Molecular mechanisms of the polymerization of fibrin and the formation of its three-dimensional network

The results of biochemical, immunochemical, and X-ray studies of the structures of fibrinogen and fibrin molecules were analyzed. The mechanisms of the successive formation of the fibrin three-dimensional network were described: the polymerization of monomeric molecules with the formation of bifilar protofibrils, the lateral association of protofibrils, and the embranchment of the forming fibrils. Data on the electron and confocal microscopy of the polymeric fibrin were considered. The role of the known polymerization centers of fibrin which participated in the formation of protofibrils and their lateral association was discussed. Data on the existence of the previously unknown polymerization centers were given. In particular, the experimental results demonstrated that one of such centers which participated in the formation of protofibrils was located in the Bβ12–46 fragment, and did not require the cleavage of fibrinopeptide B for its functioning. The results of the computer modeling of the spatial structure of the fibrin(ogen) molecule and the intermolecular interactions in the course of the fibrin polymerization were presented. The location of the αC domains in the fibrin(ogen) molecule and their role in the polymerization process were discussed. Information on the structure of the calciumbinding sites of fibrin(ogen) and the functional role of Ca²⁺ in fibrin polymerization was published. The structure of factor XIII(a) and the mechanisms of fibrin stabilization by this factor were briefly described.     

Fibrinogen sialic acid residues are low affinity calcium-binding sites that influence fibrin assembly

Calcium ions occupy low (n congruent to 10; Kd congruent to 1 mM) and high (n = 3; Kd congruent to 1 microM) affinity sites on fibrinogen and facilitate fibrin monomer polymerization. We have previously localized two of the three high affinity Ca2+ sites to gamma 311-gamma 336. However, optimal enhancement of fibrin monomer polymerization occurs only at physiological millimolar Ca2+ concentrations which are two orders of magnitude higher than the concentration required for occupancy of the high affinity Ca2+-binding sites. In this study, we show that removal of fibrinogen sialic acid residues results in loss of low affinity Ca2+-binding sites. Clotting of asialofibrinogen appears to be Ca2+-independent and results in fiber bundles thicker in diameter than normal fibrin bundles as determined by turbidometry and scanning and transmission electron microscopy. By using a Ca2+-sensitive electrode, free sialic acid is shown to bind Ca2+ (Kd congruent to 1 mM). These observations suggest that the high affinity fibrinogen D-domain Ca2+-binding sites may play a role in the tertiary structure of the D-domain, whereas, sialic acid residues are low affinity sites whose occupancy by Ca2+ at physiological calcium concentration facilitates fibrin polymerization.     

Influence of glutathione and its derivatives on fibrin polymerization

A complex relationship exists between reduced, oxidized, and nitrosated glutathione (GSH, GSSG, and GSNO, respectively). Although previous studies have demonstrated S-nitrosoglutathione (GSNO) has potent antiplatelet efficacy, little work has examined the role of GSNO and related species on subsequent aspects of coagulation (e.g., fibrin polymerization). Herein, the effects of GSH, GSSG, and GSNO on the entire process of fibrin polymerization are described. Relative to normal fibrinogen, the addition of GSH, GSSG, or GSNO leads to prolonged lag times, slower rates of protofibril lateral aggregation and the formation of clots with lower final turbidities. Dose-dependent studies indicate the influence of GSH on fibrin formation is a function of both GSH and fibrinogen concentration. Studies with Aalpha251 recombinant fibrinogen (lacking alphaC regions) showed GSH had no influence on its polymerization, suggesting the glutathione species interact within the alphaC region of fibrinogen.     

