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The origins of clot rheological behavior associated with network morphology and factor XIIIa-induced cross-linking were studied in fibrin clots. Network morphology was manipulated by varying the concentrations of fibrinogen, thrombin, and calcium ion, and cross-linking was controlled by a synthetic, active-center inhibitor of FXIIIa. Quantitative measurements of network features (fiber lengths, fiber diameters, and fiber and branching densities) were made by analyzing computerized three-dimensional models constructed from stereo pairs of scanning electron micrographs. Large fiber diameters and lengths were established only when branching was minimal, and increases in fiber length were generally associated with increases in fiber diameter. Junctions at which three fibers joined were the dominant branchpoint type. Viscoelastic properties of the clots were measured with a rheometer and were correlated with structural features of the networks. At constant fibrinogen but varying thrombin and calcium concentrations, maximal rigidities were established in samples (both cross-linked and noncross-linked) which displayed a balance between large fiber sizes and great branching. Clot rigidity was also enhanced by increasing fiber and branchpoint densities at greater fibrinogen concentrations. Network morphology is only minimally altered by the FXIIIa-catalyzed cross-linking reaction, which seems to augment clot rigidity most likely by the stiffening of existing fibers.

Fibrinogen and fibrin are essential for hemostasis and are major factors in thrombosis, wound healing, and several other biological functions and pathological conditions. The X-ray crystallographic structure of major parts of fibrin(ogen), together with computational reconstructions of missing portions and numerous biochemical and biophysical studies, have provided a wealth of data to interpret molecular mechanisms of fibrin formation, its organization, and properties. On cleavage of fibrinopeptides by thrombin, fibrinogen is converted to fibrin monomers, which interact via knobs exposed by fibrinopeptide removal in the central region, with holes always exposed at the ends of the molecules. The resulting half-staggered, double-stranded oligomers lengthen into protofibrils, which aggregate laterally to make fibers, which then branch to yield a three-dimensional network. Much is now known about the structural origins of clot mechanical properties, including changes in fiber orientation, stretching and buckling, and forced unfolding of molecular domains. Studies of congenital fibrinogen variants and post-translational modifications have increased our understanding of the structure and functions of fibrin(ogen). The fibrinolytic system, with the zymogen plasminogen binding to fibrin together with tissue-type plasminogen activator to promote activation to the active proteolytic enzyme, plasmin, results in digestion of fibrin at specific lysine residues. In spite of a great increase in our knowledge of all these interconnected processes, much about the molecular mechanisms of the biological functions of fibrin(ogen) remains unknown, including some basic aspects of clotting, fibrinolysis, and molecular origins of fibrin mechanical properties. Even less is known concerning more complex (patho)physiological implications of fibrinogen and fibrin.

