Shobhit Gogia - Academia.edu (original) (raw)

Papers by Shobhit Gogia

Blood Advances

Key Points VWF A2-domain intracellular proteolysis within ECs is enhanced upon disrupting calcium... more Key Points VWF A2-domain intracellular proteolysis within ECs is enhanced upon disrupting calcium binding (eg, in VWD type 2A mutants). VWF string cleavage on ECs is calcium independent and is strongly dependent on platelet binding.

Biorheology, Jan 19, 2015

Von Willebrand factor (VWF) is the largest glycoprotein in blood. It plays a crucial role in prim... more 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 ...

The metalloprotease ADAMTS13 regulates the size of the multimeric blood protein von Willebrand fa... more The metalloprotease ADAMTS13 regulates the size of the multimeric blood protein von Willebrand factor (VWF). Reduction or inhibition of ADAMTS13 activity in humans can lead to a bleeding disorder called Thrombotic Thrombocytopenic Purpura (TTP). In this paper, we describe a flow cytometry based assay to quantify ADAMTS13 activity in human clinical samples using an E. coli derived 77-amino acid peptide substrate that is based on a mutated form of the A2-domain of VWF. This substrate was tagged using NHS (N-hydroxysuccinimide)-fluorescein at the N-terminus and Maleimide-PEG(Polyethylene glycol)n-biotin at the C-terminus. PEG repeat unit length was 2 in the case of ‘A2-77p-short’ and it was 77 for ‘A2-77p-long’. Biotin-streptavidin bonding was used to immobilize the substrates onto streptavidin coated microspheres. In flow cytometry assays, fluorescein signal associated with A2-77p-short/A2-77p-long decreased inversely with recombinant ADAMTS13 concentration. A2-77p-long also detected ...

PLOS ONE, 2015

Protease levels in human blood are often prognostic indicators of inflammatory, thrombotic or onc... more 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

Analytical Biochemistry, 2011

The cleavage of the A2 domain of von Willebrand factor (VWF) by the metalloprotease ADAMTS13 regu... more 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 ADAMTS13mediated 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, 4 M 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.

Nature chemistry, 2015

Methods to attach polypeptides to lipid bilayers are often indirect and ineffective, and can repr... more 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...

Blood Advances

Key Points VWF A2-domain intracellular proteolysis within ECs is enhanced upon disrupting calcium... more Key Points VWF A2-domain intracellular proteolysis within ECs is enhanced upon disrupting calcium binding (eg, in VWD type 2A mutants). VWF string cleavage on ECs is calcium independent and is strongly dependent on platelet binding.

Biorheology, Jan 19, 2015

Von Willebrand factor (VWF) is the largest glycoprotein in blood. It plays a crucial role in prim... more 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 ...

The metalloprotease ADAMTS13 regulates the size of the multimeric blood protein von Willebrand fa... more The metalloprotease ADAMTS13 regulates the size of the multimeric blood protein von Willebrand factor (VWF). Reduction or inhibition of ADAMTS13 activity in humans can lead to a bleeding disorder called Thrombotic Thrombocytopenic Purpura (TTP). In this paper, we describe a flow cytometry based assay to quantify ADAMTS13 activity in human clinical samples using an E. coli derived 77-amino acid peptide substrate that is based on a mutated form of the A2-domain of VWF. This substrate was tagged using NHS (N-hydroxysuccinimide)-fluorescein at the N-terminus and Maleimide-PEG(Polyethylene glycol)n-biotin at the C-terminus. PEG repeat unit length was 2 in the case of ‘A2-77p-short’ and it was 77 for ‘A2-77p-long’. Biotin-streptavidin bonding was used to immobilize the substrates onto streptavidin coated microspheres. In flow cytometry assays, fluorescein signal associated with A2-77p-short/A2-77p-long decreased inversely with recombinant ADAMTS13 concentration. A2-77p-long also detected ...

PLOS ONE, 2015

Protease levels in human blood are often prognostic indicators of inflammatory, thrombotic or onc... more 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

Analytical Biochemistry, 2011

The cleavage of the A2 domain of von Willebrand factor (VWF) by the metalloprotease ADAMTS13 regu... more 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 ADAMTS13mediated 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, 4 M 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.

Nature chemistry, 2015

Methods to attach polypeptides to lipid bilayers are often indirect and ineffective, and can repr... more 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...