Peptide-derivatized albumins that inhibit fibrin polymerization

Synthetic peptides patterned on sequences that appear during thrombin proteolysis of fibrinogen are known to influence fibrin formation in very different ways. A-Knob sequences (GPR-) inhibit polymerization, but B-knob sequences (GHR-) can actually enhance the process. We now report that when such peptides are attached to albumin carriers, both knob conjugates inhibit fibrin formation. In contrast, the 2-aminoethylthiol-albumin conjugate control enhances the polymerization to the same degree as albumin. The peptide AHRPam, which is known to bind exclusively to the βC holes of fibrinogen/fibrin, nullifies the inhibitory effects of the GHRPYGGGCam-albumin conjugate on fibrin polymerization, indicating that the inhibition was exclusively due to interactions with βC holes. AHRPam was much less effective in countering inhibition by the GPRPGGGGCam-albumin conjugate, suggesting that the observed effects with this conjugate involve mainly the γC holes of fibrin/fibrinogen. This study demonstrates that peptides modeled on fibrin polymerization knobs tethered to albumin retain their capacity to interact with fibrinogen/fibrin and may prove useful as inhibitors of clotting in vivo.     

Interaction of fibrinogen and its derivatives with fibrin

The binding between complementary polymerization sites of fibrin monomers plays an essential role in the formation of the fibrin clot. One set of polymerization sites involved in the interaction of fibrin monomers is believed to pre-exist in fibrinogen, while the complementary set of binding sites is exposed after the cleavage of fibrinopeptides from fibrinogen. The polymerization sites present in fibrinogen and its derivatives mediate their binding to fibrin. Although the binding of fibrinogen and its derivatives to fibrin have been qualitatively studied, there has been no systematic, quantitative investigation of their interaction with forming or preformed clots. In the present study, the binding of fibrinogen and fragments DD, D1, and E1 was measured using a sonicated suspension of plasminogen- and thrombin-free human cross-linked fibrin as a model of a preformed clot. Dissociation constants of 0.056, 0.19, and 2.44 microM, and the number of binding sites corresponding to 0.10, 0.21, and 0.13/fibrin monomer unit of fibrin polymer were found for fibrinogen, fragment DD, and fragment D1, respectively. Fragment E1 did not bind to sonicated noncross-linked or cross-linked fibrin suspensions. However, it was bound to forming fibrin clots as well as to fibrin-Celite, suggesting that the binding sites on fibrin involved in the interaction with fragment E1 may have been altered upon sonication. Affinity chromatography of various fibrinogen derivatives on a fibrin-Celite column showed that only part of the bound fragment DD was displaced by arginine, whereas fragments D1 and E1 were completely eluted under the same conditions. The results indicate that interaction of fibrinogen with the preformed fibrin clots is characterized by affinity in the nanomolar range and that binding between fibrin monomers, in the process of clot formation, could be characterized by even a higher affinity.     

Coarse-grained molecular dynamics simulations of fibrin polymerization: effects of thrombin concentration on fibrin clot structure

Studies suggest that patients with deep vein thrombosis and diabetes often have hypercoagulable blood plasma, leading to a higher risk of thromboembolism formation through the rupture of blood clots, which may lead to stroke and death. Despite many advances in the field of blood clot formation and thrombosis, the influence of mechanical properties of fibrin in the formation of thromboembolisms in platelet-poor plasma is poorly understood. In this paper, we combine the concepts of reactive molecular dynamics and coarse-grained molecular modeling to predict the complex network formation of fibrin clots and the branching of fibrin monomers. The 340-kDa fibrinogen molecule was converted into a coarse-grained molecule with nine beads, and using our customized reactive potentials, we simulated the formation and polymerization process of a fibrin clot. The results show that higher concentrations of thrombin result in higher branch-point formation in the fibrin clot structure. Our results also highlight many interesting properties, such as the formation of thicker or thinner fibers depending on the thrombin concentration. To the best of our knowledge, this is the first successful molecular polymerization study of fibrin clots to focus on thrombin concentration.     

