alphaC domains of fibrinogen molecules as the structures accelerating fibrin assembly

Preparation of monomeric fibrin lacking intact alpha C-domains (monomeric X1-fragment), but fully clottable, is described. The assembly process of both monomeric fibrin and monomeric X1-fragment has been studied by electron microscopy and light scattering methods. It was shown that both proteins form similar fibrils with characteristic cross-banding. Upon dilution a sharp elevation of the differences between the assembly rates of monomeric X1-fragment and monomeric fibrin was revealed. The results obtained show that alpha C-domains take part in fibrin clot formation not as structural components but as the factor accelerating the ordered assembly of complex fibrin structure. The possible mechanism of alpha C-domains participation in fibrin clot formation are regarded.     

Department of Protein Structure and Function--Volodimir Oleksandrovich Bielitser School of the Ukrainian Academy of Sciences. Studies on structure of fibrin fiber structure and mechanism (1975-1987

In this short historical review the records about foundation and research activity of the Department of Structure and Function of Protein--school of V. A. Belitser, Member of the National Academy of Sciences of Ukraine are presented. V. A. Belitser was the founder and indispensable chief of the department since the date of its creation (1944) till 1987. The main research interests (1975-1987) of the department were focused at the investigation of structure, biological function of the fibrinogen-fibrin system, mechanisms of the network assembly and of the fibrin fibers structure. Studying the molecular mechanisms of the fibrin fiber assembly, it was shown that the specificity of the building structure was shown is determined by the specific reactive sites with strong affinity of the molecules. The activity of the sites was investigated on protein molecules as well as the fragments. The physical nature of the bonds created by the active sites, that appearing during in the process of fibrinogen activation by thrombin, was revealed. Examination of the fibrin assembly in cooperation with electronmicroscopists and studies of the complex formation between active fragments and fibrin monomer were summarized. Both the fibrin monomer polymerization and protofibril lateral association are presented as two stages in the assembly of the fibrin network. In the research of the domain fibrinogen structure the specific sites of the fibrin assembly in each of the domains were found. COOH-terminal regions of the A alpha-chains play independent part in the fibrinogen and fibrin. That is why it is relevant to consider them as alpha C-domains. In the free fibrinogen molecules (in solution) these domains are responsible for globular shape, they are linked to domains D intramolecularly. When fibrin assembly takes place, alpha C-domains play significant carriage role in fibrin molecules interaction, linking to domains D intermolecularly. The model of the fibrinogen molecule structure and the general scheme of the fibrin fibers network formation were proposed. Physico-chemical basics of a biological structure assembly were elucidated using the process of the fibrin self-assembly as an example. Much attention was devoted to the problems of practical medicine. The quantitative methods of fibrinogen, soluble fibrin and active fibrin/fibrinogen fragments estimation in blood plasma were developed.     

In vitro models of angiogenesis and vasculogenesis in fibrin gel

In vitro models of endothelial assembly into microvessels are useful for the study of angiogenesis and vasculogenesis. In addition, such models may be used to provide the microvasculature required to sustain engineered tissues. A large range of in vitro models of both angiogenesis and vasculogenesis have utilized fibrin gel as a scaffold. Although fibrin gel is conducive to endothelial assembly, its ultrastructure varies substantially based on the gel formulation and gelation conditions, making it challenging to compare between models. This work reviews existing models of endothelial assembly in fibrin gel and posits that differerences between models are partially caused by microstructural differences in fibrin gel.     

