Open Access

Christoph Westerhausen

• "How can the events in space and time which take place within the spatial boundary of a living organism be accounted for by physics and chemistry?" was the question brought up by Erwin Schrödinger 1944 in his book "What is Life?". A question that claims an interdisciplinary ansatz and cannot be solved without research between biology, chemistry, medical science and physics on an equal level. Motivated by this question, the present thesis treats biophysical aspects of primary hemostasis, mainly from a thermodynamic point of view. Especially the fundamental role of transitions of the protein von-Willebrand-Factor (VWF) and lipid membranes for their function in the context of blood clotting is studied. The protein VWF that plays a crucial role in hemostasis exhibits a conformation change under shear flow from a coiled to a stretched string like shape. During this process binding sites for blood platelets are exposed, leading to the formation of a plug of VWF and platelets, that closes"How can the events in space and time which take place within the spatial boundary of a living organism be accounted for by physics and chemistry?" was the question brought up by Erwin Schrödinger 1944 in his book "What is Life?". A question that claims an interdisciplinary ansatz and cannot be solved without research between biology, chemistry, medical science and physics on an equal level. Motivated by this question, the present thesis treats biophysical aspects of primary hemostasis, mainly from a thermodynamic point of view. Especially the fundamental role of transitions of the protein von-Willebrand-Factor (VWF) and lipid membranes for their function in the context of blood clotting is studied. The protein VWF that plays a crucial role in hemostasis exhibits a conformation change under shear flow from a coiled to a stretched string like shape. During this process binding sites for blood platelets are exposed, leading to the formation of a plug of VWF and platelets, that closes lesions of the vascular system. In the clinical picture of Von-Willebrand-Disease type 2A and 2B the patients tend to increased bleeding. The knowledge about the importance of the conformation for the proteins function and the shear force dependent conformation change lead to the question about the correlation of dysfunction and conformation change. Can these bleeding dysfunctions of mutated VWF be explained with altered threshold forces for the conformation change from coiled to elongated state? Surprisingly, the tendency for platelet aggregation of the two mutations is exact opposite: while 2A shows a decrease of function, VWF 2B exhibits a gain of function. In the frame of this thesis we address the question, whether a mechanical explanation, that correlates a higher/lower force threshold for the conformation change of the according mutation of VWF, applies or not. To answer this question the mechanical properties of the proteins were determined with atomic force spectroscopy. By fitting the necessary stretching force of the polymers with the worm-like-chain model, quantitative statistics on the elasticity associated persistence length are compared for wild type and mutated VWF. The interpretation of the experiments is supported by systematic simulation of force distance curves of polymers. Indeed differences can be seen: while VWF 2A needs slightly lower stretching forces than WT, VWF 2B polymers need higher stretching forces. Hence, this result fits not to the simple mechanical picture that the aggregation tendency is a consequence of differences in the necessary unfolding forces according to the ability of platelet aggregation. Instead we propose the following hypothesis may be the case: clinically the reason for the increased bleeding is the lack of high weight multimers in both mutations, the reason for this lack, however, is different. VWF 2A is stretched at lower forces and thus, the probability for cleavage by the VWF cleaving protease ADAMTS13 is increased. As a consequence of the increased cleaving probability the higher multimers are absent in these patients. On the other side VWF 2B needs higher stretching forces than WT. Although the high weight multimers do exist in patients with the mutation 2B, due to an increased binding affinity to platelets the high weight multimers are not available for primary hemostasis at sites of injury, leading to the same clinical effect. What is the role of the VWF conformation for its interaction with phospholipid membranes? After activation (stretching) VWF is prepared to bind to the membrane of the endothelia cells. As this adhesion is a crucial step in hemostasis, the interaction of wildtype VWF and mutations of type 2A and 2B with lipid interfaces were studied in Langmuir monolayer experiments. The, at first glance, strongly altered compressibility of the system, independent of the type of mutation, was relativized by comparison with calculations of a two component system (VWF and lipids). Detailed studies show apart from an ideal superposition some less drastic effects of globular VWF with lipid monolayers. Comparing these results with the interaction of fully elongated VWF with lipid membranes indicates that the shear force induced conformation change of VWF also switches the interaction with phospholipid membranes without special binding sites on the membrane side. In the frame of these studies thermodynamic considerations show, how information about the binding affinity between VWF and lipid monolayers in the two liquid phases can be extracted. How is the phase state affected by simple protein mimetics? After the interaction of membranes and VWF has been studied dependent on the state of the protein, the question appears about the interaction dependent on the state of the membrane. The manipulation of the state of biological membranes by the adhesion of proteins like VWF, is studied with well characterized nanoparticles as simple protein mimetics. Besides this reason the impact of NP on cell membranes of course is from high sociocultural relevance due to the increasing addition of NP for example in food and cosmetica. To overcome such barriers in nature, there are different uptake pathways that are partly associated with the presence of particular proteins. To illuminate such processes, from a physicists point of view, the impact of NP on the thermodynamical properties of phospholipid bilayers with a focus on the main phase transition was studied. The consequences are rather physical explanations of the observes effects, e.g. induced curvature, than biochemical ones. In calorimetric measurements size dependent effects of silica NP on various solid-supported phosphatidylcholine membranes were found. A detailed model combing thermodynamical and mechanical aspects explains the effects. Besides the mainly curvature induced effect, a chemical effect, namely a NP concentration dependent melting point depression was observed and explained. Furthermore in fluorescence microscopic studies of the temperature induced phase transition of giant unilamellar vesicles morphological changes occur. These experiments open ways to think about endocytosis supported by proteins rather from a physicists than from a biochemists point of view. What is the role of the phase state of the membrane for the activity of the VWF cleaving protease ADAMTS13? After activation VWF is not only more likely to bind but also more likely to interact with the cleaving protease ADAMTS13. Knowing that the conformation of VWF switches interaction strength with lipid membranes, and that protein mimetics can change the state of the lipid membrane, the question comes up, whether the state of the membrane also affects this mechanism in the blood clotting process. The optimal function of VWF in vivo depends on the cleavage of the unrolled polymers in shorter fractions by the enzyme ADAMTS13. In physiology the unrolled VWF is mainly adhered to the endothelial cells and thus, the reaction takes place in the vicinity of biological membranes. Hence, a possible mechanism affected by the membrane state is the enzyme activity. To testify the impact of the membrane state, ADAMTS13 was bound to phosphatidylcholine vesicles, the catalytic rate was measured temperature dependent and compared with the activity of unbound enzyme in lipid-free samples. In contrast to the activity of free ADAMTS13, the activity of membrane bound enzyme shows more than a simple linear temperature dependence. Astonishingly the phase transition temperature divides two temperature ranges where the temperature dependence of the rate of this, naturally not membrane associated enzyme, changes by one order of magnitude. Besides the common method for the determination of the enzyme activity from the maximal rate of kinetic curves an alternative method is shown. Deviations between theory and measurement, concerning the rate especially at the phase transition temperature are discussed in the controversy of membrane fluidity or phase state as crucial parameter for catalysis. In summary this work shows how the consequences of statistical physics, thermodynamics, in an interdisciplinary context can contribute to the answer of Schrödingers' question.…