Interaction of an analog of the E1 site of tetrapeptide Gly-Pro-Arg-Pro with the D1 site of two forms of monomeric fibrin

Two monomeric fibrin forms differing in a set of polymerization sites (fibrin desAA and fibrin-desAABB) are inhibited to a different extent by tetrapeptide Gly-Pro-Arg-Pro which simulates a moiety of polymerization site E1. The lesser sensitivity of fibrin-desAABB polymerization to the inhibiting tetrapeptide is due to the presence of active site E2 in it. A shape of the concentration dependence curve of the inhibitory effect of tetrapeptide Gly-Pro-Arg-Pro on the polymerization of both fibrin types is similar to the previously found curve for fibrinogen and its fragments--specific inhibitors of polymerization. Ca2+ intensifies inhibition of fibrin-desAABB polymerization by tetrapeptide Gly-Pro-Arg-Pro twice as much as that of fibrin-desAA evidently due to the peptide blockage of sites D2. An increase of the ionic strength from 0.15 to 0.3 enhances the inhibitory effect of the tetrapeptide on polymerization of two monomeric fibrin forms.     

The incipient stage in thrombin-induced fibrin polymerization detected by FCS at the single molecule level.

We used fluorescence correlation spectroscopy (FCS) to study the activation of fibrinogen by thrombin and the subsequent aggregation of fibrin monomers into fibrin polymers at a very low and at physiological fibrinogen concentrations. In the labeling procedure used the fibrinogen was randomly labeled and the label was bound to the fibrinopeptide A and/or to the part of fibrinogen which after activation takes part in fibrin formation. We measured a diffusion coefficient for fibrinogen of 2.48 x 10(-7) +/- 0.10 x 10(-7) cm2/s. After activation with thrombin both fibrinopeptide A and fibrin polymerization products could be demonstrated. From our findings we suggest a model for the formation of a three-dimensional network as two parallel processes, elongation and branching and that fibrin oligomers are not only intermediates in the polymerization process but also are substrates for branching.     

The interference of plasmic degradation products of human crosslinked fibrin with clot formation

The role of plasmic degradation products of human crosslinked fibrin on polymerization of fibrin monomer and clot formation was studied. Both reactions were inhibited by Fragment DD, which formed a complex with fibrin monomer in a molar ratio 1 : 1. The rate of polymerization was slightly increased by Fragment E but it was not affected by (DD)E complex and Fragment A. Approximately the same amount of fibrin was formed in the presence and absence of Fragments A, E and the complex. It was concluded that of the degradation products of crosslinked fibrin, only Fragment DD is a potent anticoagulant at physiologic pH. The (DD)E complex is inert and Fragments A and E have only marginal effects.     

The interference of plasmic degradation products of human crosslinked fibrin with clot formation.

The role of plasmic degradation products of human crosslinked fibrin on polymerization of fibrin monomer and clot formation was studied. Both reactions were inhibited by Fragment DD, which formed a complex with fibrin monomer in a molar ratio 1 : 1. The rate of polymerization was slightly increased by Fragment E but it was not affected by (DD)E complex and Fragment A. Approximately the same amount of fibrin was formed in the presence and absence of Fragments A, E and the complex. It was concluded that of the degradation products of crosslinked fibrin, only Fragment DD is a potent anticoagulant at physiologic pH. The (DD)E complex is inert and Fragments A and E have only marginal effects.     

Fibrin assembly after fibrinopeptide A release in model systems and human plasma studied with magnetic birefringence.

Magnetically induced birefringence was used to monitor fibrin polymerization after the release of the small negatively charged A fibrinopeptides from human fibrinogen by the action of the snake-venom-derived enzymes reptilase and ancrod. A range of conditions was investigated. Fibrin polymerization in solutions of purified fibrinogen shows a distinct break near the gelation point. On addition of Ca2+ or albumin the lag period is shortened, fibre thickness is increased and the break in assembly almost vanishes, probably because both of these additives promote lateral aggregation. There are minor differences in the kinetics, depending on the venom enzyme used. The kinetics of fibrin assembly in model systems containing either Ca2+ or albumin and in human plasma with a largely dormant coagulation cascade are very similar. Therefore in the latter condition there is no significant alteration in the assembly process due to interaction between fibrin or the venom enzymes and any of the plasma proteins. When the cascade is activated, the polymerization progress curves have a character that resembles a combination of the reactions observed when the venom enzymes and endogenously generated thrombin separately induce coagulation, except for a region near gelation where, paradoxically, polymerization appears to be slower on activation. The low-angle neutron-diffraction patterns from oriented gels made with thrombin or reptilase are identical. Therefore at low resolution the packing of the monomers within fibres is the same when fibrinopeptide A only or both fibrinopeptides A and B are removed.     

