Purification and Expression of VWF Fragments

Ristocetin was purchased from MP Biomedicals. Most recombinant VWF fragments and type 2B mutants were expressed from baby hamster kidney cells as described^{19 (link),27 (link)}. Plasma-derived VWF was reconstituted in phosphate-buffered saline (PBS) from outdated lyophilized Humate-P (CSL-Behring). Monoclonal antibodies 6G1, CR1, and 11A8 have been described^{19 (link),47 (link)}. ARC1172^{55 (link)} was synthesized by Integrated DNA Technologies. Mammalian cells were maintained in DMEM/F12 culture media (ThermoFisher), with 10% Foundation-B fetal bovine serum (Gemini Bio-Products, Sacramento, CA), and 1% penicillin/streptomycin (ThermoFisher). Bovine serum albumin, fraction V was purchased from Fisher Scientific. Tobacco etch virus (TEV) protease was produced with plasmid pD2087 in BL21pRIL cells and purified as described^{79 (link)}. The Human GPIb-IX complex was purified from outdated and deidentified leuko-reduced apheresis-derived platelets as described^{80 (link)}.

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Arce N.A., Cao W., Brown A.K., Legan E.R., Wilson M.S., Xu E.R., Berndt M.C., Emsley J., Zhang X.F, & Li R. (2021). Activation of von Willebrand factor via mechanical unfolding of its discontinuous autoinhibitory module. Nature Communications, 12, 2360.

Publication 2021

Ristocetin Humate-P DMEM/F12 culture media Foundation-B fetal bovine serum penicillin/streptomycin Bovine serum albumin,

Apheresis Baby Bovine serum albumin Cells Culture media Fetal bovine serum Hamster Human Humate p Kidney Mammalian Monoclonal antibodies Penicillin Phosphate Plasma Plasmid Platelets Ristocetin Saline Streptomycin Tobacco etch virus protease

Corresponding Organization :

Other organizations : Emory University, Children's Healthcare of Atlanta, Lehigh University, University of Nottingham, Curtin University

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Variable analysis

independent variables

Ristocetin
Recombinant VWF fragments and type 2B mutants
Plasma-derived VWF
Monoclonal antibodies 6G1, CR1, and 11A8
ARC1172

dependent variables

Not explicitly mentioned

control variables

Phosphate-buffered saline (PBS)
DMEM/F12 culture media
10% Foundation-B fetal bovine serum
1% penicillin/streptomycin
Bovine serum albumin, fraction V
Tobacco etch virus (TEV) protease
Human GPIb-IX complex

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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