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  1. Article ; Online: Role of fluid shear stress in regulating VWF structure, function and related blood disorders.

    Gogia, Shobhit / Neelamegham, Sriram

    Biorheology

    2015  Volume 52, Issue 5-6, Page(s) 319–335

    Abstract: Von Willebrand factor (VWF) is the largest glycoprotein in blood. It plays a crucial role in primary hemostasis via its binding interaction with platelet and endothelial cell surface receptors, other blood proteins and extra-cellular matrix components. ... ...

    Abstract Von Willebrand factor (VWF) is the largest glycoprotein in blood. It plays a crucial role in primary hemostasis via its binding interaction with platelet and endothelial cell surface receptors, other blood proteins and extra-cellular matrix components. This protein is found as a series of repeat units that are disulfide bonded to form multimeric structures. Once in blood, the protein multimer distribution is dynamically regulated by fluid shear stress which has two opposing effects: it promotes the aggregation or self-association of multiple VWF units, and it simultaneously reduces multimer size by facilitating the force-dependent cleavage of the protein by various proteases, most notably ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type repeats, motif 1 type 13). In addition to these effects, fluid shear also controls the solution and substrate-immobilized structure of VWF, the nature of contact between blood platelets and substrates, and the biomechanics of the GpIbα-VWF bond. These features together regulate different physiological and pathological processes including normal hemostasis, arterial and venous thrombosis, von Willebrand disease, thrombotic thrombocytopenic purpura and acquired von Willebrand syndrome. This article discusses current knowledge of VWF structure-function relationships with emphasis on the effects of hydrodynamic shear, including rapid methods to estimate the nature and magnitude of these forces in selected conditions. It shows that observations made by many investigators using solution and substrate-based shearing devices can be reconciled upon considering the physical size of VWF and the applied mechanical force in these different geometries.
    MeSH term(s) ADAM Proteins/metabolism ; ADAMTS13 Protein ; Blood Platelets/physiology ; Hematologic Diseases/metabolism ; Hematologic Diseases/pathology ; Humans ; Hydrodynamics ; Models, Theoretical ; Platelet Glycoprotein GPIb-IX Complex/chemistry ; Platelet Glycoprotein GPIb-IX Complex/metabolism ; Protein Structure, Quaternary ; Shear Strength ; von Willebrand Factor/chemistry ; von Willebrand Factor/metabolism
    Chemical Substances Platelet Glycoprotein GPIb-IX Complex ; adhesion receptor ; von Willebrand Factor ; ADAM Proteins (EC 3.4.24.-) ; ADAMTS13 Protein (EC 3.4.24.87) ; ADAMTS13 protein, human (EC 3.4.24.87)
    Language English
    Publishing date 2015
    Publishing country Netherlands
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Review
    ZDB-ID 82015-5
    ISSN 1878-5034 ; 0006-355X
    ISSN (online) 1878-5034
    ISSN 0006-355X
    DOI 10.3233/BIR-15061
    Database MEDical Literature Analysis and Retrieval System OnLINE

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    In stock of ZB MED Cologne/Königswinter

    Zs.A 394: Show issues Location:
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    Jg. 1995 - 2021: Lesesall (1.OG)
    ab Jg. 2022: Lesesaal (EG)

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  2. Article ; Online: Detection of Plasma Protease Activity Using Microsphere-Cytometry Assays with E. coli Derived Substrates: VWF Proteolysis by ADAMTS13.

    Gogia, Shobhit / Lo, Chi Y / Neelamegham, Sriram

    PloS one

    2015  Volume 10, Issue 5, Page(s) e0126556

    Abstract: Protease levels in human blood are often prognostic indicators of inflammatory, thrombotic or oncogenic disorders. The measurement of such enzyme activities in substrate-based assays is complicated due to the low prevalence of these enzymes and steric ... ...

