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Franz Kaiser «  Daniel Steppich »  Sabine Wüst
Daniel Steppich
The Physics of Von Willebrand Factor
Supervisor: Prof. Dr. Achim Wixforth [Experimental physics I]
Date of oral examination: 02/10/2009
121 pages, english
Haemostasis is a complex cascade of physiological processes, which induce the stop of blood loss at a site of vascular damage. In addition to various kinds of cells and proteins, this process involves numerous chemical and/or mechanical stimuli. In this thesis, the focus is set on the action of the blood clotting factor Von Willebrand Factor (VWF), which plays a pivotal role during both primary and secondary haemostasis under elevated shear flow conditions. The observation of the necessity for a critical shear stress to mechanically unroll the multimeric protein VWF from a globular into an activated unrolled conformation led to a variety of biologically and medically motivated questions: Which forces retain VWF in its globular conformation? What influence does a surface have on VWF activation? How does a change of the streaming properties at a site of vascular damage affect VWF binding? How do VWF networks react to external stress? What parameters can modify the critical shear rate within wound healing? Do VWF molecules physically bind to endothelial cells and platelets besides the biological concept of specific lock- and-key interactions? If so, which membrane and protein factors mediate this interaction? Is such a physical attachment strong enough to span extended and haemostatically active networks? Following the course of wound healing from VWF-VWF- to VWF-membrane-interactions, these open questions are approached from a physical perspective and are evaluated with regard to their medical impact within this thesis. In particular, the cumulative effect of stream line perturbations in close vicinity to an injured vessel wall and the influence of the surface itself on the critical shear rate were found to significantly affect network formation. Compared to bulk conditions, the combined slowing down of both rotational and translational VWF movement near a planar surface decreases the critical shear rate by up to 60%25 and hence facilitates VWF activation. Furthermore, modified streaming properties and vortex formation around protruding parts of damaged endothelium or extracellular matrix may directly result in an accumulation of the protein and contribute to an enhanced network formation potential. The elucidation of the interior dynamics of these extended networks represented an exquisite experimental challenge. The development of a Surface Acoustic Wave (SAW) driven microfluidic reactor integrated into an optical accessible Atomic Force Microscope (AFM) was a prerequisite for studying both the dynamics of the network formation process and its response on minute mechanical manipulation with an AFM tip. Application of pulling forces to VWF conglomerates in this effective hybrid system and monitoring their relaxation behaviour enabled the development of a %94bundle jamming%94 model for crosslinked protein fibres emphasizing the role of both memory effects and strong coupling between single molecules inside protein networks. This coupling is described by an interaction potential U. It exactly determines the order of magnitude of the critical shear rates to fit the conditions in our blood vessels. In a series of experiments with varying solvent polarity, strong evidence was given that U is governed by hydrophobic interactions. From a physiological perspective, a prominent parameter for fine-tuning U and hence VWF%92s critical shear rate is a change in the pH of the solution. In this respect, it is a spectacular finding that the critical shear rate for unrolling the VWF molecule exhibits a clear maximum at blood pH ~ 7.4 and can be sensitively manipulated by minute pH variations in the range of %B10,2. Any deviation to both acidic and basic pH values results in a distinct decrease of the critical shear rate. Its consequences on the dynamic course of blood clotting are discussed as local changes from pH ~ 7.4 may occur at vessel lesions or stenosis. The origin of VWF%92s maximal stability against external forces under normal blood conditions is related to its minimal solubility at its effective isoelectric point which seems to be evolutionary adjusted exactly to blood pH. Our hybrid system allowed to establish a phase diagram, which displays VWF%92s activity as a function of both shear rate and pH. This phase diagram represents the condensed information for the response of VWF under different haemostatic conditions. The investigation of the adhesion process of VWF to artificial phospholipid membranes identified the membrane phase state, membrane defects and domain boundaries as the main contributions for tight bonding and network formation. Atomic Force Microscopy (AFM) imaging under physiological buffer conditions revealed diverse arrangements of both single and activated VWF networks on varying membrane substrates. Advanced tip chemistry further enabled the binding of large multimers onto the AFM cantilever and the determination of extremely high interaction forces in the range of hundreds of pN of VWF with membranes in series of AFM Force Spectroscopy measurements. Both AFM imaging and Force Spectroscopy distinguished a striking influence of the hydrophobic core of the membrane to the interaction potential. The molecular binding mechanism itself seems to be relatively insensitive to VWF conformation while only the total amount of binding sites increases considerably in the activated state. Shear stress on the other hand does not exclusively affect the VWF multimer but also its dimeric building bock. Structural analysis elucidated a pronounced susceptibility of single dimers to hydrodynamic forces. This will be the first report on structural changes inside the molecular basis of VWF upon mechanical forces. Previous results concerning VWF activation will have to be reconsidered on this molecular level as structural changes in its subunit will affect the stability of the whole protein under flow conditions and hence the stretching process itself.