Blood clots perform an essential mechanical task, yet the mechanical behavior of fibrin fibers, which form the structural framework of a clot, is largely unknown. By using combined atomic force-fluorescence microscopy, we determined the elastic limit and extensibility of individual fibers. Fibrin fibers can be strained 180% (2.8-fold extension) without sustaining permanent lengthening, and they can be strained up to 525% (average 330%) before rupturing. This is the largest extensibility observed for protein fibers. The data imply that fibrin monomers must be able to undergo sizeable, reversible structural changes and that deformations in clots can be accommodated by individual fiber stretching.

In the past few years a great deal of progress has been made in studying the mechanical and structural properties of biological protein fibers. Here, we compare and review the stiffness (Young's modulus, E) and breaking strain (also called rupture strain or extensibility, epsilon(max)) of numerous biological protein fibers in light of the recently reported mechanical properties of fibrin fibers. Emphasis is also placed on the structural features and molecular mechanisms that endow biological protein fibers with their respective mechanical properties. Generally, stiff biological protein fibers have a Young's modulus on the order of a few Gigapascal and are not very extensible (epsilon(max) < 20%). They also display a very regular arrangement of their monomeric units. Soft biological protein fibers have a Young's modulus on the order of a few Megapascal and are very extensible (epsilon(max) > 100%). These soft, extensible fibers employ a variety of molecular mechanisms, such as extending amorphous regions or unfolding protein domains, to accommodate large strains. We conclude our review by proposing a novel model of how fibrin fibers might achieve their extremely large extensibility, despite the regular arrangement of the monomeric fibrin units within a fiber. We propose that fibrin fibers accommodate large strains by two major mechanisms: (1) an alpha-helix to beta-strand conversion of the coiled coils; (2) a partial unfolding of the globular C-terminal domain of the gamma-chain.

We identified the two-stranded fibrin protofibril and studied its structure in electron micrographs of negatively stained specimens. Based on these images and on considerations of symmetry, we constructed a model of the protofibril in which the two strands oftrinodular fibrin molecules are related by a twofold screw axis between the strands and two-fold axes perpendicular to them. The two strands are held together by staggered lateral contacts between the central nodules of one strand and outer nodules of the other. The molecules within a strand are joined by longitudinal contacts between outer nodules. This interpretation of the structure of protofibrils is supported by images of trimer complexes whose preparation and structure are described here, in which the central nodule of a fibrin monomer is attached to the crosslinked outer nodules of two other molecules. We conclude that the association of protofibrils to form thicker fibers must involve a second type of lateral contact, probably between outer nodules ofadjacent, in-register strands. In total, we identify three intermolecular contacts involved in the polymerization of fibrin.The plasma protein fibrinogen, the major structural component of the blood clot, is an elongated molecule whose shape is a trinodular rod, 45 nm long (1, 2). Evidence has been presented recently for a finer subdivision into seven nodules (3), but the description of two 7-nm-diameter outer nodules connected by thin linking rods to a 5-nm-diameter central nodule is sufficient for the present work. Fibrinogen is composed of two identical half molecules, which are probably related by an exact or approximate two-fold axis through the central nodule (4, 5). The two-fold molecular symmetry is consistent with the overall shape of the molecule, with the identification of the nodules with specific parts of the sequence (6, 7), and with the measurements of periodicities in fibrin and crystalline aggregates (3,4,8). The central nodule is the site of activation by the protease thrombin, which cleaves off the small fibrinopeptides to form fibrin monomer. The fibrin monomer is essentially identical in structure to fibrinogen, but instead of being soluble, it spontaneously polymerizes to form fibrin fibers.The most informative structural feature seen in electron micrographs offibrin fibers is a pattern oftransverse bands, which repeat every 22.5 nm or exactly one-half the length of the molecule. The generally accepted interpretation of this banding pattern is that the rod-like molecules are parallel to the fiber axis; the molecules are arranged end-to-end to form strands, which are one molecule thick; and alternate strands are staggered by one-half the length of the molecule (8).Ferry identified a two-stranded polymer of fibrin, which he called a protofibril, and proposed a two-step mechanism for the polymerization of fibrin (9). In the first step, fibrin monomers polymerize to form protofibrils; in the second step, these protofibrils associate laterally to form the thicker fibrin fibers. ...

We report protocols and techniques to image and mechanically manipulate individual fibrin fibers, which are key structural components of blood clots. Using atomic force microscopy-based lateral force manipulations we determined the rupture force, FR, f fibrin fibers as a function of their diameter, D, in ambient conditions. As expected, the rupture force increases with increasing diameter; however, somewhat unexpectedly, it increases as FR approximately D1.30+/-0.06. Moreover, using a combined atomic force microscopy-fluorescence microscopy instrument, we determined the light intensity, I, of single fibers, that were formed with fluorescently labeled fibrinogen, as a function of their diameter, D. Similar to the force data, we found that the light intensity, and thus the number of molecules per cross section, increases as I approximately D1.25+/-0.11. Based on these findings we propose that fibrin fibers are fractals for which the number of molecules per cross section increases as about D1.3. This implies that the molecule density varies as rhoD approximately D -0.7, i.e., thinner fibers are denser than thicker fibers. Such a model would be consistent with the observation that fibrin fibers consist of 70-80% water and only 20-30% protein, which also suggests that fibrin fibers are very porous.

A stable population of fibrinogen dimers cross-linked by Factor XIIIa has been prepared and examined in the electron microscope. The trinodular fibrinogen molecules are cross-linked through their outer nodules in an end-to-end, non-overlapping fashion. These dimers form normal banded fibers after treatment with the clotting enzyme, thrombin.