    Abstract Protease levels in human blood are often prognostic indicators of inflammatory, thrombotic or oncogenic disorders. The measurement of such enzyme activities in substrate-based assays is complicated due to the low prevalence of these enzymes and steric hindrance of the substrates by the more abundant blood proteins. To address these limitations, we developed a molecular construct that is suitable for microsphere-cytometer based assays in the milieu of human blood plasma. In this proof of principle study, we demonstrate the utility of this substrate to measure metalloprotease ADAMTS13 activity. The substrate, expressed in E. coli as a fusion protein, contains the partial A2-domain of von Willebrand factor (VWF amino acids 1594-1670) that is mutated to include a single primary amine at the N-terminus and free cysteines at the C-terminus. N-terminus fluorescence conjugation was possible using NHS (N-hydroxysuccinimide) chemistry. Maleimide-PEG(Polyethylene glycol)n-biotin coupling at the C-terminus allowed biotinylation with variable PEG spacer lengths. Once bound to streptavidin-bearing microspheres, the substrate fluorescence signal decreased in proportion with ADAMTS13 concentration. Whereas recombinant ADAMTS13 activity could be quantified using substrates with all PEG repeat-lengths, only the construct with the longer 77 PEG-unit could quantify proteolysis in blood plasma. Using this longer substrate, plasma ADAMTS13 down to 5% of normal levels could be detected within 30 min. Such measurements could also be readily performed under conditions resembling hyperbilirubinemia. Enzyme catalytic activity was tuned by varying buffer calcium, with lower divalent ion concentrations enhancing cleavage. Overall, the study highlights the substrate design features important for the creation of efficient proteolysis assays in the setting of human plasma. In particular, it emphasizes the need to introduce PEG spacers in plasma-based experiments, a design attribute commonly ignored in immobilized peptide-substrate assays.
    MeSH term(s) ADAM Proteins/metabolism ; ADAMTS13 Protein ; Biotinylation ; Escherichia coli/metabolism ; Flow Cytometry ; Humans ; Microspheres ; Peptide Fragments/metabolism ; Peptide Hydrolases/blood ; Proteolysis ; Recombinant Fusion Proteins/metabolism ; Substrate Specificity ; von Willebrand Factor/metabolism
    Chemical Substances Peptide Fragments ; Recombinant Fusion Proteins ; von Willebrand Factor ; Peptide Hydrolases (EC 3.4.-) ; ADAM Proteins (EC 3.4.24.-) ; ADAMTS13 Protein (EC 3.4.24.87) ; ADAMTS13 protein, human (EC 3.4.24.87)
    Language English
    Publishing date 2015
    Publishing country United States
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
    ISSN 1932-6203
    ISSN (online) 1932-6203
    DOI 10.1371/journal.pone.0126556
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  3. Article ; Online: Detection of Plasma Protease Activity Using Microsphere-Cytometry Assays with E. coli Derived Substrates

    Shobhit Gogia / Chi Y Lo / Sriram Neelamegham

    PLoS ONE, Vol 10, Iss 5, p e

    VWF Proteolysis by ADAMTS13.

    2015  Volume 0126556

    Abstract: Protease levels in human blood are often prognostic indicators of inflammatory, thrombotic or oncogenic disorders. The measurement of such enzyme activities in substrate-based assays is complicated due to the low prevalence of these enzymes and steric ... ...