Effect of homo poly(L-amino acids) on fibrin assembly: role of charge and molecular weight

Positively charged molecules such as protamine, leukocyte cationic protein, and the carboxyl terminus of platelet factor 4 have been shown to increase fibrin fiber thickness. Synthetic homo poly(L-amino acids) were used to explore the role of charge and molecular weight of cationic molecules on fibrin assembly. The effects of poly(L-lysine) (PLL), poly(L-glutamic acid) (PLG), poly(L-aspartic acid) (PLA), poly(L-histidine) (PLH), and poly(L-arginine) (PLArg) on the assembly and structure of fibrin gels were studied by using light-scattering techniques. At a PLG (Mr 60,000) concentration of 80 micrograms/mL and a PLA (Mr 20,000) concentration of 64 microgram/mL, neither of these negatively charged polymers produced a detectable change in either fibrin assembly kinetics or final structure. Positively charged PLArg (16 micrograms/mL) caused a 30% increase in fibrin fiber mass/length ratio without calcium. In contrast, PLH (16 micrograms/mL), also positively charged, had no effect in the absence of CaCl2 but produced a 40% increase in fiber mass/length ratio with 5 mM CaCl2. At concentrations as low as 1 microgram/mL, positively charged PLL increased the initial fibrin assembly kinetics and led to larger fiber mass/length ratio. The impact on fibrin mass/length ratio was equivalent for three different molecular weight preparations of PLL (Mr 25,000, 90,000, and 240,000). The lack of a molecular weight effect on fiber thickness and the low polymer concentrations required to produce the perturbation argue against an excluded volume effect as the mechanism by which lateral fiber growth is augmented. Mechanisms by which poly(L-amino acids) may perturb fibrin assembly are discussed.     

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.     

Fibrin protofibril and fibrinogen binding to ADP-stimulated platelets: evidence for a common mechanism

The molecular basis of platelet-fibrin binding has been elucidated by studying interactions between platelets and protofibrils, soluble two-stranded polymers of fibrin which are intermediates on the fibrin assembly pathway. The fibrinogen degradation product, fragment D, has been used to block fibrin assembly, thus enabling the preparation of stable solutions of short protofibrils, composed of fewer than twenty fibrin monomer molecules per polymer. Fibrin protofibrils bound to ADP-activated platelets in a time- and concentration-dependent process which was effectively blocked by excess unlabelled fibrinogen, i.e., the binding was specific and appeared to involve a common receptor. ADP-stimulated cells bound approx. 3 micrograms of fibrin protofibrils/10(8) platelets, compared to 4 micrograms of fibrinogen/10(8) cells, following a 30-min incubation period at room temperature. Binding of both ligands was inhibited by high concentrations of fragment D, further indicating a similar mechanism. The kinetic data obtained were well described by an apparent first-order mechanism in which the rate constant for fibrin protofibril binding was found to be 5-fold slower than that measured for fibrinogen. Two monoclonal antibodies, each directed against the platelet glycoprotein IIb-IIIa complex, inhibited the binding of fibrin protofibrils and fibrinogen in a similar, concentration-dependent manner, providing strong evidence for a common receptor. Binding of GPRP-fibrin (soluble fibrin oligomers formed in the presence of 1 mM Gly-Pro-Arg-Pro) to ADP-stimulated platelets was also inhibited by a monoclonal antibody directed against the GPIIb-IIIa complex. Neither fibrin protofibrils nor fibrinogen bound to Glanzmann's thrombasthenic platelets, which lack normal quantities of functional glycoprotein IIb-IIIa complex, further supporting the hypothesis that fibrinogen and fibrin bind to a common platelet receptor present on the glycoprotein IIb-IIIa complex.     

Fibrin but not adsorbed fibrinogen supports fibronectin assembly by spread platelets. Effects of the interaction of alphaIIb beta3 with the C terminus of the fibrinogen gamma-chain

