title: Shear-Induced Unfolding and Enzymatic Cleavage of Full-Length VWF Multimers.
abstract: Proteolysis of the multimeric blood coagulation protein von Willebrand Factor (VWF) by ADAMTS13 is crucial for prevention of microvascular thrombosis. ADAMTS13 cleaves VWF within the mechanosensitive A2 domain, which is believed to open under shear flow. In this study, we combine fluorescence correlation spectroscopy (FCS) and a microfluidic shear cell to monitor real-time kinetics of full-length VWF proteolysis as a function of shear stress. For comparison, we also measure the Michaelis-Menten kinetics of ADAMTS13 cleavage of wild-type VWF in the absence of shear but partially denaturing conditions. Under shear, ADAMTS13 activity on full-length VWF arises without denaturing agent as evidenced by FCS and gel-based multimer analysis. In agreement with Brownian hydrodynamics simulations, we find a sigmoidal increase of the enzymatic rate as a function of shear at a threshold shear rate γ˙1/2 = 5522/s. The same flow-rate dependence of ADAMTS13 activity we also observe in blood plasma, which is relevant to predict hemostatic dysfunction.
authors: Faculty of Physics and Center for NanoScience, Ludwig Maximilian University, Munich, Germany.
authors: Svenja
authors: S
authors: Lippok
authors: Department of Physics, Freie Universität Berlin, Berlin, Germany.
authors: Matthias
authors: M
authors: Radtke
authors: Department of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
authors: Tobias
authors: T
authors: Obser
authors: Faculty of Physics and Center for NanoScience, Ludwig Maximilian University, Munich, Germany.
authors: Lars
authors: L
authors: Kleemeier
authors: Department of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
authors: Reinhard
authors: R
authors: Schneppenheim
authors: Coagulation Lab, Medilys Laborgesellschaft Hamburg, Hamburg, Germany.
authors: Ulrich
authors: U
authors: Budde
authors: Department of Physics, Freie Universität Berlin, Berlin, Germany.
authors: Roland R
authors: RR
authors: Netz
authors: Faculty of Physics and Center for NanoScience, Ludwig Maximilian University, Munich, Germany. Electronic address: raedler@lmu.de.
authors: Joachim O
authors: JO
authors: Rädler
mesh_terms: D051722:ADAM Proteins / Q000378:metabolism; D057446:Hydrodynamics; D009154:Mutation; D055503:Protein Multimerization; D058767:Protein Unfolding; D059748:Proteolysis; D014841:von Willebrand Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D014841:von Willebrand Factor; D051722:ADAM Proteins