We show that the nonlinear mechanical response of networks formed from un-cross-linked fibrin or collagen type I continually changes in response to repeated large-strain loading. We demonstrate that this dynamic evolution of the mechanical response arises from a shift of a characteristic nonlinear stress-strain relationship to higher strains. Therefore, the imposed loading does not weaken the underlying matrices but instead delays the occurrence of the strain stiffening. Using confocal microscopy, we present direct evidence that this behavior results from persistent lengthening of individual fibers caused by an interplay between fiber stretching and fiber buckling when the networks are repeatedly strained. Moreover, we show that covalent cross-linking of fibrin or collagen inhibits the shift of the nonlinear material response, suggesting that the molecular origin of individual fiber lengthening may be slip of monomers within the fibers. Thus, a fibrous architecture in combination with constituents that exhibit internal plasticity creates a material whose mechanical response adapts to external loading conditions. This design principle may be useful to engineer novel materials with this capability.ECM | nonlinear rheology | factor XIII | blood clot N etworks of stiff biopolymer fibers are a major component of the structural architecture of multicellular organisms; their unique material properties provide rigidity and protect structural integrity. These networks are particularly important in the extracellular matrix (ECM) where they provide mechanical support to living cells and form many of the load-carrying structures in the body. One important example is fibrin, which forms the underlying scaffold of blood clots and the provisional matrix (1). Another important example is collagen type I, the major structural constituent of all connective tissue, tendons, ligaments, and bone (2). Because the in vivo structure of these fiber networks is so complex, investigations of in vitro networks of both proteins have been used to explore their structure and unique mechanical properties, and to elucidate their underlying design principles. Interestingly, fibrin and collagen exhibit many similar features: Both proteins self-assemble into thick, hierarchically ordered, rather stiff fibers through electrostatic and hydrophobic interactions (3, 4); these fibers associate into sparse, 3D networks that possess unusual mechanical properties not seen in synthetic polymers. In both cases, these networks display highly nonlinear mechanics and stiffen significantly as the strain increases (5-8). In addition, they are both also viscoelastic: They partially store elastic energy and partially relax internal stress through dissipative processes (9-12). All of these properties are delicately influenced by the structure of the networks, by the molecular interactions between monomers, and by the addition of covalent cross-links (7,13,14). This creates a delicate interplay between the viscoelastic and nonlinear mechanical properties of these ne...

Thrombin exosite 1 binds the predominant ␥ A /␥ A -fibrin form with low affinity. A subpopulation of fibrin molecules, ␥ A /␥-fibrin, has an extended COOH terminus ␥-chain that binds exosite 2 of thrombin. Bivalent binding to ␥ A /␥-fibrin increases the affinity of thrombin 10-fold, as determined by surface plasmon resonance. Because of its higher affinity, thrombin dissociates 7-fold more slowly from ␥ A /␥-fibrin clots than from ␥ A /␥ A -fibrin clots. After 24 h of washing, however, both ␥ A /␥-and ␥ A /␥ A -fibrin clots generate fibrinopeptide A when incubated with fibrinogen, indicating the retention of active thrombin. Previous studies demonstrated that heparin heightens the affinity of thrombin for fibrin by simultaneously binding to fibrin and exosite 2 on thrombin to generate a ternary heparinthrombin-fibrin complex that protects thrombin from inhibition by antithrombin and heparin cofactor II. In contrast, dermatan sulfate does not promote ternary complex formation because it does not bind to fibrin. Heparin-catalyzed rates of thrombin inhibition by antithrombin were 5-fold slower in ␥ A /␥-fibrin clots than they were in ␥ A /␥ A -fibrin clots. This difference reflects bivalent binding of thrombin to ␥ A /␥-fibrin because (a) it is abolished by addition of a ␥-chaindirected antibody that blocks exosite 2-mediated binding of thrombin to the ␥-chain and (b) the dermatan sulfate-catalyzed rate of thrombin inhibition by heparin cofactor II also is lower with ␥ A /␥-fibrin than with ␥ A /␥ A -fibrin clots. Thus, bivalent binding of thrombin to ␥ A /␥-fibrin protects thrombin from inhibition, raising the possibility that ␥ A /␥-fibrin serves as a reservoir of active thrombin that renders thrombi thrombogenic.

Thrombin bound to soluble fibrin degradation products is primarily responsible for the increase in FPA levels that occurs when a clot undergoes t-PA-induced lysis. Like clot-bound thrombin, thrombin bound to fibrin derivatives is protected from inhibition by heparin but susceptible to inactivation by direct thrombin inhibitors. These findings help to explain the superiority of direct thrombin inhibitors over heparin as adjuncts to thrombolytic therapy.

Histidine-rich glycoprotein (HRG) circulates in plasma at a concentration of 2M and binds plasminogen, fibrinogen, and thrombospondin. Despite these interactions, the physiologic role of HRG is unknown. Previous studies have shown that mice and humans deficient in HRG have shortened plasma clotting times. To better understand this phenomenon, we examined the effect of HRG on clotting tests. HRG prolongs the activated partial thromboplastin time in a concentrationdependent fashion but has no effect on tissue factor-induced clotting, localizing its effect to the contact pathway. Plasma immunodepleted of HRG exhibits a shortened activated partial thromboplastin time that is restored to baseline with HRG replenishment. To explore how HRG affects the contact pathway, we examined its binding to factors XII, XIIa, XI, and XIa. HRG binds factor XIIa with high affinity, an interaction that is enhanced in the presence of Zn 2؉ , but does not bind factors XII, XI, or XIa. In addition, HRG inhibits autoactivation of factor XII and factor XIIa-mediated activation of factor XI. These results suggest that, by binding to factor XIIa, HRG modulates the intrinsic pathway of coagulation, particularly in the vicinity of a thrombus where platelet release of HRG and Zn 2؉ will promote this interaction.(Blood. 2011;117(15): 4134-4141) IntroductionDespite the capacity of the intrinsic pathway to enhance thrombin generation, patients deficient in factor XII (FXII) do not bleed. 1 Even patients with FXI deficiency rarely have hemorrhagic complications, except with surgery or major trauma. 2 Because of these observations, it is well accepted that the contact system plays little part in hemostasis; and, by extension, it was also thought to be unimportant for thrombosis. However, a number of recent studies challenge this thinking. First, mice with deficiencies of highmolecular-weight kininogen (HK), bradykinin B2 receptor, FXII, or FXI are protected against injury-induced thrombosis, and an antibody against FXI inhibits thrombus formation in a baboon arteriovenous shunt model. [3][4][5][6][7] These observations raise the possibility that the contact pathway contributes to thrombogenesis. 8 Second, potential physiologic activators of FXII have been identified, which could initiate the contact system at sites of vascular injury. In addition to glycosaminoglycans and collagen, novel activators include polyphosphates and RNA. 9,10 Polyphosphates, which are released from the dense granules of platelets activated at sites of injury, trigger coagulation in a FXII-dependent fashion. Likewise, RNA released from the damaged vessel wall also can activate FXII, and RNAase administration to animals attenuates thrombosis at sites of injury, observations that have sparked a renewed interest in the contact pathway. 10 Consequently, it is important to better understand this pathway and how it is regulated.Histidine-rich glycoprotein (HRG) is an abundant plasma protein whose role is largely unknown. 11 HRG circulates at a concentration of approximately 2M. I...