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  • Atomic force fluorescence microscopy reveals elasticity of blood clot fibers

BioPhotonics
Oct 2006
Raquel Harper

Blood clots are important for stopping the flow of blood and sealing wounds after an injury. However, if they block arteries, they can cause conditions such as heart attacks and strokes. Although scientists know that blood clots consist of platelets and a meshwork of fibrin fibers, the mechanical properties of these fibers have not been understood. Researchers have struggled to find a technique that could simultaneously view the fibers at high resolution while mechanically manipulating them.

Martin Guthold, an assistant professor of physics at Wake Forest University in Winston-Salem, N.C., and his colleagues combined two microscopy techniques and discovered that they could acquire quality images of fibrin fibers being mechanically stretched. The images revealed some unusual and surprising characteristics of the blood clot fibers.

The researchers used an inverted optical fluorescence microscope from Carl Zeiss MicroImaging GmbH of Germany to view the sample from below while manipulating it from above. Guthold explained that the fluorescence microscope allowed them to acquire images quickly. To manipulate the fibrin fibers, which are about 100 nm in diameter, they also simultaneously used an atomic force microscope from Veeco Instruments Inc. of Woodbury, N.Y.

They used the sharp needle attached to the cantilever of the atomic force microscope to pull on a fiber, stretching it as far sideways as possible. The fibers were suspended across 12-μm-wide channels and anchored on the ridges at each end — just like a bridge over a gorge.

They discovered that the fibrin fibers could be stretched to more than four times their original length before rupturing. A few could even be stretched more than six times their length. And the fibers could stretch to three times their length and still return to their original length without damage. This was extraordinary for a few reasons. First, no other naturally occurring fiber had been found to stretch this far. Spider web silk can be stretched to only three times its length before rupturing. The elasticity of actin and microtubules (the fibers in our cytoskeleton) is about 1.2 times their length, and of elastin (the fiber in our arteries and skin), about 2.5 times its length.


Researchers combined fluorescence and atomic force microscopy to view blood clot fibers’ elasticity and extensibility. The series of images shows an individual blood clot fiber being stretched to 180 percent, which is 2.8 times its length.


The scientists also found that the cross-linking between the fibrin fiber monomers — the covalent bonds that link the fiber monomers together when a protein called factor XIII is added to the reaction — displayed completely opposite extensibility and elasticity properties from other naturally occurring fibers.

Most fibers, such as rubber, collagen and keratin, are less stretchable when they consist of cross-linked fibers than they are when consisting of uncross-linked fibers. For example, consider a rubber car tire — it’s rather stiff and not very stretchable. This is because it has lots of cross-links between its fiber monomers. But compare the tire to a rubber band, which is very stretchable. The rubber band doesn’t have as many cross-links holding the individual fibers together.

The unusual finding was that the cross-linked fibrin fibers stretched farther than the uncross-linked fibers — opposite to other fibers, such as rubber. And because these fibers are naturally cross-linked in humans, when a blood clot forms, these individual fibers can stretch a long way to stem the flow of blood. Knowing this part of the blood clot process helps the researchers understand the mechanical behavior of single fibrin fibers during a heart attack or stroke.


The individual fiber imaged on the facing page is part of this blood clot. The scientists also were surprised that these individual fibers could stretch farther than networks of fibers (clots). Although individual fibrin fibers have more than 330 percent extensibility, as a network they have only between 100 and 200 percent. The researchers believe this suggests that clot rupture may not come from individual fibers, as previously assumed.


Guthold and his colleagues plan to measure how much force is required to rupture fibrin fibers. They hope to look at various mutants of fibrin fibers that come from patients with clotting disorders to see if the mutants display different characteristics. They plan to use this knowledge to construct a model of the blood clot.

Science, Aug. 4, 2006, p. 634.


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