Effect of fibronectin on fibrinogen conversion into fibrin

The effects of fibronectin on fibrinogen clotting induced by thrombin or reptilase and on fibrin monomer polymerization in a pure system in the absence of factor XIIIa were studied. It was shown that within a broad range of concentrations and molar ratios of the mixed proteins, fibronectin does not alter significantly the fibrinogen clotting time either under thrombin or under reptilase action. The effect of fibronectin on the fibrin self-assembly consists in a slight acceleration of this process, whose degree is directly dependent on the fibronectin/fibrin monomer molar ratio as well as on the absolute fibrin monomer content at a constant molar ratio. The stimulating effect of fibronectin is amplified by Ca2+. The experimental results suggest that fibronectin can noncovalently bind the fibrin monomer and/or intermediate polymers in the non-enzymatic phase of fibrinogen conversion to fibrin.     

The role of complementary E2 and D2 centers in the reaction between fibrin and fibrinogen

The effect of fibrinogen on the two steps of polymerization of two fibrin forms differing in the set of polymerization sites (fibrin-desAA and fibrin-desAABB) was studied. It was shown that fibrinogen inhibited the protofibril growth and fibril formation at the stage of lateral aggregation more effectively with fibrin-desAABB than with fibrin desAA. When the fibrinogen D2-site was blocked by tetrapeptide Gly-His-Arg-Pro, the key structure of the E2-site, the inhibitory activity of fibrinogen diminished. A conclusion is drawn that the high susceptibility of fibrin-desAABB to fibrinogen is due to the interaction of the E2-active site with the D2-site of the fibrinogen molecule. The concentration dependence of the tetrapeptide Gly-His-Arg-Pro-induced inactivation of fibrinogen and the effects of temperature and Ca2+ on the tetrapeptide interaction with fibrinogen were investigated.     

Interaction between complementary polymerization sites in the structural D and E domains of human fibrin.

Plasmic degradation products of human fibrin, fragments DD, D, and E, bind to fibrin. It has been inferred from this observation that the binding occurs by attraction of complementary sites located in the NH2- and COOH-terminal domains of the fibrin molecule. The interaction between fragments D1 and E1 has been investigated in this work since it represents the first step in the process of fibrin clot formation. Fragment D1, that was initially as active as fragment DD, lost most of its anticoagulant activity after purification by cation-exchange chromatography. The lability of fragment D1 function explained the previous unsuccessful attempts to form a complex between fragments D1 and E1. The loss of fragment D1 anticoagulant activity was not associated with the cleavage of the gamma 63-85 chain segment, since fragments D1A and D1 identically inhibited the fibrin monomer polymerization rate. In order to demonstrate the formation of a complex between fragments D1 and E1, three lines of experiments were advanced. First, the anticoagulant activity of fragment D1 was neutralized by fragment E1 in a dose-dependent manner, demonstrating that the association between these fragments involved polymerization sites. Second, two products, D1.E1 and D1.E1.D1, were stabilized in a reaction with bifunctional cross-linking reagents, proving the formation of D.E complexes in aqueous solution. Third, immobilized fragment D1 bound fragments E1 and E2, but not fragment E3, showing that fragments E1 and E2 attached via a polymerization site to the complementary one in fragment D1, since this association was disrupted by fibrin polymerization inhibitory peptide GPRP. These results provided direct evidence for specific binding between the structural D and E domains of fibrin mediated through complementary polymerization sites. Thus, the initial formation of fibrin clot fibers appears to be driven by specific association of these sites.     

