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Blood clot mechanics studied with AFM

Apr 2007
A specific region of the blood clot structural protein actively participates in clot stretching

David L. Shenkenberg

Blood clots are necessary to stop bleeding, but a clot in the wrong place can cause a heart attack or stroke. On one hand, very stiff clots are more likely to cause problems and potential death, but, on the other hand, clots that are too soft may not stop bleeding effectively. To understand why some clots become abnormally soft or stiff, it could help to study the mechanical properties of fibrinogen molecules, the precursors of the principal structural component of blood clots.

A fibrinogen molecule contains a globular region at each end and one in its center. These three spherical portions of the molecule are connected by structures that resemble springs, called coiled-coils. Any of the globular regions or the coiled-coils could account for the elasticity and rigidity of fibrinogen. Researchers at the University of Pennsylvania in Philadelphia recently used atomic force microscopy (AFM) to discover which of these structures governs the mechanical properties of fibrinogen.

Researchers used atomic force microscopy to unfold fibrinogen molecules, which contain springlike coiled-coils connected to globular regions. Images reprinted with permission of Biophysical Journal.

To examine the mechanical properties of fibrinogen, they stretched the cross-linked fibrinogen molecules between an AFM probe and a mica surface. They chose this method because it can pull on large proteins with high enough forces and because it does not require complex chemistry to attach molecules to the probe or the surface — the molecules just stick there. The researchers employed silicon nitride cantilevers and a Veeco AFM and used software written in-house to calculate peak forces and the stretching dimensions. The AFM equipment was not limiting in these experiments, but the nature of the molecules was.

In early experiments, the researchers pulled on fibrinogen monomers to unfold them, but found that they needed to use a chain of fibrinogen molecules to obtain reproducible data and to reliably detect unfolding. They used an enzymatic reaction to cross-link fibrinogen monomers, enabling creation of a linear chain of the molecules. This is the same chemical reaction that naturally occurs in humans to strengthen blood clots. They stopped the reaction with a blood thinner that inhibits the cross-linking reaction once they had the desired length of molecular aggregates.

The forcible extension of the fibrinogen molecules resulted in a graph with many small peaks, a pattern that resembled sawteeth. To create that pattern, one structure in fibrinogen had to become 23 nm longer when unfolded. They verified their experimental data by performing Monte Carlo simulations, which produced nearly identical results and confirmed the measurements.

The forcible extension of fibrinogen with atomic force microscopy produced these nonlinear, sawtooth-shaped patterns. The top two extension curves are separated from the bottom curve for clarity.

To determine which structure unfolded, the investigators compared the 23-nm length change shown on the AFM graph with known amino acid composition and x-ray crystallography data depicting the lengths of all the regions of the molecule. The comparison demonstrated that only the coiled-coils could have exhibited that length change. The researchers presented their results in the March 2007 issue of the Biophysical Journal.

A molecular spring?

Study author John W. Weisel said that the results showed for the first time that the coiled-coils are integral to the flexibility of fibrinogen, not merely passive structures, and that the coiled-coils behave like nonlinear springs when extended. In other words, they stretch like springs, but the unfolding occurs step-by-step, instead of smoothly like a typical spring. However, he said that they still do not know whether the process is reversible or whether the spring can be compressed again. He explained that the spring analogy applies also to fibers of cross-linked fibrin, a protein derived from fibrinogen, because they are some of the stretchiest biological materials known.

To understand what causes the coiled-coils to unfold in a nonlinear fashion, the researchers compared the forced unfolding of fibrinogen with that of myosin II, a muscle protein that also contains coiled-coils. Myosin II consists of a relatively straight 150-nm double-helical coiled-coil, and it unfolds in a much more gradual manner, with its force extension curve reaching a plateau.

Several irregularities in the structure of fibrinogen could explain why the molecule unfolds in a step-by-step manner. Fibrinogen coiled-coils consist of 17-nm segments that are triple-helical with quadruple-helical ends that interrupt the structure. Each segment is bent in the middle, rather than straight. Although there is a sequence of amino acids that is typical of the coiled-coil, some of the sequence of fibrinogen has an irregular pattern. Any of these structures could contribute to the coiled-coils’ step-by-step extension, but Weisel said that their precise roles are not immediately obvious.

According to Weisel, the other region of fibrinogen that is most likely playing a role in its unfolding is a domain that flanks the coiled-coils and connects to the central region of the molecule. He said that his group is currently performing genetic deletion experiments to examine whether this domain aids or hinders coiled-coil unfolding.

The scientists believe that computer modeling efforts ultimately will be necessary to have a complete understanding of blood clot mechanics because of the nonlinearity of the unfolding of coiled-coils as well as previous work that demonstrated nonlinear dynamics of fibrin gels.

Weisel said that they are now trying to see whether the coiled-coils unfold when whole clots are deformed. They also are investigating how fibrin polymerizes to make clots, how clots dissolve and how platelets aggregate. On the clinical side, they are exploring the effects of binding of sugars to fibrinogen that occurs in diabetes, and the nitration and oxidation of certain amino acids that occur in smokers or in those with coronary artery disease.

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