We investigated the assembly of soluble fibronectin by lysophosphatidic acid-activated platelets adherent to fibrinogen or fibrin. More fibronectin was assembled by activated platelets spread on fibrin matrices than by platelets spread on adsorbed fibrinogen. The difference between platelets adherent to fibrinogen and fibrin occurred under both static and flow conditions. Similar differences were seen in binding of the 70-kDa N-terminal fragment of fibronectin that recognizes fibronectin assembly sites on adherent cells. Antibody and peptide blocking studies demonstrated that alphaIIb beta3 integrin mediates platelet adhesion to fibrinogen, whereas both alphav beta3 and alphaIIb beta3 mediate platelet adhesion to fibrin. The hypothesis that engagement of the C-terminal QAGDV sequence of the fibrinogen gamma-chain by alphaIIb beta3 inhibits the ability of the platelet to assemble fibronectin was tested by several experiments. Activated platelets adherent to adsorbed mutant fibrinogen lacking the QAGDV sequence (gammadelta5FG) were assembly-competent, as were platelets adherent to adsorbed normal fibrinogen that had been pretreated with the 7E9 antibody to the C terminus of the gamma-chain. Moreover, adsorbed normal fibrinogen but not gammadelta5FG suppressed the ability of co-adsorbed fibronectin to direct assembly of soluble fibronectin by spread platelets. The suppressive effect was lost when a surface of co-adsorbed fibronectin and fibrinogen was pretreated with 7E9. These results support a model in which the engagement of alphaIIb beta3 by the C-terminal sequence of the fibrinogen gamma-chain initiates signals that suppress subsequent fibronectin assembly by spread platelets. This interaction is less dominant when platelets adhere to fibrin, resulting in enhanced fibronectin assembly.     

The effect of fibrin structure on fibrinolysis.

Fibrin structure contributes to the regulation of the fibrinolytic rate. As the fibrin fiber size is decreased, the fibrinolytic rate also decreases. Fibrin structure was altered by either changing the ratio of thrombin to fibrinogen, i.e. altering the assembly rate or by adding a fibrin assembly inhibitor, iopamidol. Changes in the fibrinolytic rate were followed by measuring the time dependence of the decrease in the fiber mass/length ratio during fibrinolysis. A measure of the overall fibrinolytic rate was determined from the decrease in the mass/length ratio versus time. An 8-fold reduction in the fibrinolytic rate was seen on decreasing the mass/length ratio from 2.7 x 10(12) daltons/cm to 0.5 x 10(12) daltons/cm. It is shown that thin fibrin fibers have a decreased rate of conversion of plasminogen to plasmin by tissue plasminogen activator and that thin fibrin fibers are lysed more slowly than thick fibrin fibers.     

Localization of the domains of fibrin involved in binding to platelets

The molecular basis of platelet-fibrin interactions has been investigated by using synthetic peptides as potential inhibitors of fibrin protofibril and fibrinogen binding to ADP-stimulated platelets, adhesion of fibrin fibers to the platelet surface, and platelet-mediated clot retraction. Synthetic peptides of sequence RGDS and HHLGGAKQAGDV, corresponding to regions of the fibrinogen alpha- and gamma-chains previously identified as platelet recognition sites, inhibited the binding of radiolabelled soluble fibrin oligomers to ADP-stimulated platelets with IC50 values of 10 and 40 microM, respectively. Synthetic GPRP and GHRP, corresponding to the N-terminal tripeptide sequence of the fibrin alpha-chains and the tetrapeptide sequence of the beta-chains, respectively, were minimally effective in blocking soluble fibrin polymer binding to ADP-stimulated platelets. Platelet functions which are unique to the three-dimensional fibrin network were examined by measurements of the extent of adhesion of fluorophore-labelled fibrin to platelets with a microfluorimetric technique and by light scattering measurements of the time course of clot retraction. Inhibition of fibrin-platelet adhesion by RGDS, HHLGGAKQAGDV and GHRP exhibited a similar, linear dependence reaching 1/2 maximum at about 200 microM, suggesting nonspecific effects. GPRP inhibited fibrin assembly but did not appear to have specific effects on fibrin-platelet adhesion. Only RGDS effected clot retraction, causing a 4-6-fold decrease in rate at 230 microM. These results indicate that fibrinogen and fibrin protofibrils, which are obligatory intermediates on the fibrin assembly pathway, share a set of common platelet recognition sites located at specific regions of the alpha- and gamma-chains of the multinodular fibrin(ogen) molecules. The RGDS site is also involved in mediating interactions between the three-dimensional fibrin network and stimulated platelets.     

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.     

