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.

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...

• In contracted clots and thrombi, erythrocytes are compressed to close-packed polyhedral structures with platelets and fibrin on the surface.• Polyhedrocytes form an impermeable seal to stem bleeding and help prevent vascular obstruction but confer resistance to fibrinolysis.Contraction of blood clots is necessary for hemostasis and wound healing and to restore flow past obstructive thrombi, but little is known about the structure of contracted clots or the role of erythrocytes in contraction. We found that contracted blood clots develop a remarkable structure, with a meshwork of fibrin and platelet aggregates on the exterior of the clot and a close-packed, tessellated array of compressed polyhedral erythrocytes within. The same results were obtained after initiation of clotting with various activators and also with clots from reconstituted human blood and mouse blood. Such close-packed arrays of polyhedral erythrocytes, or polyhedrocytes, were also observed in human arterial thrombi taken from patients. The mechanical nature of this shape change was confirmed by polyhedrocyte formation from the forces of centrifugation of blood without clotting. Platelets (with their cytoskeletal motility proteins) and fibrin(ogen) (as the substrate bridging platelets for contraction) are required to generate the forces necessary to segregate platelets/ fibrin from erythrocytes and to compress erythrocytes into a tightly packed array. These results demonstrate how contracted clots form an impermeable barrier important for hemostasis and wound healing and help explain how fibrinolysis is greatly retarded as clots contract. (Blood. 2014;123(10):1596-1603 IntroductionBlood clotting is a necessary part of hemostasis in which platelets aggregate to form a temporary sealant and fibrinogen is converted to a network of fibrin polymers to stem bleeding, yet both of these processes are also linked to thrombosis. [1][2][3][4] The resulting viscoelastic gel then contracts through the action of cytoplasmic motility proteins inside platelets, such that fluid (serum) is expelled, a process called clot contraction or retraction. Clots made from platelet-rich plasma (PRP) generate a bulk contractile force that begins shortly after the clot is formed and increases over minutes to hours to a maximum of about 1500 to 4500 dyn/cm 2 . 5,6 The function of clot contraction is not fully known, but it appears to reinforce hemostasis by forming a seal, promote wound healing by approximating the edges, and restore blood flow by decreasing the area obstructed by intravascular clots. [6][7][8] Although erythrocytes are a major component of blood clots, little is known about their participation in clot contraction. Historically, the presence of erythrocytes in contracted blood clots has been recognized and sometimes exploited; for example, during the time of medical bloodletting, the size of the contracted clot from blood removed from the patient was used as a measure of erythrocyte mass to determine when the procedure should cease. 6 Moreover, erythrocyte...

Research on all stages of fibrin polymerization, using a variety of approaches including naturally occurring and recombinant variants of fibrinogen, x-ray crystallography, electron and light microscopy, and other biophysical approaches, has revealed aspects of the molecular mechanisms involved. The ordered sequence of fibrinopeptide release is essential for the knob-hole interactions that initiate oligomer formation and the subsequent formation of 2-stranded protofibrils. Calcium ions bound both strongly and weakly to fibrin(ogen) have been localized, and some aspects of their roles are beginning to be discovered. Much less is known about the mechanisms of the lateral aggregation of protofibrils and the subsequent branching to yield a 3-dimensional network, although the aC region and B:b knob-hole binding seem to enhance lateral aggregation. Much information now exists about variations in clot structure and properties because of genetic and acquired molecular variants, environmental factors, effects of various intravascular and extravascular cells, hydrodynamic flow, and some functional consequences. The mechanical and chemical stability of clots and thrombi are affected by both the structure of the fibrin network and cross-linking by plasma transglutaminase. There are important clinical consequences to all of these new findings that are relevant for the pathogenesis of diseases, prophylaxis, diagnosis, and treatment. (Blood. 2013;121(10):1712-1719 IntroductionFibrin polymer is an end product of the enzymatic cascade of blood clotting. In vivo formation of the polymeric fibrin network, along with platelet adhesion and aggregation, are the key events in salutary stopping of bleeding at the site of injury (hemostasis) as well as in pathological vascular occlusion (thrombosis). Fibrin polymerization comprises a number of consecutive reactions, each affecting the ultimate structure and properties of the fibrin scaffold. These properties determine the development and outcomes of various diseases, such as heart attack, ischemic stroke, cancers, trauma, surgical and obstetrical complications, hereditary and acquired coagulopathies, and thrombocytopathies. In addition to providing a better understanding of the pathogenesis of such disorders, the knowledge of the molecular mechanisms of fibrin formation provides a foundation for new diagnostic tools and therapeutic approaches. Fibrinopeptide releaseFibrinogen is 45 nm-long and made up of 6 paired polypeptide chains (Aa Bb g) 2 held together by 29 disulfide bonds. There are now crystal structures of large parts of fibrinogen, including the g-and b-nodules, the coiled coil, and the central nodule.1 Still missing in the crystal structures are the flexible NH 2 -terminal (N-terminal) ends of the Aa-chains and the carboxyl-terminal (C-terminal) portion of the Aa-chain, but modern simulation techniques make possible partial computational reconstructions of these parts of fibrin(ogen) (Figure 1).Fibrin polymerization is initiated by the thrombin cleavage of fibrinopeptides A (...

A variety of methods exist for the design or selection of antibodies and other proteins that recognize the water-soluble regions of proteins; however, companion methods for targeting transmembrane (TM) regions are not available. Here, we describe a method for the computational design of peptides that target TM helices in a sequence-specific manner. To illustrate the method, peptides were designed that specifically recognize the TM helices of two closely related integrins (alphaIIbbeta3 and alphavbeta3) in micelles, bacterial membranes, and mammalian cells. These data show that sequence-specific recognition of helices in TM proteins can be achieved through optimization of the geometric complementarity of the target-host complex.