    Abstract Protease levels in human blood are often prognostic indicators of inflammatory, thrombotic or oncogenic disorders. The measurement of such enzyme activities in substrate-based assays is complicated due to the low prevalence of these enzymes and steric hindrance of the substrates by the more abundant blood proteins. To address these limitations, we developed a molecular construct that is suitable for microsphere-cytometer based assays in the milieu of human blood plasma. In this proof of principle study, we demonstrate the utility of this substrate to measure metalloprotease ADAMTS13 activity. The substrate, expressed in E. coli as a fusion protein, contains the partial A2-domain of von Willebrand factor (VWF amino acids 1594-1670) that is mutated to include a single primary amine at the N-terminus and free cysteines at the C-terminus. N-terminus fluorescence conjugation was possible using NHS (N-hydroxysuccinimide) chemistry. Maleimide-PEG(Polyethylene glycol)n-biotin coupling at the C-terminus allowed biotinylation with variable PEG spacer lengths. Once bound to streptavidin-bearing microspheres, the substrate fluorescence signal decreased in proportion with ADAMTS13 concentration. Whereas recombinant ADAMTS13 activity could be quantified using substrates with all PEG repeat-lengths, only the construct with the longer 77 PEG-unit could quantify proteolysis in blood plasma. Using this longer substrate, plasma ADAMTS13 down to 5% of normal levels could be detected within 30 min. Such measurements could also be readily performed under conditions resembling hyperbilirubinemia. Enzyme catalytic activity was tuned by varying buffer calcium, with lower divalent ion concentrations enhancing cleavage. Overall, the study highlights the substrate design features important for the creation of efficient proteolysis assays in the setting of human plasma. In particular, it emphasizes the need to introduce PEG spacers in plasma-based experiments, a design attribute commonly ignored in immobilized peptide-substrate assays.
    Keywords Medicine ; R ; Science ; Q
    Subject code 500
    Language English
    Publishing date 2015-01-01T00:00:00Z
    Publisher Public Library of Science (PLoS)
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  4. Article: Role of calcium in regulating the intra- and extracellular cleavage of von Willebrand factor by the protease ADAMTS13.

    Gogia, Shobhit / Kelkar, Anju / Zhang, Changjie / Dayananda, Kannayakanahalli M / Neelamegham, Sriram

    Blood advances

    2017  Volume 1, Issue 23, Page(s) 2063–2074

    Abstract: von Willebrand factor (VWF) and the metalloprotease a disintegrin and metalloprotease with thrombospondin type 1 motif 13 (ADAMTS13) are present both within endothelial cells (ECs) and in peripheral blood. Calcium concentrations are lower in ... ...

    Abstract von Willebrand factor (VWF) and the metalloprotease a disintegrin and metalloprotease with thrombospondin type 1 motif 13 (ADAMTS13) are present both within endothelial cells (ECs) and in peripheral blood. Calcium concentrations are lower in intracellular compartments (80-400 μM) compared with the extracellular milieu (∼1.25 mM). Because low calcium favors VWF A2-domain proteolysis by ADAMTS13, the dependence of proteolysis rates on calcium was assayed both within ECs and in blood. Confocal microscopy studies demonstrate partial perinuclear colocalization of VWF with ADAMTS13 in human umbilical vein ECs (HUVECs). Consequently, low levels (5%-10%) of VWF cleavage products were detected in HUVEC lysates and also culture-supernatant following EC stimulation. This proteolysis occurred before disulfide bond formation. Compared with wild-type VWF A2-domain, calcium-binding mutants including the common von Willebrand disease (VWD) type 2A R1597W mutant were expressed in an open conformation in ECs and were highly susceptible to intracellular proteolysis. Fluorescence resonance energy transfer measurements demonstrate strong calcium-dependent VWF-A2 conformation changes at concentrations <500 μM, with unfolding rates being fourfold higher for monomeric VWF A2-domain compared with multimeric, full-length VWF. Under shear, physiological levels of ADAMTS13 did not cleave VWF strings on HUVECs, unless platelets were attached to stretch these strings under flow. Further, VWF-platelet string cleavage under shear proceeded with equal efficiency in the absence and presence of calcium at shear stress ≥1 dyn/cm
    Language English
    Publishing date 2017-10-24
    Publishing country United States
    Document type Journal Article
    ZDB-ID 2876449-3
    ISSN 2473-9537 ; 2473-9529
    ISSN (online) 2473-9537
    ISSN 2473-9529
    DOI 10.1182/bloodadvances.2017009027
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  5. Article ; Online: Escherichia coli-derived von Willebrand factor-A2 domain fluorescence/Förster resonance energy transfer proteins that quantify ADAMTS13 activity.

    Dayananda, Kannayakanahalli M / Gogia, Shobhit / Neelamegham, Sriram

    Analytical biochemistry

    2010  Volume 410, Issue 2, Page(s) 206–213

    Abstract: The cleavage of the A2 domain of von Willebrand factor (VWF) by the metalloprotease ADAMTS13 regulates VWF size and platelet thrombosis rates. Reduction or inhibition of this enzyme activity leads to thrombotic thrombocytopenic purpura (TTP). We ... ...

