Native Fibrin Research Articles

Variability in individual native fibrin fiber mechanics

Fibrin fibers are important structural elements in blood coagulation. They form a mesh network that acts as a scaffold and imparts mechanical strength to the clot. A review of published work measuring the mechanics of fibrin fibers reveals a range of values for fiber extensibility. This study investigates fibrinogen concentration as a variable responsible for variability in fibrin mechanics. It expands previous work to describe the modulus, strain hardening, extensibility, and the force required for fiber failure when fibers are formed with different fibrinogen concentrations using lateral force atomic force microscopy. Analysis of the mechanical properties showed fibers formed from 1 mg ml-1and 2 mg ml-1fibrinogen had significantly different mechanical properties. To help clarify our findings we developed two behavior profiles to describe individual fiber mechanics. The first describes a fiber with low initial modulus and high extensible, that undergoes significant strain hardening, and has moderate strength. Most fibers formed with 1 mg ml-1fibrinogen had this behavior profile. The second profile describes a fiber with a high initial modulus, minimal strain hardening, high strength, and low extensibility. Most fibrin fibers formed with 2 mg ml-1fibrinogen were described by this second profile. In conclusion, we see a range of behaviors from fibers formed from native fibrinogen molecules but various fibrinogen concentrations. Potential differences in fiber formation are investigated with SEM. It is likely this range of behaviors also occursin vivo. Understanding the variability in mechanical properties could contribute to a deeper understanding of pathophysiology of coagulative disorders.

PEG-fibrin conjugates: the PEG impact on the polymerization dynamics.

Fibrin and its modifications, particularly those with functionalized polyethylene glycol (PEG), remain highly attractive as a biomaterial in drug delivery and regenerative medicine. Despite the extensive knowledge of fibrinogenesis, there is little information on the processes occurring after its modification. Previously, we found structural differences between native fibrin and its conjugates with PEG that allows us to hypothesize that a combination of methods such as terahertz (THz) pulsed spectroscopy and rheology may contribute to the characterization of gelation and reveal the effect of PEG on the polymerization dynamics. Compared to native fibrin, PEGylated fibrins had a homogenously soft surface; PEGylation also led to a significant decrease in the gelation time: from 42.75 min for native fibrin to 31.26 min and 35.09 min for 5 : 1 and 10 : 1 PEGylated fibrin, respectively. It is worth noting that THz pulsed spectroscopy makes it possible to reliably investigate only the polymerization process itself, while it does not allow us to observe statistically significant differences between the distinct PEGylated fibrin gels. The polymerization time constant of native fibrin measured by THz pulsed spectroscopy was 14.4 ± 2.8 min. However, it could not be calculated for PEGylated fibrin because the structural changes were too rapid. These results, together with those previously reported, led us to speculate that PEG-fibrin conjugates formed homogenously distributed highly water-shelled aggregates without bundling compared to native fibrin, ensuring rapid gelation and stabilization of the system without increasing its complexity.

Chitosan Tubes Prefilled with Aligned Fibrin Nanofiber Hydrogel Enhance Facial Nerve Regeneration in Rabbits.

Facial nerves are fragile and easily injured, for example, by traffic accidents or operations. Facial nerve injury drastically reduces the quality of life in affected patients, and its treatment presents clinical challenges. A promising therapeutic strategy includes nerve conduits with appropriate fillers capable of guiding nerve regeneration. In this study, a three-dimensional hierarchically aligned fibrin nanofiber hydrogel (AFG) assembled via electrospinning and molecular self-assembly was first used to mimic the architecture of the native fibrin cable, which is similar to the nerve extracellular matrix (ECM). AFG as a substrate in chitosan tubes (CST) was used to bridge a 7 mm-long gap in a rabbit buccal branch facial nerve defect model. The results showed that AFG and CST showed good compatibility to support the adhesion, activity, and proliferation of Schwann cells (SCs). Further morphological, histological, and functional analyses demonstrated that the regenerative outcome of AFG-prefilled CST was close to that of autologous nerve grafts and superior to that of CST alone or CSTs prefilled with random fibrin nanofiber hydrogel (RFG), which indicated that AFG-prefilled CST markedly improved axonal regeneration with enhanced remyelination and functional recovery, thus showing great potential for clinical application for facial nerve regeneration treatments.

