Thomas A. J. McKinnon

Endothelial von Willebrand factor regulates angiogenesis

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.

A novel binding site for ADAMTS13 constitutively exposed on the surface of globular VWF

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.

Characterization of W1745C and S1783A: 2 novel mutations causing defective collagen binding in the A3 domain of von Willebrand factor

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.

The plasma von Willebrand factor O-glycome comprises a surprising variety of structures including ABH antigens and disialosyl motifs.

Summary.  Background: von Willebrand factor (VWF) is a key component for maintenance of normal hemostasis. Its glycan moieties, accounting for about 20% of its molecular weight, have been shown to affect many of its properties. Previous studies reported correlations between VWF secretion, half‐life and the nature or presence of its N‐glycans, and more importantly between VWF plasma level and the type of N‐linked ABH antigens. Despite the presence of 10 predicted O‐glycosylation sites, the O‐glycome remains poorly characterized, impairing the complete elucidation of its influence on VWF functions. So far only a single glycan structure, a disialyl core 1 glycan, has been identified. Objectives: To define an exhaustive profile of the VWF O‐glycan structures to help the understanding of their role in VWF regulation and properties. Methods: Plasma‐derived VWF O‐linked sugars were isolated and analyzed using state‐of‐the‐art mass spectrometry methodologies. Results and conclusions: We provide here a detailed analysis of the human plasma‐derived VWF O‐glycome. Eighteen O‐glycan structures including both core 1 and core 2 structures are now demonstrated to be present on VWF. Amongst the newly determined structures are unusual tetra‐sialylated core 1 O‐glycans and ABH antigen‐containing core 2 O‐glycans. In conjunction with current models explaining VWF activity, knowledge of the complete O‐glycome will facilitate research aimed at providing a better understanding of the influence of glycosylation on VWF functions.

Mapping the N-glycome of human von Willebrand factor.

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 Asn(2635) 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.

Expression of terminal alpha2-6-linked sialic acid on von Willebrand factor specifically enhances proteolysis by ADAMTS13.

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.

Cellular and molecular basis of von Willebrand disease: studies on blood outgrowth endothelial cells

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 characterized; 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 (BOECs) from peripheral blood of 4 type 1 VWD and 4 type 2 VWD patients and 9 healthy controls. We confirmed the endothelial lineage of BOECs, then measured VWF messenger RNA (mRNA) and protein levels (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, BOECs 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 BOECs from 3 type 2 VWD patients, supporting the dysfunctional VWF model. However, 1 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 cellular mechanisms of the disease.

Identification of functionally important residues in TFPI Kunitz domain 3 required for the enhancement of its activity by protein S

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, in the present study, 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 in which no protein S enhancement of TFPI E226Q was detected. Surface plasmon resonance demonstrated concentration-dependent binding of protein S to wild-type TFPI, but almost no binding to TFPI E226Q. We conclude that the TFPI Kunitz domain 3 residue Glu226 is essential for TFPI enhancement by protein S.

The O‐linked glycans of human von Willebrand factor modulate its interaction with ADAMTS‐13

O‐linked glycans (OLGs) are clustered on either side of the von Willebrand factor (VWF) A1 domain and modulate its interaction with platelets; however, their influence on the VWF interaction with ADAMTS‐13 is unknown.

Circulating but not immobilized N-deglycosylated von Willebrand factor increases platelet adhesion under flow conditions

The role of von Willebrand factor (VWF) as a shear stress activated platelet adhesive has been related to a coiled-elongated shape conformation. The forces dominating this transition have been suggested to be controlled by the proteins polymeric architecture. However, the fact that 20% of VWF molecular weight originates from glycan moieties has so far been neglected in these calculations. In this study, we present a systematic experimental investigation on the role of N-glycosylation for VWF mediated platelet adhesion under flow. A microfluidic flow chamber with a stenotic compartment that allows one to mimic various physiological flow conditions was designed for the efficient analysis of the adhesion spectrum. Surprisingly, we found an increase in platelet adhesion with elevated shear rate, both qualitatively and quantitatively fully conserved when N-deglycosylated VWF (N-deg-VWF) instead of VWF was immobilized in the microfluidic channel. This has been demonstrated consistently over four orders of magnitude in shear rate. In contrast, when N-deg-VWF was added to the supernatant, an increase in adhesion rate by a factor of two was detected compared to the addition of wild-type VWF. It appears that once immobilized, the role of glycans is at least modified if not-as found here for the case of adhesion-negated. These findings strengthen the physical impact of the circulating polymer on shear dependent platelet adhesion events. At present, there is no theoretical explanation for an increase in platelet adhesion to VWF in the absence of its N-glycans. However, our data indicate that the effective solubility of the protein and hence its shape or conformation may be altered by the degree of glycosylation and is therefore a good candidate for modifying the forces required to uncoil this biopolymer.