Speaker: Gijsje Koenderink
Department: Systems Biophysics, AMOLF
Subject: Biophysics of fibrin clots
Location: Erasmus MC
Author: Nemo Andrea
Gijsje Koenderink is head of the systems biophysics department at the AMOLF research institute, based in Amsterdam. She was going to discuss the topic of fibrin clots, as part of the hematology lectures at the Erasmus Medical Centre. As the more reductionist approach of AMOLF and the highly applied nature of a good fraction of research at the Erasmus MC inherently attract a different crowd, the lecture was presentedin a fashion that focused on the findings, rather than theoretical justifications.
She opened up her talk with a general introduction regarding the research carried out at AMOLF, introducing topics such as their minimal systems and the reductionist approach to understanding the interactions and behavior of these systems. Part of the focus of research at AMOLF is into the mechanical properties of cells and other biological structures. Cells display behavior that is known as strain stiffening, where the cells become stiffer as the strain on said cells increases. This holds true for many biopolymer networks too. At the same time, these networks can exhibit dynamic behavior, meaning that these structures can be both very rigid but also very flexible/dynamic, which is behavior not commonly seen in synthetic materials.
In line with the reductionist approach, research done at AMOLF makes use of purified fibrin with only a few of the commonly associated factors such as thrombin and Factor XIII to serve as a model system for fibrin behavior. Fibrinogen is a glycoprotein with long unstructured tails that, upon cleavage by thrombin, can self-assemble into polymers. After cleavage, fibrinogen is known as fibrin. These polymers form dense networks that form, with the aid of platelets, blood clots that are essential for healing of wounds. Problems in this mechanism of wound healing, which could be with the system being too active (spontaneous clot formation in the absence of wounds – thrombosis) or too inactive (lack of clot formation – e.g. von Willebrand disease) are important areas of active research. Recently, the long unstructured regions of fibrin have been visualized by AFM.
Height mode AFM images of a single fibrin molecule in top left and growing fibril in the other images. Taken from .
These fibrin networks are not passive structures, but are under stress from various forces, as would be expected from the biological role of clotting. Examples of forces are the shear flow induced by the flow of blood, forces from migrating cells and platelets. These networks also have to be degradable, and are thus also subject to enzymatic degradation, which differs per region and condition. Fibrin networks naturally form in networks where the fibers are randomly aligned, but will line up when the network is stretched. To quantify mechanical property of polymer networks, rheological measurements are indispensable. Using a rheometer, it was shown that, as one would expect, fibrin networks also exhibit strain stiffening. This matches the behavior of another common ECM protein: collagen.
Naturally, predicting strain stiffening is not a new challenge in the field of polymer physics, and, like with collagen, existing theory can explain a good portion of the measured strain stiffening of fibrin. When the strain on the networks get exceedingly large however, the theoretical model fails to accurately predict the stress. Thus, an extension of the model was required for the high strain regime. One of the hypotheses as to why the model fails to predict all regions of strain was that fibrin fibers are themselves elastic. In the theory used for the strain stiffening, the assumption is made that the polymers are inelastic. In order to test whether fibrin is itself elastic they turned to optical tweezers. They combined optical tweezers with a multi-channel microfluidic flow device in order to efficiently capture single fibrin molecules and measure the behavior of such a molecule under forces.
From these measurements that fibrin exhibits elastic behavior, in that it will return to its original length and elastic modulus when it is released from a stretched position (i.e. it is not deformed, no hysteresis). Naturally, then the question arises: what part of the molecule mediates this reversible stretching. Atomic force measurements then determined that the alpha coiled coil regions of the protein are highly stretchable. To test whether they could see this conformational change in protein structure leading to lengthening happen, they turned to X-ray diffraction. Using a synchrotron x-ray beam, they were able to view changes to the protein as it unfolds. Traditionally, you need a crystal of your protein in order to determine the structure using x-ray diffraction technique, but fibrins natural periodic spacing in polymer network was sufficient to determine spacing of the protein structural elements. In low strain regimes, the X ray diffraction pattern clearly showed the known spacing between the subunits of fibrin, but when large strain was applied to the network (in the region where polymer theory failed to predict the behavior, and stretching of the proteins was expected) these specific patterns disappeared, confirming that the protein indeed undergoes conformational changes under high strain.
Remarkably, they found that while extension of a fibrin network is quite reversible, compression is not. After having been compressed and decompressed, the networks become significantly stiffer than in their pre-compression state. They hypothesized that this was because upon compression, the fibrin molecules become more closely spaced and can undergo more interactions and form new connections, which would strengthen the network. This would naturally require the protein to be able to form more interactions than the known binding patterns. In order to test this they used an optical trap setup where they held 2 fibrin molecules in 2 traps and lined them up in a cross pattern. It was then observed that these molecules ‘stuck’ to each other and required significant force to be separated again. Thus, it seems feasible that this is the mechanism that explains the stiffening after compression.
It was apparent that this lecture was meant for the researchers at the Erasmus Medical Centre, rather than at more biophysically oriented researchers, as no formulas or theories were discussed on the slides. It was interesting to see that techniques like optical tweezers are relatively unknown among the researchers at the EMC, seeing as they are prominently featured in the nanobiology programme. Curiously, when I signed up for this lecture, I did not know the speaker, but as part of my bachelor end project I am speaking with people at AMOLF, so it was interesting to see one of the people I met there back at the EMC. A small world.
 Visualization of fibrinogen αC regions and their arrangement during fibrin network formation by high-resolution AFM. J Thromb Haemost 2015; 13: 570–9.
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