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SHENC – shear flow regulation of hemostasis

The first step in closing wounds is the aggregation of the von Willebrand factor glycoprotein with blood platelets, which plugs the hole in the blood vessel and stops blood from leaking out. Using computer simulations and artificial blood vessel experiments, researchers from Mannheim have shown that this aggregation is a reversible process that depends on the shear forces resulting from the flow of the blood. This also prevents the clogging of healthy blood vessels.

High shear forces occur when blood flows through the fine branches of the arteries and capillaries. These forces can potentially disrupt the soft walls that are lined with endothelial cells, resulting in minor bleeding that needs to be stopped immediately to prevent it from becoming life threatening. Von Willebrand factor (VWF) helps platelets stick to damaged blood vessels and is therefore a key protein in a force-sensing reaction cascade that triggers primary hemostasis, i.e. platelet plug formation.

Aggregation of von Willebrand factor (VWF). © www.haemophilie-portal.de

VWF is a rather unusual protein and is named after the Finnish doctor Erik Adolf von Willebrand. It is synthesised by endothelial cells and stored in special organelles known as Weibel-Palade bodies. [VWF is also produced by megakaryocytes, which are bone marrow cells responsible for the production of blood thrombocytes (platelets), and released by the thrombocytes during secondary hemostasis, i.e. the formation of a stable fibrin clot.] VWF monomers, including the sugar side chains, have a molecular weight of around 360 kDa. VWF dimers, which are found in the Weibel-Palade bodies of the endothelial cells, can assemble into long three-dimensional chains with a molecular weight of around 20,000 kDa. 

Chemical signals, e. g. those resulting from injured blood vessel walls, lead to the release of VWF multimers from the endothelial cells. VWFs are the largest proteins that occur in the bloodstream and have a shear-flow sensitive structure. Blood that seeps from a wound leads to high shear forces and the VWF multimer responds to this shear by expanding to an elongated form (several hundred micrometres long), thereby exposing particular binding sites for collagen and platelet receptors. VWF can thus adhere to the vessel wall and bind to thrombocytes. In the flowing blood, the shear forces lead to the aggregation of VWF multimers, which adhere to thrombocytes and form large aggregates which serve as a blood clot and close the wound.

Polymer-colloid aggregates

Prof. Dr. Stefan W. Schneider © UMM

Dr. Volker Huck and Professor Dr. Stefan W. Schneider from the Department of Experimental Dermatology at the Mannheim Medical Faculty of Heidelberg University focused on the question as to how this process enables the relatively quick closure of wounds without interfering with blood flow and without clogging healthy blood vessels. They simulated and measured the flow-driven self-assembly of polymer (VWF molecules) -colloid (thrombocytes) composites in the presence of shear flow. They found that the composite morphology was tunable and completely reversible: from a specific flow pressure onwards, the VWFs unfold, cross-link and adhere to the colloids. Decreasing shear flow leads to the disintegration of the aggregates. 

Computer simulation of the reversible aggregation of VWF multimers (red) and colloids (blue) under the influence of shear forces. Explanation in the text. © Universitätsmedizin Mannheim
The researchers from Mannheim reported in Nature Communications on the experimental verification of their theoretical model. They used an artificial blood vessel – a flow chamber system – in which they monitored the flow behaviour of VWF multimers and thrombocytes using a combination of interference reflection contrast microscopy and fluorescence microscopy. They found that the “self-assembly” process could also be reversed in the in vitro blood vessel system; decreasing flow velocity leads to the disintegration of the aggregates. These observations help explain why healthy, uninjured blood vessels do not clog. An aggregate of cross-linked VWF multimers and platelets only leads to soft blood clots in contact with the wound where high shear forces exist. This leads to secondary hemostasis and permanent wound closure. Aggregates that flow past the wound disintegrate as the shear forces decrease with the distance from the wound.

SHENC – shear flow regulation of hemostasis

Reversible polymer-colloid aggregation in an artificial blood vessel system. Explanation in the text. © Universitätsmedizin Mannheim
Stefan Schneider’s team is part of the research group “SHENC - Shear flow regulation of HEmostasis - bridging the gap between Nanomechanics and Clinical presentation” which was established by the German Research Foundation (DFG) in 2011. Researchers from 12 laboratories in Germany and Austria bring their medical and physiological competences into the consortium with the goal of clarifying the characteristics of VWF and its pivotal role during primary hemostasis using different methodological approaches from the fields of experimental and theoretical biophysics, molecular biology and nanotechnology. The researchers’ main focus is to obtain insights into the shear flow regulation of hemostasis with the aim of bridging the gap between the structure of VWF, its response to nanomechanical stress and its clinical presentation in both physiological and pathophysiological conditions, as the acronym SHENC implies.
DFG research unit FOR 1543: "SHENC – Shear Flow Regulation of Hemostasis“. © DFG

The SHENC consortium also involves Dr. Frauke Gräter’s Molecular Biomechanics research group at the Heidelberg Institute for Theoretical Studies. Gräter had already previously worked with Schneider’s team at the Mannheim University Hospital. Using computer simulations, Gräter and her team succeeded in determining in the nanometre scale the mechanical forces that act on the VWF protein as a result of the shear flow of the blood. They were able to show that shear forces also play a role in the disintegration of the VWF aggregates: tensile forces cause the protein to partially unfold, thereby leading to the exposure of a binding site for ADAMTS 13, a metalloprotease enzyme that cleaves von Willebrand factor. The SHENC researchers from Heidelberg also simulate pathological VWF variants. The clarification of the basic biomechanical and molecular mechanisms of the control mechanisms in the VWF system is an important prerequisite for the identification and modulation of the crucial steps of primary hemostasis. Knowing how the highly-sensitive equilibrium of VWF activation, aggregation with thrombocytes and VWF degradation is maintained in primary hemostasis or disturbed in case of disease, might lead to new approaches for the diagnosis and therapy of pathological situations. 

Publication:
Hsieh Chen, Mohammad A. Fallah, Volker Huck, Jennifer I. Angerer, Armin J. Reininger, Stefan W. Schneider, Matthias F. Schneider, Alfredo Alexander-Katz : Blood-clotting-inspired reversible polymer-colloid composite assembly in flow. Nature Communications 2013. DOI: 10.1038/ncomms2326

Website address: https://www.gesundheitsindustrie-bw.de/en/article/news/shenc-shear-flow-regulation-of-hemostasis