Abstract
Although we know a great deal about the structure, properties and many functions of fibrin(ogen), we still know very little about the microscopic and molecular origins of the clot’s mechanical properties, even though they are necessary for its functions, since hemostasis is essentially a mechanical process. In addition, it has been shown that individuals who have myocardial infarction at an early age tend to form very stiff clots. We have carried out studies at different levels of structure and integrated the results through a model that demonstrates that fibrin clot mechanical properties are manifestations of the observed mechanical characteristics of fibrin(ogen) molecules. By stretching whole fibrin clots with an extensional rheometer, we observed fibrin’s remarkable extensibility with a mechanical response that was initially linear with an increase in stiffness at larger elongation, above two-fold. These results are consistent with the large extensibility that has been observed in single fibrin fibers and may also play a role in the mechanics of blood clots at high strain, as in arterial blood flow. Furthermore, we found that protein structural transitions are required even at lower elongations. Some of the corresponding structural changes in the clots with stretching up to about four-fold were observed by electron microscopy. Scanning electron microscopy of the clots revealed extensive reorientation of the fibers making up the clots in the direction of applied stress. The orientational order was quantified from the scanning electron microscope images using a custom, automated image analysis algorithm that calculates a network order parameter, revealing a high degree of alignment for stretched, initially unoriented fibrin gels. Crosssections of stretched clots were examined by transmission electron microscopy. The most striking change observed was a huge (up to 10-fold) decrease in volume with stretching, with aggregation or bundling of fibers. Basic features of the mechanics of single fibrin fibers are known. These measurements have recently been extended to the level of single molecules using atomic force microscopy. When factor XIIIa-ligated fibrinogen oligomers were stretched by atomic force microscopy, the coiled-coils were found to unfold first under force. Until now, these observations at the molecular and fiber levels have not been correlated with the behavior of whole fibrin clots. These levels of structure were bridged through small angle X-ray fiber diffraction patterns obtained from fibrin clots, since the primary peaks in the X-ray diffraction pattern correspond to the characteristic 22.5 nm repeat distance in fibrin fibers arising from the molecular packing. In contrast to some earlier reported results, there was no change in periodicity with stretching. Instead, these peaks broadened as the sample was stretched, consistent with structural disruptions like protein unfolding while the position of the 22.5 nm peak corresponding to the fibrin repeat remained constant. Since all of these measurements are quantitative, we developed a constitutive model, including all of the features observed, that suggests that the whole clot and fiber mechanical properties are a consequence of coiled-coil unfolding. All together, this study has allowed us to develop a truly multiscale understanding of fibrin mechanics that reveals how clots or thrombi, even though they are made up of relatively stiff fibers, can still have large extensibility that allows them to withstand large strains and open and permeable structures such that they are readily lysed. Understanding how the network, fiber, and molecular properties give rise to fibrin mechanics could contribute to designs of tougher or more extensible clots or lead to new strategies for breaking up clots or making them less occlusive.
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