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Forschungsarbeit

Unravelling the Secrets of Blood Clotting

by Daniel Michael Steppich 

Blood clotting and wound healing is an essential task of our microvascular system. Especially in small arterioles and veins there is continuous damage to the vessel wall simply caused by high streaming velocities and the blood pressure due to the pumping of the heart. At the same time nature provided our body with a protein in the blood stream called “von-Willebrand-Factor” (VWF), which serves as an omnipresent and instantaneous repair mechanism for sealing vessel lesions. At sites of elevated shear flow VWF is transformed from a coiled into an elongated conformation comprising a multitude of binding sites for parts of the extracellular matrix initiating wound healing.

 

a: complex interaction within a blood vessel[Bildunterschrift / Subline]: Figure 1: a) The sketch illustrates the complex interaction within a blood vessel near a site of vascular damage. Among a multitude of blood constituents erythrocytes (red), platelets (yellow) and VWF molecules (blue) face a discontinuity of the endothelial cell layer covering the vessel wall. VWF molecules mechanically unroll at this vessel lesion, bind to the exposed extracellular matrix und initiate wound healing.
b: artificial blood vessel on a biochip[Bildunterschrift / Subline]: b) The photograph shows an artificial blood vessel on a biochip [side length ~ 1cm]. The red fluid is constraint within a hydrophobic barrier into variable geometries (notice the mimicking of vessel bifurcations). A SAW pumping system generates various flow conditions indicated by the arrows.

By means of a newly designed Surface Acoustic Wave (SAW) driven pumping device we can mimic blood flow conditions on a two dimensional biochip. Using this setup we identified the shear forces of the blood stream itself to be responsible for the mechanical unrolling and therefore the activation of single VWF proteins [PNAS]. On the other hand, the forces needed to activate VWF are fine-tuned by various other parameters such as temperature, pH, salt concentration, etc. Varying these parameters the process of VWF unrolling and therefore network formation can be influenced significantly. From a medical perspective any change in the clotting properties of VWF has strong implications at sites of vessel occlusions or pathological or inflamed tissue. After controlling the network formation process itself, we focused on the interior dynamics and binding behaviour of VWF bundles and conglomerates. Via mechanical deformation of VWF bundles we were able to elucidate and describe the dynamic binding behaviour of VWF molecules inside elongated VWF bundles. This is one of the key questions in understanding the process of network formation and therefore wound healing itself [Bundle Relaxation].

Figure 2: Ultralarge VWF network comprising various elongated fibres and bundles [side length ~ 500µm][Bildunterschrift / Subline]: Figure 2: Ultralarge VWF network comprising various elongated fibres and bundles [side length ~ 500µm]

Additionally to the network formation process we focus on the interaction of VWF molecules with the endothelial cell wall. As model systems we use both complete cell layers and artificial phospholipid bilayers to mimic and analyse the binding behaviour in terms of electric charge, hydrophobicity of the membrane or the phase state of the membrane and the protein. By fluorescence spectroscopy (figure 3) and Atomic Force Microscopy (AFM) we could unravel preferred conditions for VWF binding to these model membranes.

Figure 3: Fluorescence image of a VWF network on a DSPC/DOTAP membrane. The lipid structure separates into two physes. Binding of the huge VWF network (red) shows no correlation with the underlying membrane domain structure (green). (Scale bar 20µm)[Bildunterschrift / Subline]: Figure 3: Fluorescence image of a VWF network on a DSPC/DOTAP membrane. The lipid structure separates into two physes. Binding of the huge VWF network (red) shows no correlation with the underlying membrane domain structure (green). (Scale bar 20µm)

Motivated by concrete medical questions this work will help to understand the physical concepts of blood clotting and wound healing on a very basic level. This basic physical level is essential for an understanding and in the end for curing the Von Willebrand Disease, the most common inherited bleeding disorder.


References:

S. W. Schneider, S. Nuschele, A. Wixforth, C. Gorzelanny, A. Alexander-Katz, R. R. Netz, M. F. Schneider, Shear-induced unfolding triggers adhesion of von Willebrand factor fibers, PNAS (2007) vol. 104 (19) 7899–7903

A. Alexander-Katz, M. F. Schneider, S.W. Schneider, A. Wixforth, R. R. Netz, Shear-Flow-Induced Unfolding of Polymeric Globules, Phys. Rev. Lett. (2006) 97, 138101

Daniel M. Steppich, Jennifer I. Angerer, Kumudesh Sritharan, Stefan W. Schneider, Stefan Thalhammer, Achim Wixforth, Alfredo Alexander-Katz, Matthias F. Schneider, Relaxation of ultralarge VWF bundles in a microfluidic–AFM hybrid reactor, Biochemical and Biophysical Research Communications 369 (2008) 507-512


Daniel Michael Steppich
* 1979

Stationen
  • 2000- 2005
  • Dipl.-Phys. at the University of Augsburg
  • Seit 2006
  • PhD thesis in the framework of the international doctorate program „Material Science of Complex Interfaces“ (CompInt) at the University of Augsburg
  • 2006
  • Visiting scientist at the University of Linz (Austria, 2 months)
  • 2007
  • Guest scientist at Yale University (New Haven, CT, USA, 3months)

Veröffentlichungen
  • D. M. Steppich, J. I. Angerer, Kumudesh Sritharan, S.W. Schneider, S. Thalhammer, A. Wixforth, A. Alexander-Katz, M. Schneider, Relaxation of ultralarge VWF bundles in a microfluidic–AFM hybrid reactor, Biochemical and Biophysical Research Communications
  • D.M. Steppich, S. Thalhammer, A. Wixforth, M.F. Schneider, Nanomechanics and Microfluidics as a Tool for Unravelling Blood Clotting Disease. In: B. Bhushan, Applied Scanning Probe Methods Vol. 11-13
  • Beate Griepernau, Siman Leis, Matthias F. Scheider, Martin Sikor, Daniel M. Steppich, Rainer A. Böckmann, 1-Alkanols and membranes: A story of attraction, Biochimica et Biophysica Acta 1768 (2007) 2899–2913
  • D.M. Steppich, J. Opfer, J.I. Angerer, A. Grießer, S.W. Schneider, S. Nuschele, A. Wixforth, A. Alexander-Katz, T. Franke, R.R. Netz, M.F. Schneider,Von Willebrand Factor’s activation and degradation are regulated by mechanical stress and pH
  • D. M. Steppich, J. I. Angerer, S. Boessinger, A. Schmid, T. Obser, A. Wixforth, M. F. Schneider, A Molecular Binding Model of von Willebrand Factor (VWF) to Cell Membranes