Binding and conformational changes of VWF under shear
Final Report Abstract
We studied, using a combined approach of fluorescence correlation spectroscopy (FCS) and microfluidics, the physical polymer properties of the Von Willebrand Factor (VWF) and how force induced conformational changes relate to its enzymatic degradation. VWF is a multimeric protein and acts as a mechanosensor responding with a size-dependent globule-stretch transition to increasing shear rates. Using quantitative gel analysis, FCS and total internal reflection fluorescence microscopy we analyzed the size distribution of recombinant VWF and VWF-eGFP in collaboration with the groups of M. Benoit, U. Budde and R. Schneppenheim. We found an exponentially decaying size distribution of multimers for recombinant VWF as well as for VWF derived from blood samples in accordance with the notion of a step-growth polymerization. Importantly, the distribution is solely described by one parameter, the extent of polymerization. The later was e.g. found to be reduced in the case of the pathologically relevant mutant VWF-IIC. The VWF-specific protease ADAMTS13 systematically shifts the VWF size distribution toward smaller sizes. This dynamic evolution was monitored using FCS quantifying the relation of ADAMTS13 concentration to the degree of VWF breakdown. Proteolysis of the multimeric blood coagulation protein VWF by ADAMTS13 occurs within the mechanosensitive A2 domain, which is believed to open under shear flow. We therefore, in a second study, combined FCS and a microfluidic shear set-up to monitor real-time kinetics of full-length VWF proteolysis as a function of shear stress. Under shear, ADAMTS13 activity on full-length VWF arises without denaturing agent as evidenced by FCS and gel-based multimer analysis. The sigmoidal increase of the enzymatic rate as a function of shear was in agreement with Brownian hydrodynamics simulations carried out in the group of Roland Netz. The shear-induced degradation was also observed in full blood plasma. FCS measurements also proved useful to quantify the binding of the protein disulfide isomerase PDIA1 to VWF, which allowed to clarify the mechanism of VWF dimerization as published by M. Brehm. In a collaboration with Frauke Gräter and Camilo Aponte-Santamaria we showed that mutations in the VWF A2 domain facilitate the ADAMTS13 cleavage process. Specifically, in experiment and computer simulation, the mutation G1629E was shown to increase VWF cleavage via site-specific thermodynamic destabilization even in the absence of forces or denaturants. In summary, FCS was applied and further developed to address and quantify VWF conformational changes and the concomitant enzymatic degradation and binding behavior, both in reconstituted systems in buffer as well as in full blood plasma.
Publications
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Exponential size distribution of von Willebrand factor. Biophys J. 2013 Sep 3;105(5):1208-16
Lippok S, Obser T, Müller JP, Stierle VK, Benoit M, Budde U, Schneppenheim R, Rädler JO
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Internal Tension in a Collapsed Polymer under Shear Flow and the Connection to Enzymatic Cleavage of von Willebrand Factor. The European Physical Journal E 39, no. 3 (2016)
Radtke M, Lippok S, Rädler JO, Netz RR
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Shear-Induced Unfolding and Enzymatic Cleavage of Full-Length VWF Multimers. Biophys. J. 110, no. 3 (2016): 545–54
Lippok S, Radtke M, Obser T, Kleemeier L, Schneppenheim R, Budde U, Netz RR, Rädler JO
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Von Willebrand Factor is dimerized by Protein Disulfide Isomerase. Blood, 2016 Mar 3;127(9):1183-91
Lippok S, Kolšek K, Löf A, Eggert D, Vanderlinden W, Müller JP, König G, Obser T, Röhrs K, Schneppenheim S, Budde U, Baldauf C, Aponte-Santamaría C, Gräter F, Schneppenheim R, Rädler JO, Brehm MA
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(2017). Mutation G1629E Increases von Willebrand Factor Cleavage via a Cooperative Destabilization Mechanism. Biophys. J. 112(1):57-65
Aponte-Santamaría C, Lippok S, Mittag JJ, Obser T, Schneppenheim R, Baldauf C, Gräter F, Budde U, Rädler JO