Abstract-Abnormal fibrin architecture is thought to be a determinant factor of hypofibrinolysis. However, because of the lack of structural knowledge of the process of fibrin digestion, relationships between fibrin architecture and hypofibrinolysis remain controversial. To elucidate further structural and dynamic changes occurring during fibrinolysis, cross-linked plasma fibrin was labeled with colloidal gold particles, and fibrinolysis was followed by confocal microscopy. Morphological changes were characterized at fibrin network and fiber levels. The observation of a progressive disaggregation of the fibrin fibers emphasizes that fibrinolysis proceeds by transverse cutting rather than by progressive cleavage uniformly around the fiber. Plasma fibrin clots with a tight fibrin conformation made of thin fibers were dissolved at a slower rate than those with a loose fibrin conformation made of thicker (coarse) fibers, although the overall fibrin content remained constant. Unexpectedly, thin fibers were cleaved at a faster rate than thick ones. A dynamic study of FITC-recombinant tissue plasminogen activator distribution within the fibrin matrix during the course of fibrinolysis showed that the binding front was broader in coarse fibrin clots and moved more rapidly than that of fine plasma fibrin clots. These dynamic and structural approaches to fibrin digestion at the network and the fiber levels reveal aspects of the physical process of clot lysis. Furthermore, these results provide a clear explanation for the hypofibrinolysis related to a defective fibrin architecture as described in venous thromboembolism and in premature coronary artery disease. Key Words: fibrin Ⅲ fibrinolysis Ⅲ confocal microscopy T he fibrin matrix has a much more complicated role than that of providing the scaffolding of the thrombus or being the target of fibrinolysis. Abnormal fibrin structure in vitro has been related to in vivo premature coronary artery disease in young patients and to severe venous thromboembolic disease in patients with dysfibrinogenemias. 1,2 In those situations, an abnormal fibrin matrix made up of abnormally thin fibers has been shown to promote hypofibrinolysis and embolization. Although much is known about the molecular basis of fibrinolysis, relationships between fibrin conformation and fibrinolysis need to be clarified.Fibrin actively regulates its self-dissolution through numerous interactions with fibrinolytic and antifibrinolytic components. Activation of plasminogen by tissue plasminogen activator (tPA) that is initiated on the conversion of fibrinogen into fibrin is a critical step that is affected by fibrin structure. The theory of a decrease of plasminogen binding to fibrin 3 has been strengthened from observations showing that clots with a fine fibrin (tight) conformation display a slower lysis rate than those with a coarse fibrin (loose) conformation. 2,4,5 So far, neither a molecular nor a structural basis has been detected for these differences. Moreover, a recent report demonstrates that under othe...

Blood clots and thrombi consist primarily of a mesh of branched fibers made of the protein fibrin. We propose a molecular basis for the marked extensibility and negative compressibility of fibrin gels based on the structural and mechanical properties of clots at the network, fiber, and molecular levels. The force required to stretch a clot initially rises linearly and is accompanied by a dramatic decrease in clot volume and a peak in compressibility. These macroscopic transitions are accompanied by fiber alignment and bundling after forced protein unfolding. Constitutive models are developed to integrate observations at spatial scales that span six orders of magnitude and indicate that gel extensibility and expulsion of water are both manifestations of protein unfolding, which is not apparent in other matrix proteins such as collagen.Fibrin clots are proteinaceous gels that polymerize in the blood as a consequence of biochemical cascades at sites of vascular injury. Together with platelets, this meshwork stops bleeding and supports active contraction during wound healing (1,2). Fibrin also provides a scaffold for thrombi, clots that block blood vessels and cause tissue damage, leading to myocardial infarction, ischemic stroke, and other cardiovascular diseases (3). To maintain hemostasis while minimizing the impact of thrombosis, fibrin must have suitable stiffness and plasticity (4), but also sufficient permeability so that the network can be effectively decomposed (lysed) by proteolytic enzymes (5,6). It is challenging to meet all of these conditions because open scaffolds would be expected to break at low strains, as is true for collagen gels (7). To address how fibrin clots are both permeable and highly extensible, we studied fibrin structures across multiple spatial scales, from whole clots to single fibers and single molecules (Fig. 1).Fibrin clots were made from purified human fibrinogen under conditions (8) that resulted in the formation of long, straight fibers, similar to those found in physiological clots. To simplify the interpretation, the clots were covalently ligated with the use of a transglutaminase (blood clotting factor XIIIa), as naturally occurs in the blood, which prevents protofibrils from sliding past one another, thus eliminating persistent creep (9).Measurements of the extensibility of 2-mm-diameter fibrin clots ( Fig. 2A) demonstrated that the clots could be stretched to more than three times their relaxed length before breaking, with an average stretch of 2.7 ± 0.15-fold (n =6)(10). This is comparable to the single-fiber extensibility that is observed when a fibrin fiber is laterally stretched with an atomic force microscope (11). Qualitatively, the resulting force-strain curve for fibrin is similar to those observed for rubbers and other materials made from flexible chains (12). However, for fibrin clots, which are made of longer, straighter fibers than the thermally fluctuating polymer chains in rubber, models of rubber-like elasticity predict a branching density that is wro...