Hematopoietic Stem Cell Cytokines and Fibroblast Growth factor-2 Stimulate Human Endothelial Cell-Pericyte Tube Co-Assembly in 3D Fibrin Matrices under Serum-Free Defined Conditions

We describe a novel 3D fibrin matrix model using recombinant hematopoietic stem cell cytokines under serum-free defined conditions which promotes the assembly of human endothelial cell (EC) tubes with co-associated pericytes. Individual ECs and pericytes are randomly mixed together and EC tubes form that is accompanied by pericyte recruitment to the EC tube abluminal surface over a 3-5 day period. These morphogenic processes are stimulated by a combination of the hematopoietic stem cell cytokines, stem cell factor, interleukin-3, stromal derived factor-1α, and Flt-3 ligand which are added in conjunction with fibroblast growth factor (FGF)-2 into the fibrin matrix. In contrast, this tube morphogenic response does not occur under serum-free defined conditions when VEGF and FGF-2 are added together in the fibrin matrices. We recently demonstrated that VEGF and FGF-2 are able to prime EC tube morphogenic responses (i.e. added overnight prior to the morphogenic assay) to hematopoietic stem cell cytokines in collagen matrices and, interestingly, they also prime EC tube morphogenesis in 3D fibrin matrices. EC-pericyte interactions in 3D fibrin matrices leads to marked vascular basement membrane assembly as demonstrated using immunofluorescence and transmission electron microscopy. Furthermore, we show that hematopoietic stem cell cytokines and pericytes stimulate EC sprouting in fibrin matrices in a manner dependent on the α5β1 integrin. This novel co-culture system, under serum-free defined conditions, allows for a molecular analysis of EC tube assembly, pericyte recruitment and maturation events in a critical ECM environment (i.e. fibrin matrices) that regulates angiogenic events in postnatal life.     

A model for the role of hyaluronic acid and fibrin in the early events during the inflammatory response and wound healing

A model is presented outlining the molecular and cellular events that occur during the early stages of the wound healing process. The underlying theme is that there is a specific binding interaction between fibrin, the major clot protein, and hyaluronic acid (HA), a constituent of the wound extracellular matrix. This binding interaction, which could also be stabilized by other cross-linking components, provides the driving force to organize a three-dimensional HA matrix attached to and interdigitated with the initial fibrin matrix. The HA-fibrin matrix plays a major role in the subsequent tissue reconstruction processes. We suggest that HA and fibrin have both structural and regulatory functions at different times during the wound healing process. The concentration of HA in blood and in the initial clot is very low. This is consistent with the proposed interaction between HA and fibrin(ogen), which could interfere with either fibrinogen activation or fibrin assembly and cross-linking. We propose that an activator (e.g. derived from a plasma precursor, platelets or surrounding cells) is produced during the clotting reaction and then stimulates one or more blood cell types to synthesize and secrete HA into the fibrin matrix of the clot. We predict that HA controls the stability of the matrix by regulating the degradation of fibrin. The new HA-fibrin matrix increases or stabilizes the volume and porosity of the clot and then serves as a physical support, a scaffold through which cells trapped in the clot or cells infiltrating from the peripheral edge of the wound can migrate. The HA-fibrin matrix also actively stimulates or induces cell motility and activates and regulates many functions of blood cells, which are involved in the inflammatory response, including phagocytosis and chemotaxis. The secondary HA-fibrin matrix itself is then modified as cells continue to migrate into the wound, secreting hyaluronidase and plasminogen activator to degrade the HA and fibrin. At the same time these cells secrete collagen and glycosaminoglycans to make a more differentiated matrix. The degradation products derived from both fibrin and HA are, in turn, important regulatory molecules which control cellular functions involved in the inflammatory response and new blood vessel formation in the healing wound. The proposed model generates a number of testable experimental predictions.     

Multidomain structure of fibrinogen and its transformations

Generalized concepts of some structural peculiarities of fibrinogen, its transformation into fibrin and assembly have been considered on the basis of author's and published data. The role of local conformational changes in different areas of fibrinogen molecule and of separate reaction centers in formation of single- and double-stranded rod-like equilibrium fibrin oligomers and flexible branched copolymers of fibrinogen with fibrin E fragment has been considered. The mechanism of compactization has been discussed.     

