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 ␣v␤3 levels and increased angiopoietin (Ang)-2 release. ECs expanded from blood- IntroductionAngiogenesis, the formation of new vessels from pre-existing ones, occurs physiologically in specific circumstances such as wound healing and the menstrual cycle. Dysregulated angiogenesis contributes to the pathogenesis of many disorders, including diabetes, cancer, and macular degeneration (reviewed in Carmeliet 1 ). Angiogenic factors such as vascular endothelial growth factor (VEGF) and the angiopoietins (Ang) orchestrate signaling pathways that promote endothelial cell (EC) migration, proliferation, and ultimately the formation of a new vessel. VEGF-A is a major regulator of angiogenesis (reviewed in Grothey and Galanis 2 ) and acts on ECs mainly through VEGF receptor-2 (VEGFR-2), a tyrosine kinase receptor (reviewed in Olsson 3 ), to promote endothelial proliferation, migration, and sprouting of tip cells in the early stages of angiogenesis (reviewed in Gerhardt 4 ). Ang-1 and Ang-2, which bind to the endothelial Tie-2 receptor, act in the later stages of blood vessel formation and are essential for the maturation of a stable vascular network and for the maintenance of endothelial integrity (reviewed in Thomas and Augustin 5 ). Ang-1 and Ang-2 were originally identified as agonist and antagonist of Tie-2 signaling, respectively, with Ang-1 supporting EC survival and endothelial integrity 6 and Ang-2 promoting blood vessel destabilization and regression. 7 However, recent data suggest a more complex picture that includes cross-talk between the VEGF and the Ang pathways. 8 Growth factor signaling pathways are influenced by surface adhesion molecules that mediate cell-cell or cell-matrix interactions, particularly by members of the integrin superfamily. The integrin that has received most attention in ECs is ␣v␤3 (reviewed in Hodivala-Dilke 9 ), which mediates binding to several extracellular matrix proteins and growth factor receptors including VEGFR-2, thus influencing VEGFR-2 signaling (reviewed in Somanath et al 10 ). ␣v␤3 plays a complex role in angiogenesis. Although the origina...

No 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. IntroductionVon Willebrand factor (VWF) is a large multimeric plasma glycoprotein essential to normal hemostasis, first acting as the carrier molecule for procoagulant factor VIII (FVIII), extending its half-life within the circulation by protecting it from proteolytic degradation, and second, supporting platelet adhesion to thrombogenic surfaces at sites of vascular injury. 1,2 Synthesis of VWF is limited to megakaryocytes and endothelial cells. 3 The pre-pro-VWF molecule comprises a 22-amino acid signal peptide, a 741-amino acid propeptide, and the 2050-amino acid mature subunit. The pro-VWF monomer is composed of 4 types of domains (A-D) arranged as follows: NH 2 -D1-D2-DЈ-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-CK-COOH. VWF multimers are formed by C-and N-terminal intermolecular disulphide bonds, with the largest multimers exceeding 2 ϫ 10 4 kDa and being the most hemostatically active. Within the circulation, the multimeric size of VWF is controlled by the plasma metalloprotease ADAMTS13, which cleaves VWF at the Y1605-M1606 bond within the A2 domain, reducing multimeric size and thus regulating its adhesive function. 4 During synthesis, VWF undergoes extensive posttranslational modification resulting in the addition of 12 N-linked and 10 O-linked glycosylation sites per mature monomer. 5 The overall structural composition of the glycans has been determined, but their exact functional significance is poorly understood. There is some evidence to suggest they protect the molecule from proteolytic degradation and are required for dimerization and subsequent multimerization. [6][7][8] Significantly, a small proportion of the N-linked glycans on VWF present the ABO(H) blood group sugars, 9,10 and the importance of this is highlighted by the well-established ...

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. (Blood.

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.

