title: Unfolding the A2 domain of von Willebrand factor with the optical trap.
abstract: Von Willebrand factor (VWF) is a multimeric plasma glycoprotein involved in both hemostasis and thrombosis. VWF conformational changes, especially unfolding of the A2 domain, may be required for efficient enzymatic cleavage in vivo. It has been shown that a single A2 domain unfolds at most probable unfolding forces of 7-14 pN at force loading rates of 0.35-350 pN/s and A2 unfolding facilitates A2 cleavage in vitro. However, it remains unknown how much force is required to unfold the A2 domain in the context of a VWF multimer where A2 may be stabilized by other domains like A1 and A3. With the optical trap, we stretched VWF multimers and a poly-protein (A1A2A3)3 that contains three repeats of the triplet A1A2A3 domains at constant speeds of 2000 nm/s and 400 nm/s, respectively, which yielded corresponding average force loading rates of 90 and 22 pN/s. We found that VWF multimers became stiffer when they were stretched and extended by force. After force increased to a certain level, sudden extensional jumps that signify domain unfolding were often observed. Histograms of the unfolding force and the unfolded contour length showed two or three peaks that were integral multiples of approximately 21 pN and approximately 63 nm, respectively. Stretching of (A1A2A3)3 yielded comparable distributions of unfolding force and unfolded contour length, showing that unfolding of the A2 domain accounts for the behavior of VWF multimers under tension. These results show that the A2 domain can be indeed unfolded in the presence of A1, A3, and other domains. Compared with the value in the literature, the larger most probable unfolding force measured in this study suggests that the A2 domain is mechanically stabilized by A1 or A3 although variations in experimental setups and conditions may complicate this interpretation.
authors.affiliation: Department of Biomedical Engineering, Washington University, St. Louis, Missouri, USA.
authors.forename: Junyi
authors.initials: J
authors.lastname: Ying
authors.forename: Yingchen
authors.initials: Y
authors.lastname: Ling
authors.forename: Lisa A
authors.initials: LA
authors.lastname: Westfield
authors.forename: J Evan
authors.initials: JE
authors.lastname: Sadler
authors.forename: Jin-Yu
authors.initials: JY
authors.lastname: Shao
mesh_terms: D000595:Amino Acid Sequence; D001696:Biomechanical Phenomena; D002460:Cell Line; D006801:Humans; D008969:Molecular Sequence Data; D052898:Optical Tweezers; D020815:Polyproteins / Q000138:chemical synthesis / Q000737:chemistry; D017510:Protein Folding; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D014841:von Willebrand Factor / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D020815:Polyproteins; D014841:von Willebrand Factor
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