Optical-Tweezer Single-Molecule Assay for ADAMTS13-Mediated Proteolysis of the von Willebrand Factor A2 Domain.

Weixuan Chen, Wenpeng Cao, X Long Zheng, X Frank Zhang

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Article in Methods in molecular biology (Clifton, N.J.), 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.

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4 authors.

Weixuan ChenDepartment of Biomedical Engineering, University of Massachusetts, Amherst, MA, USA.

Wenpeng CaoDepartment of Bioengineering, Lehigh University, Bethlehem, PA, USA.

X Long ZhengDepartment of Pathology and Laboratory Medicine, The University of Kansas Medical Center, Kansas City, KS, USA.

X Frank ZhangDepartment of Biomedical Engineering, University of Massachusetts, Amherst, MA, USA. frank.zhang@umass.edu.

Funding

Pathogenesis of thrombotic microangiopathiesR01HL164016 · NHLBI · UNIVERSITY OF KANSAS MEDICAL CENTER · PI X. Long Zheng · 2023 to 2026

$2.1M

Mechanism underlying cofactor-dependent proteolysis of von Willebrand FactorR01HL157975 · NHLBI · UNIVERSITY OF KANSAS MEDICAL CENTER · PI ZHANG, XIAOHUI FRANK, ZHENG, X. LONG · 2022 to 2025

$1.8M

NHLBI NIH HHS R01 HL157975NHLBI NIH HHS R01 HL164016

6 · The paper itself

Abstract

Optical tweezers enable real-time visualization of mechanosensitive enzyme-substrate reactions at the level of single protein molecules. We present a protocol that quantifies the force-dependent cleavage of the von Willebrand factor (VWF) A2 domain by the metalloprotease ADAMTS13, in the absence or presence of its physiological cofactor factor VIII (FVIII). Recombinant A2 domains are site-specifically conjugated to double-stranded DNA handles via SpyTag-SpyCatcher chemistry and tethered between two beads. Controlled stretching by optical tweezers unfolds the A2 substrate, exposing the Tyr1605-Met1606 bond for enzymatic cleavage, whereas sequential extension-relaxation cycles in an ADAMTS13-containing buffer reveal stepwise tether-length reductions that correspond to individual cleavage events. Dwell-time analysis across multiple enzyme concentrations yields single-molecule Michaelis-Menten parameters (k

Indexed as

ADAMTS13 ProteinOptical TweezersSingle Molecule Imagingvon Willebrand FactorFactor VIIIHumansProtein DomainsProteolysisRecombinant ProteinsADAMTS13 ProteinADAMTS13 protein, humanFactor VIIIRecombinant Proteinsvon Willebrand FactorADAMTS13DNA handlesHemostasisMechanoenzymatic cleavageProtein unfoldingSingle-molecule optical tweezervon Willebrand Factor

Identifiers

PMID41917363

DOI10.1007/978-1-0716-5218-3_15

PMCPMC13335629

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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the Socratic graph.