    Abstract The cleavage of the A2 domain of von Willebrand factor (VWF) by the metalloprotease ADAMTS13 regulates VWF size and platelet thrombosis rates. Reduction or inhibition of this enzyme activity leads to thrombotic thrombocytopenic purpura (TTP). We generated a set of novel molecules called VWF-A2 FRET (fluorescence/Förster resonance energy transfer) proteins, where variants of yellow fluorescent protein (Venus) and cyan fluorescent protein (Cerulean) flank either the entire VWF-A2 domain (175 amino acids) or truncated fragments (141, 113, and 77 amino acids) of this domain. These proteins were expressed in Escherichia coli in soluble form, and they exhibited FRET properties. Results show that the introduction of Venus/Cerulean itself did not alter the ability of VWF-A2 to undergo ADAMTS13-mediated cleavage. The smallest FRET protein, XS-VWF, detected plasma ADAMTS13 activity down to 10% of normal levels. Tests of acquired and inherited TTP could be completed within 30 min. VWF-A2 conformation changed progressively, and not abruptly, on increasing urea concentrations. Although proteins with 77 and 113 VWF-A2 residues were cleaved in the absence of denaturant, 4M urea was required for the efficient cleavage of larger constructs. Overall, VWF-A2 FRET proteins can be applied both for the rapid diagnosis of plasma ADAMTS13 activity and as a tool to study VWF-A2 conformation dynamics.
    MeSH term(s) ADAM Proteins/blood ; ADAM Proteins/chemistry ; ADAMTS13 Protein ; Blood Chemical Analysis/methods ; Blood Coagulation/physiology ; Cell Line ; Escherichia coli/metabolism ; Fluorescence ; Fluorescence Resonance Energy Transfer/methods ; Humans ; Protein Conformation ; Purpura, Thrombotic Thrombocytopenic/blood ; Purpura, Thrombotic Thrombocytopenic/diagnosis ; von Willebrand Diseases/blood ; von Willebrand Diseases/diagnosis ; von Willebrand Factor/biosynthesis ; von Willebrand Factor/chemistry ; von Willebrand Factor/genetics
    Chemical Substances von Willebrand Factor ; ADAM Proteins (EC 3.4.24.-) ; ADAMTS13 Protein (EC 3.4.24.87) ; ADAMTS13 protein, human (EC 3.4.24.87)
    Language English
    Publishing date 2010-12-10
    Publishing country United States
    Document type Journal Article ; Research Support, N.I.H., Extramural
    ZDB-ID 1110-1
    ISSN 1096-0309 ; 0003-2697
    ISSN (online) 1096-0309
    ISSN 0003-2697
    DOI 10.1016/j.ab.2010.12.005
    Database MEDical Literature Analysis and Retrieval System OnLINE

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    In stock of ZB MED Cologne/Königswinter

    Zs.A 389: Show issues Location:
    Je nach Verfügbarkeit (siehe Angabe bei Bestand)
    bis Jg. 1994: Bestellungen von Artikeln über das Online-Bestellformular
    Jg. 1995 - 2021: Lesesall (1.OG)
    ab Jg. 2022: Lesesaal (EG)

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  6. Article ; Online: Functionalization of cobalt porphyrin-phospholipid bilayers with his-tagged ligands and antigens.

    Shao, Shuai / Geng, Jumin / Ah Yi, Hyun / Gogia, Shobhit / Neelamegham, Sriram / Jacobs, Amy / Lovell, Jonathan F

    Nature chemistry

    2015  Volume 7, Issue 5, Page(s) 438–446

    Abstract: Methods to attach polypeptides to lipid bilayers are often indirect and ineffective, and can represent a substantial bottleneck in the formation of functionalized lipid-based materials. Although the polyhistidine tag (his-tag) has been transformative in ... ...

