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Fibrin and Blood Clotting

Collaborators: Martin Guthold (Wake Forest Physics), Susan Lord (Pathology), Jennifer Mullin, Dorothy Erie (Chemistry).

This collaboration uses the nanoManipulator core project. It will also make use of the Mixture of Model and Experiment core project to extract curvature and coverage information.

Overview: Fibrin fibers are the major structural component of blood clots. Determining the mechanical properties of these fibers will provide new insights into the wound healing process and will advance our understanding of heart attacks and strokes. We are measuring the rupture force and other mechanical properties of individual fibrin fibers (Fig. FB1). The nanoManipulator interface is used to locate the desired modification site, and its polyline virtual tip is used to effect the modification. Images of two manipulations of these fibers are shown in the figure below: (A) & (B) and (D) & (E). (A) and (D) show the fibers before the manipulation during which a lateral force was applied to the fiber until it ruptured. Images (B) and (E) depict the fiber after the manipulation and (C) and (F) show the respective lateral force traces that were recorded during the manipula-tions.

The lateral force traces show two steps and two peaks. The two steps are due to a reversal in the direction of tip travel (i.e., from left to right and then from right to left) and correspond to the friction between the substrate and the tip. They are not of interest in the current study. However, the two peaks in the lateral force traces, a common feature in the measurements of all ~30 fibers, yield some mechanical properties of fibrin.

Manipulation of fibrin: (A-C) Manipulation of fibrin fiber 1, ambient condition (diameter: 150nm). Fibrin before (A) and after (B) manipulation. (C) Lateral force trace for manipulation of fiber 1. (D-F) Manipulation of fibrin fiber 2, ambient conditions (diameter: 180nm). Fibrin before (D) and after (E) manipulation. The fiber moved back towards its original location, indicative of an elastic deformation. (F) Lateral force trace for manipulation of fiber 2. (G) Rupture force vs. diameter of fiber (logarithmic scale). The deduced exponent is about 1.4. (H) Fibrin fibers on grid surface imaged in buffer. Scale bar in all images is 1 mm.

By comparing the images with the lateral force traces one can see that the first peak occurs when the tip contacts the fiber and partially detaches it from the surface. As the tip continues its travel, the fiber becomes taut again and is being stretched out. At this point, the force again increases rapidly until the rupture point. From such curves the rupture force of human fibrin was determined.

Furthermore, the manipulation depicted in (D) and (E) suggests that the deformation in the fibrin fiber is (at least partially) elastic. The fiber was initially bent further than the bend that is seen in image (E). In fact, it had been bent all the way to the end of the scratch that is left by the tip in the surface (image (E), arrow). The fiber moved back towards its straight configuration due to a restoring force. Such behavior indicates that the bending of the fiber was elastic (possibly in addition to a permanent plastic deformation).

The dependence of the rupture force on the diameter of the fiber (using logarithmic axis) is shown in (G). The rupture force increases proportional to R^1.4. The image in (H) shows fibrin fibers that were deposited onto a silicon grid (1mm troughs and 2mm wide and 100nm tall ridges) and imaged in buffer (10mM CaCl2, 130mM NaCl, 20mM Hepes, pH 7.4). The goal is to have fibers spanned over the troughs, so that the surface does not interfere with measurements on the fibers. [Guthold2000b]

Update:
Our combination of a fluorescence optical microscope and an atomic force microscope allows the simultaneous application of forces while imaging the structure of a biological specimen in liquid. The nanoManipulator allows us to easily measure the mechanical properties such as stiffness and breaking strain in samples such as fibrin fibers. We have created surfaces with ridges that allow the fibrin fibers to lay across a trough in the surface, with 10 microns of the fiber suspended in the liquid. With the ends of the fiber attached at either end to the substrate, the AFM tip can be moved through the fiber to stretch and finally break the fiber. The simultaneous imaging of the fiber during stretching allows the measurement of the strain of the fiber. We label the fibrin fiber with quantum dots that act as fluorescent labels to make the fiber visible in the microscope. Imaging of the individual quantum dots labeling the fiber will allow the tracking of strain with the potential for detecting the stretching of individual fibrin proteins within the 100nm diameter fiber.

At left, a labeled fibrin fiber is imaged lying suspended in fluid, attached to the structured substrate “hills”. (Right) Fibrin fiber, labeled with quantum dot fluorescence markers, is stretched to over 350% without breaking. Tracking of the discrete markers will allow us to measure internal strains.

References: Guthold, M., W. Liu, B. Stephens, S. T. Lord, R. R. Hantgan, D. A. Erie, R. M. Taylor II and R. Superfine (2004). "Visualization and Mechanical Manipulations of Individual Fibrin Fibers." Biophys. J. 87(6): 4226-4236
W. Liu,, Jawerth, L., M. Falvo, R. Superfine, S. Lord, M. Guthold, “Extensibility of Fibrin Fibers.” (Submitted.)