This was confirmed in purified FXa inhibition assays where no protein S enhancement of TFPI E226Q was detected. Surface plasmon resonance demonstrated concentration-dependent binding of protein S to WT TFPI, but almost no binding to TFPI E226Q. We conclude that TFPI Kunitz domain 3 residue Glu226 is essential for TFPI enhancement by protein S.
[Show abstract][Hide abstract] ABSTRACT: vWF (von Willebrand factor) is a key component for maintenance of normal haemostasis, acting as the carrier protein of the coagulant Factor VIII and mediating platelet adhesion at sites of vascular injury. There is ample evidence that vWF glycan moieties are crucial determinants of its expression and function. Of particular clinical interest, ABH antigens influence vWF plasma levels according to the blood group of individuals, although the molecular mechanism underlying this phenomenon remains incompletely understood. The present paper reports analyses of the human plasma vWF N-glycan population using advanced MS. Glycomics analyses revealed approximately 100 distinct N-glycan compositions and identified a variety of structural features, including lactosaminic extensions, ABH antigens and sulfated antennae, as well as bisecting and terminal GlcNAc residues. We estimate that some 300 N-glycan structures are carried by human vWF. Glycoproteomics analyses mapped ten of the consensus sites known to carry N-glycans. Glycan populations were found to be distinct, although many structural features were shared across all sites. Notably, the H antigen is not restricted to particular N-glycosylation sites. Also, the Asn2635 site, previously designated as unoccupied, was found to be highly glycosylated. The delineation of such varied glycan populations in conjunction with current models explaining vWF activity will facilitate research aimed at providing a better understanding of the influence of glycosylation on vWF function.
[Show abstract][Hide abstract] ABSTRACT: We have examined the effect of the O-linked glycan (OLG) structures of VWF on its interaction with the platelet receptor glycoprotein Ibα. The 10 OLGs were mutated individually and as clusters (Clus) on either and both sides of the A1 domain: Clus1 (N-terminal side), Clus2 (C-terminal side), and double cluster (DC), in both full-length-VWF and in a VWF construct spanning D' to A3 domains. Mutations did not alter VWF secretion by HEK293T cells, multimeric structure, or static collagen binding. The T1255A, Clus1, and DC variants caused increased ristocetin-mediated GPIbα binding to VWF. Platelet translocation rate on OLG mutants was increased because of reduced numbers of GPIbα binding sites but without effect on bond lifetime. In contrast, OLG mutants mediated increased platelet capture on collagen under high shear stress that was associated with increased adhesion of these variants to the collagen under flow. These findings suggest that removal of OLGs increases the flexibility of the hinge linker region between the D3 and A1 domain, facilitating VWF unfolding by shear stress, thereby enhancing its ability to bind collagen and capture platelets. These data demonstrate an important functional role of VWF OLGs under shear stress conditions.
[Show abstract][Hide abstract] ABSTRACT: The 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.
[Show abstract][Hide abstract] ABSTRACT: We examined the role that N-linked glycans play in the synthesis and expression of von Willebrand Factor (VWF). Blocking the addition of N-linked glycans (NLGs) or inhibiting initial glycan processing prevented secretion of VWF. To determine whether specific glycosylation sites were important, the 16 VWF N-linked glycosylation sites were mutated followed by expression in HEK293T cells. Four NLG mutants affected VWF expression: N99Q (D1 domain), N857Q (D' domain), N2400Q (B1 domain), and N2790Q (CK domain) either abolished or reduced secretion of VWF and this was confirmed by metabolic labeling. Multimer analysis of mutant N2790Q cell lysate revealed an increase in VWF monomers, which was also observed when the isolated CK domain was expressed with N2790 mutated. Immunofluorescence microscopy showed that mutants N99Q, N857Q, and N2790Q were primarily retained within the ER, producing only few pseudo Weibel-Palade bodies over longer time periods compared with wtVWF. All the variants also showed an increase in free thiol reactivity. This was greatest with N857Q and D4-C2 NLG mutants, which had approximately 6-fold and 3- to 4-fold more free thiol reactivity than wtVWF. These data provide further evidence of the critical role that individual N-linked glycans play in determining VWF synthesis and expression.
[Show abstract][Hide abstract] ABSTRACT: von Willebrand factor (VWF) multimeric composition is regulated in plasma by ADAMTS13. VWF deglycosylation enhances proteolysis by ADAMTS13. In this study, the role of terminal sialic acid residues on VWF glycans in mediating proteolysis by ADAMTS13 was investigated. Quantification and distribution of VWF sialylation was examined by sequential digestion and high-performance liquid chromatography analysis. Total sialic acid expression on VWF was 167nmol/mg, of which the majority (80.1%) was present on N-linked glycan chains. Enzymatic desialylation of VWF by alpha2-3,6,8,9 neuraminidase (Neu-VWF) markedly impaired ADAMTS13-mediated VWF proteolysis. Neu-VWF collagen binding activity was reduced to 50% (+/- 14%) by ADAMTS13, compared with 11% (+/- 7%) for untreated VWF. Despite this, Neu-VWF exhibited increased susceptibility to other proteases, including trypsin, chymotrypsin, and cathepsin B. VWF expressing different blood groups exhibit altered ADAMTS13 proteolysis rates (O > or = B > A > or = AB). However, ABO blood group regulation of ADAMTS13 proteolysis was ablated on VWF desialylation, as both Neu-O-VWF and Neu-AB-VWF were cleaved by ADAMTS13 at identical rates. These novel data show that sialic acid protects VWF against proteolysis by serine and cysteine proteases but specifically enhances susceptibility to ADAMTS13 proteolysis. Quantitative variation in VWF sialylation therefore represents a key determinant of VWF multimeric composition and, as such, may be of pathophysiologic significance.
