Shear-induced Disulfide Bond Formation Regulates Adhesion Activity of von Willebrand Factor

Choi, Hiuwan; Aboulfatova, Khatira; Pownall, Henry J.; Cook, Richard G.; Dong, Jing

doi:10.1074/jbc.m704047200

Cited by 102 publications

(151 citation statements)

References 43 publications

Supporting

Mentioning

140

Contrasting

Unclassified

Order By: Relevance

“…As a control and calibrant we expressed the Y1584C variant of VWF that has an extra unpaired cysteine residue per monomer. Consistent with more recent reports, evidence of unpaired cysteine residues was obtained with purified plasma derived VWF, 29 ( Figure 7A). Recombinant wtVWF also reacted similarly with MPB, demonstrating the presence of free thiols.…”

Section: Mutation Of Vwf N-linked Glycan Sites Affects Disulphide Bonsupporting

confidence: 79%

Specific N-linked glycosylation sites modulate synthesis and secretion of von Willebrand factor

McKinnon¹,

Goode²,

Birdsey

et al. 2010

Blood

View full text Add to dashboard Cite

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. (Blood. 2010;116(4):640-648) Introductionvon Willebrand factor (VWF) is a large multimeric plasma glycoprotein essential for normal hemostasis, acting firstly by supporting platelet adhesion to surfaces at sites of vascular injury and secondly as the carrier molecule for pro-coagulant Factor VIII (FVIII). 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 a 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-D'-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-CK-COOH. 1 VWF multimers are formed by C-and N-terminal intermolecular disulphide bonds, 4,5 with the largest multimers exceeding 2 ϫ 10 4 kDa and having the greatest adhesive activity. 6 During synthesis, VWF undergoes extensive posttranslational modification resulting in the addition of 12 N-linked and 10 O-linked glycosylation sites per mature monomer. 7 Furthermore, the propeptide also contains 4 potential N-linked glycosylation sites although it is not known if these are used. In total, carbohydrate accounts for approximately 20% of the molecular weight of VWF. 8 N-linked glycosylation is one of the most common co/ posttranslational modifications. It is characterized by the addition of a carbohydrate moiety to the protein via a beta-glycosidic linkage between an N-acetylglucosamine residue and the ␦-amide of an asparagine residue in the sequence NXS/T (or in some cases NXC), 9 where X is any amino acid except proline. 10 This takes place after translocation into the endoplasmic reticulum (ER) when the enzyme oligosaccharyltransferase (OST) transfers a preformed 14-saccharide core...

show abstract

Section: Mutation Of Vwf N-linked Glycan Sites Affects Disulphide Bonsupporting

confidence: 79%

Specific N-linked glycosylation sites modulate synthesis and secretion of von Willebrand factor

McKinnon¹,

Goode²,

Birdsey

et al. 2010

Blood

View full text Add to dashboard Cite

show abstract

“…VWF circulates in blood as multimers with variable masses and adhesive activities (larger multimers are more adhesive). VWF multimers circulating in the plasma can be converted into more adhesive forms by exposure to pathological high levels of fluid shear stress [3]. In contrast, ultralarge forms of VWF multimers (ULVWF) secreted from the Weibel-Palade bodies of endothelial cells, and anchored to the surface of these cells, are intrinsically hyperadhesive and form strong bonds with GP Ib-IX-V complexes on platelets [4,5].…”

mentioning

confidence: 99%

“…In addition to the disulfide bonds that link VWF monomers into VWF multimers, the large VWF multimers have exposed free thiols [3]. These exposed thiols may enable the large multimers to form mixed disulfide bonds and associate laterally into even larger fibrillar forms [3].…”

mentioning

confidence: 99%

“…It has been shown that VWF multimers change from a globular to an elongated conformation under shear stress [10][11][12] and remain * chkiang@rice.edu in their activated, more adhesive state for hours [13]. Prolonged activation may coincide with the lateral association of VWF multimers into a fibrillar form of VWF [3,8,14].…”

mentioning

confidence: 99%

“…During production of VWF multimers, two monomers (each containing propeptides) initially join at their C-termini via disulfide bonds to form a dimer. The dimers (with propeptides eliminated by furin cleavage) then polymerize at their N-termini to form multimers [1][2][3]6,7]. Each 250-kDa VWF monomeric subunit contains repetitive domains: D -D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-CK (Fig.…”

mentioning

confidence: 99%

See 2 more Smart Citations

Single-molecule force measurements of the polymerizing dimeric subunit of von Willebrand factor

Wijeratne

Yeh

et al. 2016

Phys. Rev. E

Self Cite

View full text Add to dashboard Cite

Von Willebrand factor (VWF) multimers are large adhesive proteins that are essential to the initiation of hemostatic plugs at sites of vascular injury. The binding of VWF multimers to platelets, as well as VWF proteolysis, is regulated by shear stresses that alter VWF multimeric conformation. We used single molecule manipulation with atomic force microscopy (AFM) to investigate the effect of high fluid shear stress on soluble dimeric and multimeric forms of VWF. VWF dimers are the smallest unit that polymerizes to construct large VWF multimers. The resistance to mechanical unfolding with or without exposure to shear stress was used to evaluate VWF conformational forms. Our data indicate that, unlike recombinant VWF multimers (RVWF), recombinant dimeric VWF (RDVWF) unfolding force is not altered by high shear stress (100 dynes/cm 2 for 3 min at 37 • C). We conclude that under the shear conditions used (100 dynes/cm 2 for 3 min at 37 • C), VWF dimers do not self-associate into a conformation analogous to that attained by sheared large VWF multimers.

show abstract

A novel gelatin sponge for accelerated hemostasis

Hajosch

Suckfuell

Oesser³

et al. 2010

J Biomed Mater Res

View full text Add to dashboard Cite

To more effectively manage the substantial bleeding encountered during surgical procedures in oto-rhino-laryngology, we developed a novel hemostatic sponge made of pharmaceutical grade, chemically cross-linked gelatin. The sponge is characterized by a high pore density, reduced ligaments, and a high nanoscale roughness of lamella surfaces in the matrix. In vitro blood uptake assays revealed a very rapid absorption of human blood, which was two to three times faster than that measured with comparative hemostyptic devices. In an in vitro hemorrhage model using human veins, the novel gelatin sponge matrix induced hemostasis less than a minute after bleeding was induced. The sponge was shown to bring about rapid hemostasis when it was administered in a young patient suffering from acute bleeding of a pharyngeal angiofibroma, even though the patient had been treated with an anticoagulant because of a transient ischemic attack. As the gelatin matrix of the sponge is biocompatible and resorbable, the hemostyptic device could be left in place and was shown to be resorbed within 2 weeks. We hypothesize that the excellent hemostatic performance of the sponge might be linked to enhanced capillary effects in conjunction with optimized anchoring of fibrinogen on the nano-rough material surface, as suggested by scanning electron microscopy. The novel gelatin sponge appears to be a promising hemostatic matrix, which could be of great benefit for patients suffering from epistaxis and other acute injuries resulting in severe bleeding.

show abstract

Shear-induced Disulfide Bond Formation Regulates Adhesion Activity of von Willebrand Factor

Cited by 102 publications

References 43 publications

Specific N-linked glycosylation sites modulate synthesis and secretion of von Willebrand factor

Specific N-linked glycosylation sites modulate synthesis and secretion of von Willebrand factor

Single-molecule force measurements of the polymerizing dimeric subunit of von Willebrand factor

A novel gelatin sponge for accelerated hemostasis

Contact Info

Product

Resources

About