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.

journal_name

Biophys J

journal_title

Biophysical journal

authors

Lippok S,Radtke M,Obser T,Kleemeier L,Schneppenheim R,Budde U,Netz RR,Rädler JO

doi

10.1016/j.bpj.2015.12.023

subject

Has Abstract

pub_date

2016-02-02 00:00:00

pages

545-554

issue

3

eissn

0006-3495

issn

1542-0086

pii

S0006-3495(15)04766-9

journal_volume

110

pub_type

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