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 4 Individuals with the very rare Bombay phenotype are non-Secretors and also fail to express H transferase (FUT 1). 5 Such people cannot synthesize A or B antigenic structures regardless of their ABO blood group genotype, and ABH antigens are absent from both their erythrocytes and secretions. 6 Para-Bombay individuals also fail to express H transferase, but do express the FUT2 (Secretor)-encoded ␣-1,2-fucosyltransferase, so that ABH antigens are present in their secretions but not on erythrocytes. 6,7 It is well established that ABO blood group exerts a major quantitative effect on plasma von Willebrand factor (VWF) levels, with significantly lower levels in group O individuals. 8,9 Moreover, ABH antigenic determinants have been identified on the N-linked glycans of circulating VWF according to the blood group of the individual. 10 However, the mechanism through which these glycans influence plasma-VWF antigen (VWF:Ag) levels remains unclear. Animal studies have shown that VWF glycans may influence rate of hepatic clearance, 11 and previous data suggested it may be mediated by the H antigen. 12 On the other hand, Bowen recently reported that VWF of different ABO blood groups exhibited different susceptibility to specific cleavage by ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type-1 repeats-13) (O Ն B Ͼ A Ն AB). 13 To further investigate how glycan expression on VWF influences plasma VWF:Ag levels, we have collected plasma samples from a series of Bombay and para-Bombay individuals. As these individuals lack the H antigen, they provide a critical test of current hypotheses. We report the novel observation that Bombay phenotype is associated with plasma-VWF levels similar or lower than group O. In addition, we demonstrate that Bombay VWF demonstrates significant increased susceptibility to cleavage by ADAMTS13, via a conformationdependent mechanism. Study design VWF glycans, VWF antigen, and VWF multim...

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 TFPIdepleted 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 concentrationdependent binding of protein S to wildtype 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. (Blood. 2012;120(25):5059-5062) IntroductionTissue factor pathway inhibitor (TFPI) is a Kunitz-type protease inhibitor consisting of an acidic aminoterminal polypeptide, followed by 3 tandem Kunitz-type domains (Kunitz domains 1, 2, and 3) and a basic carboxyterminal tail. 1,2 TFPI exerts its anticoagulant function by inhibiting tissue factor (TF)-induced coagulation in the blood. [3][4][5] Purified assays have shown that FXa inhibition by TFPI occurs in a 2-step process that can be described by the inhibition constants K i and K i *, 6 respectively, in the following equation:In 2006, Hackeng et al identified protein S as a cofactor for TFPI that is capable of reducing the K i for FXa inhibition by TFPI by 10-fold. 7 More recently, Ndonwi et al showed that protein S enhancement of TFPI is dependent on the TFPI Kunitz domain 3. 8 A TFPI R199L variant showed partial loss of protein S cofactor function compared with wild-type (WT) TFPI. 8 The present study is an investigation of the role of all surface-exposed charged residues of TFPI Kunitz domain 3. Methods Generation, expression, and purification of TFPI variantsTen composite and individual point mutations were generated: D194Q/ R195Q/R199Q, E202Q/R204Q/K218Q, K213Q/R215Q/K232Q, E226Q/ E234Q/R237Q, D194Q, R195Q, R199Q, E226Q, E234Q, and R237Q (supplemental Figure 1, available on the Blood Web site; see the Supplemental Materials link at the top of the online article). Details of vector construction, protein expression, purification, and quantification can be found in the supplemental Methods. Thrombin-generation assay determined by CATCalibrated automated thrombogram (CAT) was performed in normal or TFPI-depleted plasma (supplemental Methods), as described previously. 9,10 Purified or WT TFPI and TFPI variants in concentrated conditioned medium (0-1.5nM) were added to the plasma. Purification status did not influence inhibitory function. FXa inhibition assayFXa (0.5nM) activity was monitored by the cleavage of the chromogenic substrate S-2765 (Chromogenix) in the presence of absence of TFPI (0-4nM) and protein S (0-320nM) essentially as descr...

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 ␣2-3,6,8,9 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 > B > A > 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. (Blood. 2010;115(13): 2666-2673) Introduction von Willebrand factor (VWF) is a large multimeric plasma sialoglycoprotein that plays 2 essential roles in normal hemostasis. 1 First, it mediates platelet adhesion to exposed subendothelial tissues at sites of vascular injury. 2 Second, VWF acts as a carrier molecule for procoagulant factor VIII (FVIII), thereby protecting it from premature proteolytic degradation and clearance. 3 In vivo expression of VWF is restricted to endothelial cells (ECs) and megakaryocytes only. VWF synthesized within ECs is either secreted constitutively into the plasma or alternatively stored in specific intracellular organelles known as Weibel-Palade bodies. 4 In contrast, VWF synthesized within megakaryocytes is subsequently stored within the ␣ granules of their platelet progeny. Consequently, plasma VWF is derived chiefly from ECs. 5 Within ECs, VWF undergoes complex posttranslational modification before secretion. 6 In the endoplasmic reticulum, individual VWF monomers are assembled into dimers through the formation of C-terminal disulphide bonds. 7 Subsequently, VWF dimers form high-molecular-weight multimers in the Golgi after another round of N-terminal disulphide bond formation. 8 The multimeric composition of plasma VWF is a critical determinant of its functional activity, because larger multimers bind both collagen and platelets with significantly higher affinities and are thus more efficient in inducing platelet aggregation under high-shear conditions. 9,10 In normal plasma, VWF multimer distributio...