[Show abstract][Hide abstract] ABSTRACT: Investigation of 3 families with bleeding symptoms demonstrated a defect in the collagen-binding activity of von Willebrand factor (VWF) in association with a normal VWF multimeric pattern. Genetic analysis showed affected persons to be heterozygous for mutations in the A3 domain of VWF: S1731T, W1745C, and S1783A. One person showed compound heterozygosity for W1745C and R760H. W1745C and S1783A have not been reported previously. The mutations were reproduced by site-directed mutagenesis and mutant VWF expressed in HEK293T cells. Collagen-binding activity measured by immunosorbent assay varied according to collagen type: W1745C and S1783A were associated with a pronounced binding defect to both type I and type III collagen, whereas the principal abnormality in S1731T patients was a reduction in binding to type I collagen only. The multimer pattern and distribution of mutant proteins were indistinguishable from wild-type recombinant VWF, confirming that the defect in collagen binding resulted from the loss of affinity at the binding site and not impairment of high-molecular-weight multimer formation. Our findings demonstrate that mutations causing an abnormality in the binding of VWF to collagen may contribute to clinically significant bleeding symptoms. We propose that isolated collagen-binding defects are classified as a distinct subtype of von Willebrand disease.
[Show abstract][Hide abstract] ABSTRACT: ADAMTS13 metalloprotease regulates the multimeric size of von Willebrand factor (VWF) by cleaving the Tyr1605-Met1606 bond in the VWF A2 domain. The mechanisms of VWF recognition by ADAMTS13 have yet to be fully resolved. Most studies have focused on the role of exosites within the VWF A2 domain, involved in interaction with the ADAMTS13 spacer domain. In the present study, we expressed different C-terminal domain VWF fragments and evaluated their binding to ADAMTS13 and its truncated mutants, MDTCS and del(TSP5-CUB). Using plate binding assay and surface plasmon resonance, we identified a novel ADAMTS13 binding site (K(D) approximately 86 nM) in the region of VWF spanning residues 1874 to 2813, which includes the VWF D4 domain and that interacts with the C-terminal domains of ADAMTS13. We show that the interaction occurs even when VWF is in static conditions, assumed to be globular and where the VWF A2 domain is hidden. We demonstrate that C-terminal VWF fragments, as well as an antibody specifically directed toward the VWF D4 domain, inhibit VWF proteolysis by ADAMTS13 under shear conditions. We propose that this novel VWF C-terminal binding site may participate as the initial step of a multistep interaction ultimately leading to proteolysis of VWF by ADAMTS13.
[Show abstract][Hide abstract] ABSTRACT: We examined the role of N-linked glycan structures of VWF on its interaction with ADAMTS13. PNGase F digestion followed by lectin analysis demonstrated that more than 90% of VWF N-linked glycan chains could be removed from the molecule (PNG-VWF) without disruption of its multimeric structure or its ability to bind to collagen. PNG-VWF had an approximately 4-fold increased affinity for ADAMTS13 compared with control VWF. PNG-VWF was cleaved by ADAMTS13 faster than control VWF and was also proteolysed in the absence of urea. Occupancy of the N-linked glycan sites at N1515 and N1574 and their presentation of ABO(H) blood group sugars were confirmed with an isolated tryptic fragment. Recombinant VWF was mutated to prevent glycosylation at these sites. Mutation of N1515 did not alter ADAMTS13 binding or increase rate of ADAMTS13 proteolysis. Mutation of N1574 increased the susceptibility of VWF to ADAMTS13 proteolysis and allowed cleavage in the absence of urea. Mutation of N1574 in the isolated recombinant VWF-A2 domain also increased binding and ADAMTS13 proteolysis. These data demonstrate that the N-linked glycans of VWF have a modulatory effect on the interaction with ADAMTS13. At least part of this effect is conformational, but steric hindrance may also be important.
[Show abstract][Hide abstract] ABSTRACT: ABO blood group is an important determinant of plasma von Willebrand factor antigen (VWF:Ag) levels, with lower levels in group O. Previous reports have suggested that ABO(H) sugars affect the susceptibility of VWF to ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type-1 repeats-13) cleavage. To further test this hypothesis, we collected plasma from individuals with the rare Bombay blood group. VWF:Ag levels were significantly lower in Bombay patients (median, 0.69 IU/mL) than in groups AB, A, or B (P < .05) and lower than in group O individuals (median, 0.82 IU/mL). Susceptibility of purified VWF fractions to recombinant ADAMTS13 cleavage, assessed using VWF collagen-binding assay (VWF:CB), was increased in Bombays compared with either group O or AB. Increasing urea concentration (0.5 to 2 M) increased the cleavage rate for each blood group but eliminated the differences between groups. We conclude that reduction in the number of terminal sugars on N-linked glycan increases susceptibility of globular VWF to ADAMTS13 proteolysis and is associated with reduced plasma VWF:Ag and VWF:CB levels.