Decorin modulates fibrin assembly and structure

Emerging evidence indicates that fibrin clotting is regulated by different external factors. We demonstrated recently that decorin, a regulator of collagen fibrillogenesis and transforming growth factor-beta activity, binds to the D regions of fibrinogen (Dugan, T.A., Yang, V. W.-C., McQuillan, D.J., and H??k, M. (2003) J. Biol. Chem. 278, 13655-13662). We now report that the decorin-fibrinogen interaction alters the assembly, structure, and clearance of fibrin fibers. Relative to fibrinogen, substoichiometric amounts of decorin core protein modulated clotting, whereas an excess of an active decorin peptide was necessary for similar activity. These concentration-dependent effects suggest that decorin bound to the D regions sterically modulates fibrin assembly. Scanning electron microscopy images of fibrin clotted in the presence of increasing concentrations of decorin core protein showed progressively decreasing fiber diameter. The sequestration of Zn(2+) ions from the N-terminal fibrinogen-binding region abrogated decorin incorporation into the fibrin network. Compared with linear thicker fibrin fibers, the curving thin fibers formed with decorin underwent accelerated tissue-type plasminogen activator-dependent fibrinolysis. Collectively, these data demonstrate that decorin can regulate fibrin organization and reveal a novel mechanism by which extracellular matrix components can participate in hemostasis, thrombosis, and wound repair.     

Involvement of the alpha chain in fibrin clot formation. Effect of monoclonal antibodies.

Murine monoclonal antibodies 9C3, 7B1, and 9E9 have been obtained using native human fibrinogen as the antigen. The antibodies reacted with the epitopes in the COOH-terminal domain of the A alpha chain. Fragmentation of the A alpha chain with plasmin, and, as in the case of the 9E9 epitope, with V8 protease, followed by isolation of the smallest reacting peptides, allowed the localization of the epitopes for 9C3, 7B1, and 9E9 to the amino acid sequences of alpha 240-268, alpha 425-440, and alpha 541-574, respectively. All three monoclonal antibodies strongly inhibited the rate of fibrin polymer assembly from monomers, both in the purified system and in the human plasma. The mechanism of this strong inhibition implied a rapid formation of fibrin protofibrils, followed by capping with IgG molecules of protofibrils containing approximately ten monomers. These observations demonstrated that certain regions in the COOH terminus of the alpha chain may play an important role in the assembly of a fibrin clot, presumably being involved in lateral aggregation of protofibrils.     

Transport of probe molecules through fibrin gels as observed by means of holographic relaxation methods

Holographic relaxation spectroscopy (HRS) has been used to study transport of benzospiropyran (SP), BSA labeled with azobenzene (BSA-ABITC), and IgG-κ labeled with fluorescein (IgG-FITC) through fibrin gels formed under various conditions. The structures of the gels were controlled by means of the concentrations of fibrinogen, thrombin, and Ca²⁺ present during assembly of the fibrin. The diffusion coefficient of free dye (SP) was found to be independent of the fibrinogen concentration. The diffusion rate of labeled BSA reflected the assembly conditions of the gel for fibrinogen concentrations above approximately 6 g/L. In particular, the diffusion coefficient was higher in gels formed in the presence of 5 mM Ca²⁺. The labeled IgG showed photoinduced aggregation, as previously reported, as well as photoinduced attachment to the gel network to produce a permanent diffraction grating. Thus IgG is not a probe in the classical sense, but provides a model for protein diffusion and interactions in gels. These studies indicate that HRS is well suited to the study of molecular transport in fibrin gels.     