Characterization of the kinetic pathway for liberation of fibrinopeptides during assembly of fibrin

The time dependence of the release of fibrinopeptides from fibrinogen was studied as a function of the concentration of fibrinogen, thrombin, and Gly-Pro-Arg-Pro, an inhibitor of fibrin polymerization. The release of fibrinopeptides during fibrin assembly was shown to be a highly ordered process. Rate constants for individual steps in the formation of fibrin were evaluated at pH 7.4, 37 degrees C, gamma/2 = 0.15. The initial event, thrombin-catalyzed proteolysis at Arg-A alpha 16 to release fibrinopeptide A (kcat/Km = 1.09 X 10(7) M-1s-1) was followed by association of the resulting fibrin I monomers. Association of fibrin I was found to be a reversible process with rate constants of 1 X 10(6) M-1s-1 and 0.064 s-1 for association and dissociation, respectively. Assuming random polymerization of fibrin I monomer, the equilibrium constant for fibrin I association (1.56 X 10(7) M-1) indicates that greater than 80% of the fibrin I protofibrils should contain more than 10 monomeric units at 37 degrees C, pH 7.4, when the fibrin I concentration is 1.0 mg/ml. Association of fibrin I monomers was shown to result in a 6.5-fold increase in the susceptibility of Arg-B beta 14 to thrombin-mediated proteolysis. The 6.5-fold increase in the observed specificity constant from 6.5 X 10(5) M-1s-1 to 4.2 X 10(6) M-1s-1 upon association of fibrin I monomers and the rate constant for fibrin association indicates that most of the fibrinopeptide B is released after association of fibrin I monomers. The interaction between a pair of polymerization sites in fibrin I dimer was found to be weaker than the interaction of fibrin I with Gly-Pro-Arg-Pro and weaker than the interaction of fibrin I with fibrinogen.     

Functional role of Bbeta-chain N-terminal fragment in the fibrin polymerization process

Four mAbs of the IgG(1) class to the thrombin-treated N-terminal disulfide knot of fibrin, secreted by various hybridomas, have been selected. Epitopes for two mAbs, I-3C and III-10d, were situated in human fibrin fragment Bbeta15-26, and those for two other mAbs, I-5G and I-3B, were in fragment Bbeta26-36. Three of these mAbs, I-5G, I-3B and III-10D, as well as their Fab-fragments, decreased the maximum rate of fibrin desAA and desAABB polymerization up to 90-95% at a molar ratio of mAb (or Fab-fragment) to fibrin of 1 or 2. The fourth mAb, I-3C, did not influence the fibrin desAABB polymerization and inhibited by 50% the maximum rate of fibrin desAA polymerization. These results suggest that these mAb inhibitors block a longitudinal fibrin polymerization site. As the mAbs retard both fibrin desAABB and fibrin desAA polymerization, one can conclude that the polymerization site does not coincide with polymerization site 'B' (Bbeta15-17). To verify this suggestion, the polymerization inhibitory activity of synthetic peptides BbetaSARGHRPLDKKREEA(12-26), BbetaLDKKREEA(19-26), BbetaAPSLRPAPPPI(26-36), BbetaAPSLRPAPPPISGGGYRARPA(26-46) and BbetaGYRARPA(40-46), which imitate the various sequences in the N-terminal region of the fibrin Bbeta-chain, have been investigated. Peptides Bbeta12-26 and Bbeta26-46, but not Bbeta40-46, Bbeta19-26, and Bbeta26-36, proved to be specific inhibitors of fibrin polymerization. The IC(50) values for Bbeta12-26 and Bbeta26-46 were 2.03 x 10(-4) and 2.19 x 10(-4) m, respectively. Turbidity and electron microscopy data showed that peptides Bbeta12-26 and Bbeta26-46 inhibited the fibrin protofibril formation stage of fibrin polymerization. The conclusion was drawn that fibrin fragment Bbeta12-46 took part in fibrin protofibril formation simultaneously with site 'A' (Aalpha17-19) prior to removal of fibrinopeptide B. A model of the intermolecular connection between fragment Bbeta12-46 of one fibrin desAA molecule and the D-domain of another has been constructed.     