    Abstract Methods to attach polypeptides to lipid bilayers are often indirect and ineffective, and can represent a substantial bottleneck in the formation of functionalized lipid-based materials. Although the polyhistidine tag (his-tag) has been transformative in its simplicity and efficacy in binding to immobilized metals, the successful application of this approach has been challenging in physiological settings. Here we show that lipid bilayers containing porphyrin-phospholipid conjugates that are chelated with cobalt, but not with other metals, can effectively capture his-tagged proteins and peptides. The binding follows a Co(II) to Co(III) transition and occurs within the sheltered hydrophobic bilayer, resulting in an essentially irreversible attachment in serum or in a million fold excess of competing imidazole. Using this approach we anchored homing peptides into the bilayer of preformed and cargo-loaded liposomes to enable tumour targeting without disrupting the bilayer integrity. As a further demonstration, a synthetic protein fragment derived from the human immunodeficiency virus was bound to immunogenic liposomes for potent antibody generation for an otherwise non-antigenic peptide.
    MeSH term(s) Cobalt/chemistry ; Lipid Bilayers/chemistry ; Liposomes ; Phospholipids/chemistry ; Porphyrins/chemistry
    Chemical Substances Lipid Bilayers ; Liposomes ; Phospholipids ; Porphyrins ; Cobalt (3G0H8C9362)
    Language English
    Publishing date 2015-04-20
    Publishing country England
    Document type Journal Article ; Research Support, N.I.H., Extramural
    ZDB-ID 2464596-5
    ISSN 1755-4349 ; 1755-4330
    ISSN (online) 1755-4349
    ISSN 1755-4330
    DOI 10.1038/nchem.2236
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  7. Article: Escherichia coli-derived von Willebrand factor-A2 domain fluorescence/Förster resonance energy transfer proteins that quantify ADAMTS13 activity

    Dayananda, Kannayakanahalli M. / Gogia, Shobhit / Neelamegham, Sriram

    Analytical biochemistry

    Volume v. 410,, Issue no. 2

    Abstract: The cleavage of the A2 domain of von Willebrand factor (VWF) by the metalloprotease ADAMTS13 regulates VWF size and platelet thrombosis rates. Reduction or inhibition of this enzyme activity leads to thrombotic thrombocytopenic purpura (TTP). We ... ...

    Abstract The cleavage of the A2 domain of von Willebrand factor (VWF) by the metalloprotease ADAMTS13 regulates VWF size and platelet thrombosis rates. Reduction or inhibition of this enzyme activity leads to thrombotic thrombocytopenic purpura (TTP). We generated a set of novel molecules called VWF-A2 FRET (fluorescence/Förster resonance energy transfer) proteins, where variants of yellow fluorescent protein (Venus) and cyan fluorescent protein (Cerulean) flank either the entire VWF-A2 domain (175 amino acids) or truncated fragments (141, 113, and 77 amino acids) of this domain. These proteins were expressed in Escherichia coli in soluble form, and they exhibited FRET properties. Results show that the introduction of Venus/Cerulean itself did not alter the ability of VWF-A2 to undergo ADAMTS13-mediated cleavage. The smallest FRET protein, XS-VWF, detected plasma ADAMTS13 activity down to 10% of normal levels. Tests of acquired and inherited TTP could be completed within 30min. VWF-A2 conformation changed progressively, and not abruptly, on increasing urea concentrations. Although proteins with 77 and 113 VWF-A2 residues were cleaved in the absence of denaturant, 4M urea was required for the efficient cleavage of larger constructs. Overall, VWF-A2 FRET proteins can be applied both for the rapid diagnosis of plasma ADAMTS13 activity and as a tool to study VWF-A2 conformation dynamics.
    Keywords Venus ; energy transfer ; thrombocytopenic purpura ; enzyme inhibition ; protein synthesis ; urea ; yellow fluorescent protein ; fluorescence ; amino acids ; Escherichia coli ; enzyme activity ; metalloproteinases ; thrombosis ; blood coagulation factors
    Language English
    Document type Article
    ISSN 0003-2697
    Database AGRIS - International Information System for the Agricultural Sciences and Technology

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