A review of studies of the activation of the blood coagulation mechanism in chimpanzees (Pan troglodytes)

This paper reviews our recent studies of blood coagulation activation in the chimpanzee which were carried out employing sensitive immunoassays that measure activation markers of blood coagulation in plasma. Infused factor VIIa activated both factors IX and X in vivo; this reaction depended on the formation of the factor VIIa-tissue factor (TF) complex. The infusion of endotoxin also led to assembly of the factor VIIa-TF complex, enhancing fibrin formation. This process occurred through the intermediate action of specific cytokines.     

Platelet factor 4 (CXCL4) seals blood clots by altering the structure of fibrin

Platelet factor-4 (PF4/CXCL4) is an orphan chemokine released in large quantities in the vicinity of growing blood clots. Coagulation of plasma supplemented with a matching amount of PF4 results in a translucent jelly-like clot. Saturating amounts of PF4 reduce the porosity of the fibrin network 4.4-fold and decrease the values of the elastic and loss moduli by 31- and 59-fold, respectively. PF4 alters neither the cleavage of fibrinogen by thrombin nor the cross-linking of protofibrils by activated factor XIII but binds to fibrin and dramatically transforms the structure of the ensuing network. Scanning electron microscopy showed that PF4 gives rise to a previously unreported pattern of polymerization where fibrin assembles to form a sealed network. The subunits constituting PF4 form a tetrahedron having at its corners a RPRH motif that mimics (in reverse orientation) the Gly-His-Arg-Pro-amide peptides that co-crystallize with fibrin. Molecular modeling showed that PF4 could be docked to fibrin with remarkable complementarities and absence of steric clashes, allowing the assembly of irregular polymers. Consistent with this hypothesis, as little as 50 microm the QVRPRHIT peptide derived from PF4 affects the polymerization of fibrin.     

Deglycosylation of fibrinogen accelerates polymerization and increases lateral aggregation of fibrin fibers

Fibrinogen, the major structural precursor of blood clots, was deglycosylated by peptide-N-(N-acetyl-beta-glucosaminyl)asparagine amidase without denaturation of the polypeptide chains. Deglycosylated fibrinogen behaved normally in clinical coagulation assays, although it is less soluble than normal fibrinogen. However, the turbidity of clots formed from deglycosylated fibrinogen always rose faster and higher than that of clots from normal fibrinogen. Scanning and transmission electron microscopy demonstrated that fibrin made from clots of deglycosylated fibrinogen consisted of thicker, less-branched fiber bundles in a more porous network. Moreover, the degree of lateral aggregation was directly related to clot turbidity and inversely related to branching. Deglycosylation promoted turbidity development, lateral aggregation, and porosity of clots under all conditions tested. All other steps in the coagulation pathways appeared to be unaffected by the absence of carbohydrate. These results suggest that carbohydrate constitutively affects the behavior of deglycosylated fibrinogens by 1) contributing a repulsive force that promotes fibrinogen solubility and limits fibrin assembly and 2) sensitizing fibrin to conditions that influence assembly and clot structure.     

α-α Cross-links increase fibrin fiber elasticity and stiffness

Fibrin fibers, which are ~100 nm in diameter, are the major structural component of a blood clot. The mechanical properties of single fibrin fibers determine the behavior of a blood clot and, thus, have a critical influence on heart attacks, strokes, and embolisms. Cross-linking is thought to fortify blood clots; though, the role of α-α cross-links in fibrin fiber assembly and their effect on the mechanical properties of single fibrin fibers are poorly understood. To address this knowledge gap, we used a combined fluorescence and atomic force microscope technique to determine the stiffness (modulus), extensibility, and elasticity of individual, uncross-linked, exclusively α-α cross-linked (γQ398N/Q399N/K406R fibrinogen variant), and completely cross-linked fibrin fibers. Exclusive α-α cross-linking results in 2.5× stiffer and 1.5× more elastic fibers, whereas full cross-linking results in 3.75× stiffer, 1.2× more elastic, but 1.2× less extensible fibers, as compared to uncross-linked fibers. On the basis of these results and data from the literature, we propose a model in which the α-C region plays a significant role in inter- and intralinking of fibrin molecules and protofibrils, endowing fibrin fibers with increased stiffness and elasticity.