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The major structural component of a blood clot is a mesh of fibrin fibers. Our goal was to determine whether fibrinogen glycation and fibrin fiber diameter have an effect on the mechanical properties of single fibrin fibers. We used a combined atomic force microscopy/fluorescence microscopy technique to determine the mechanical properties of individual fibrin fibers formed from blood plasma. Blood samples were taken from uncontrolled diabetic patients as well as age-, gender-, and body-mass-index-matched healthy individuals. The patients then underwent treatment to control blood glucose levels before end blood samples were taken. The fibrinogen glycation of the diabetic patients was reduced from 8.8 to 5.0 mol glucose/mol fibrinogen, and the healthy individuals had a mean fibrinogen glycation of 4.0 mol glucose/mol fibrinogen. We found that fibrinogen glycation had no significant systematic effect on single-fiber modulus, extensibility, or stress relaxation times. However, we did find that the fiber modulus, Y, strongly decreases with increasing fiber diameter, D, as Y∝D−1.6. Thin fibers can be 100 times stiffer than thick fibers. This is unusual because the modulus is a material constant and should not depend on the sample dimensions (diameter) for homogeneous materials. Our finding, therefore, implies that fibrin fibers do not have a homogeneous cross section of uniformly connected protofibrils, as is commonly thought. Instead, the density of protofibril connections, ρPb, strongly decreases with increasing diameter, as ρPb∝D−1.6. Thin fibers are denser and/or have more strongly connected protofibrils than thick fibers. This implies that it is easier to dissolve clots that consist of fewer thick fibers than those that consist of many thin fibers, which is consistent with experimental and clinical observations.

The differences between coarse and fine fibrin clots first reported by Ferry have been interpreted in terms of nonspecific ionic strength effects for nearly 50 years and have fostered the notion that fibrin polymerization is largely controlled by electrostatic forces. Here we report spectroscopic and electron microscopy studies carried out in the presence of different salts that demonstrate that this long-held interpretation needs to be modified. In fact, the differences are due entirely to the specific binding of Cl− to fibrin fibers and not to generic ionic strength or electrostatic effects. Binding of Cl− opposes the lateral aggregation of protofibrils and results in thinner fibers that are also more curved than those grown in the presence of inert anions such as F−. The effect of Cl− is pH dependent and increases at pH>8.0, whereas fibers grown in the presence of F− remain thick over the entire pH range from 6.5 to 9.0. From the pH dependence of the Cl− effect it is suggested that the anion exerts its role by increasing the pKa of a basic group ionizing around pH 9.2. The important role of Cl− in structuring the fibrin clot also clarifies the role played by the release of fibrinopeptide B, which leads to slightly thicker fibers in the presence of Cl− but actually reduces the size of the fibers in the presence of F−. This effect becomes more evident at high, close to physiological concentrations of fibrinogen. We conclude that Cl− is a basic physiological modulator of fibrin polymerization and acts to prevent the growth of thicker, stiffer, and straighter fibers by increasing the pKa of a basic group. This discovery opens new possibilities for the design of molecules that can specifically modify the clot structure by targeting the structural domains responsible for Cl− binding to fibrin.