Studies on the mechanism of thrombin. Interaction with fibrin

Fibrin monomer Sepharose was used to investigate the interactions of thrombin with fibrin. Thrombin binding was found to be reversible and saturable and to depend on the thrombin: fibrin ratio. Scatchard analysis indicated a single class of binding sites with K alpha = 4.9 X 10(5) M-1. Ca2+ ions caused rapid desorption and elution of thrombin from fibrin monomer, and the Ca2+ concentration needed for maximal desorption depended on the fibrin:thrombin ratio. Mg2+, Mn2+, and Sr2+ also released thrombin from fibrin monomer but not as efficiently as Ca2+. These results indicate that divalent metal ions induce a physical change in fibrin monomer which results in desorption of thrombin. Thrombin binding to fibrin in a gel was compared to binding to fibrin monomer. These studies showed that as fibrin monomers polymerize to form the gel network, thrombin is released. Under static conditions the released thrombin remains associated with the gel because diffusion is limited by the gel. However, the thrombin can be readily removed when buffer is allowed to flow through the gel. These results lead to the possibility that thrombin binding to fibrin monomer and its subsequent release, either by Ca2+ or by polymerization, may have important consequences for regulating the effective thrombin concentration in vivo.     

Functional and diagnostic significance of A-, Bbeta 1-42 and Bbeta 15-42 peptide fragments of fibrin(ogen)]

A scheme of in vitro formation and hydrolysis of the fibrin clot is suggested. This scheme considers the data concerning a cofactor role of fibrin in activation of polymerization and fibrinolysis. Such parameters of a number of components as concentration of A-, B beta 1-42-and B beta 15-42 peptides and of other fragments of fibrinogen which allow characterizing a state of hyperfibrinolysis, hyperclotting or dynamic equilibrium of these systems are selected in the scheme.     

Calix[4]arene methylenebisphosphonic acids as inhibitors of fibrin polymerization

Calix[4]arenes bearing two or four methylenebisphosphonic acid groups at the macrocyclic upper rim have been studied with respect to their effects on fibrin polymerization. The most potent inhibitor proved to be calix[4]arene tetrakis-methylene-bis-phosphonic acid (C-192), in which case the maximum rate of fibrin polymerization in the fibrinogen + thrombin reaction decreased by 50% at concentrations of 0.52 × 10(-6) M (IC(50)). At this concentration, the molar ratio of the compound to fibrinogen was 1.7 : 1. For the case of desAABB fibrin polymerization, the IC(50) was 1.26 × 10(-6) M at a molar ratio of C-192 to fibrin monomer of 4 : 1. Dipropoxycalix[4]arene bis-methylene-bis-phosphonic acid (C-98) inhibited fibrin desAABB polymerization with an IC(50) = 1.31 × 10(-4) M. We hypothesized that C-192 blocks fibrin formation by combining with polymerization site 'A' (Aα17-19), which ordinarily initiates protofibril formation in a 'knob-hole' manner. This suggestion was confirmed by an HPLC assay, which showed a host-guest inclusion complex of C-192 with the synthetic peptide Gly-Pro-Arg-Pro, an analogue of site 'A'. Further confirmation that the inhibitor was acting at the initial step of the reaction was obtained by electron microscopy, with no evidence of protofibril formation being evident. Calixarene C-192 also doubled both the prothrombin time and the activated partial thromboplastin time in normal human blood plasma at concentrations of 7.13 × 10(-5) M and 1.10 × 10(-5) M, respectively. These experiments demonstrate that C-192 is a specific inhibitor of fibrin polymerization and blood coagulation and can be used for the design of a new class of antithrombotic agents.     

Spatiotemporal Characterization of a Fibrin Clot Using Quantitative Phase Imaging

Studying the dynamics of fibrin clot formation and its morphology is an important problem in biology and has significant impact for several scientific and clinical applications. We present a label-free technique based on quantitative phase imaging to address this problem. Using quantitative phase information, we characterized fibrin polymerization in real-time and present a mathematical model describing the transition from liquid to gel state. By exploiting the inherent optical sectioning capability of our instrument, we measured the three-dimensional structure of the fibrin clot. From this data, we evaluated the fractal nature of the fibrin network and extracted the fractal dimension. Our non-invasive and speckle-free approach analyzes the clotting process without the need for external contrast agents.