Self-assembled fibrinogen nanofibers support fibroblast adhesion and prevent E. coli infiltration

Fibrinogen nanofibers hold great potential for wound healing applications since they mimic the native blood clot architecture and offer important binding sites to support fibroblast adhesion and migration. Recently, we introduced a new method of salt-induced self-assembly to prepare nanofibrous fibrinogen scaffolds. Here, we present our results on the mechanical properties of these scaffolds and their interaction with 3T3 fibroblasts and E. coli bacteria, which we used as model systems for wound healing. Hydrated, nanofibrous fibrinogen scaffolds showed a Young's modulus of 1.3 MPa, which is close to the range of native fibrin. 3T3 fibroblasts adhered and proliferated well on nanofibrous and planar fibrinogen up to 72 h with a less pronounced actin cytoskeleton on nanofibers in comparison to planar fibrinogen. Fibroblasts on nanofibers were smaller with many short filopodia while larger cells with few long filopodia were found on planar fibrinogen. Live cell tracking revealed higher migration velocities on nanofibers in comparison to planar fibrinogen. The growth of E. coli bacteria on nanofibrous fibrinogen was significantly reduced as compared to agar controls with no bacteria migrating through the nanofibers. In summary, we conclude that self-assembled fibrinogen nanofibers could become highly attractive as future scaffolds for wound healing applications.

Impact of Reactive Amphiphilic Copolymers on Mechanical Properties and Cell Responses of Fibrin‐Based Hydrogels

AbstractMechanical properties of hydrogels can be modified by the variation of structure and concentration of reactive building blocks. One promising biological source for the synthesis of biocompatible hydrogels is fibrinogen. Fibrinogen is a glycoprotein in blood, which can be transformed enzymatically to fibrin playing an important role in wound healing and clot formation. In the present work, it is demonstrated that hybrid hydrogels with their improved mechanical properties, tunable internal structure, and enhanced resistance to degradation can be synthesized by a combination of fibrinogen and reactive amphiphilic copolymers. Water‐soluble amphiphilic copolymers with tunable molecular weight and controlled amounts of reactive epoxy side groups are used as reactive crosslinkers to reinforce fibrin hydrogels. In the present work, copolymers that can influence the mechanical properties of fibrin‐based hydrogels are used. The reactive copolymers increase the storage modulus of the hydrogels from 600 Pa to 30 kPa. The thickness of fibrin fibers is regulated by the copolymer concentration. It could be demonstrated that the fibrin‐based hydrogels are biocompatible and support cell proliferation. Their degradation rate is considerably slower than that of native fibrin gels. In conclusion, fibrin‐based hydrogels with tunable elasticity and fiber thickness useful to direct cell responses like proliferation and differentiation are produced.

The Structural Features of Native Fibrin and Its Conjugates with Polyethylene Glycol and Vascular Endothelial Growth Factor according to Small-Angle X-Ray Scattering

Fibrin is one of the promising biomaterials in tissue engineering for creating favorable conditions for the formation of a capillary-like vascular network. The use of native fibrin is limited due to its rapid degradation and opacity. To improve its properties, including biological ones, various modifications have been proposed, for example, using functionalized polyethylene glycol (PEG). In this work, homobifunctional (O,O''-bis[2-(N-succinimidyl-succinylamino)ethyl])polyethylene glycol of various molecular weights was used to modify fibrin. The modification of fibrin with vascular endothelial growth factor was also analyzed for possible stimulation of the formation of tubule-like structures. Small-angle X-ray scattering (SAXS) data and ab initio modeling of the three-dimensional particle shape showed that PEG–fibrin conjugates consist of filamentous particles, depending on the size of the PEG molecule, the molar ratio of fibrin to PEG, and the presence or absence of vascular endothelial growth factor.

Fibrinogen αC‐regions are not directly involved in fibrin polymerization as evidenced by a “Double‐Detroit” recombinant fibrinogen mutant and knobs‐mimic peptides

BackgroundFibrin polymerization, following fibrinopeptides A and B (FpA, FpB) cleavage, relies on newly exposed α‐ and β‐chains N‐termini (GPR, GHR; A‐, B‐knobs, respectively) engaging preexistent a and b pockets in other fibrin(ogen) molecules' γ‐ and (B)β‐chains C‐terminal regions. A role for mostly disordered (A)α‐chains C‐terminal regions “bridging” between fibrin molecules/fibrils has been proposed. ObjectivesFibrinogen Detroit is a clinically observed mutation (AαR19 → S) with nonengaging GPS A‐knobs. By analogy, a similar Bβ‐chain mutation, BβR17 → S, should produce nonengaging GHS B‐knobs. A homozygous “Double‐Detroit” mutant (AαR19 → S, BβR17 → S; DD‐FG) was developed: with A‐a and B‐b engagements endogenously blocked, other interactions would become apparent. MethodsDD‐FG, wild‐type recombinant (WT‐FG), and human plasma (hp‐FG) fibrinogen self‐association was studied by turbidimetry coupled with fibrinopeptides release high‐performance liquid chromatography (HPLC)/mass spectrometry analyses, and by light‐scattering following size‐exclusion chromatography (SE‐HPLC). ResultsIn contrast to WT‐FG and hp‐FG, DD‐FG produced no turbidity increase, irrespective of thrombin concentration. The SE‐HPLC profile of concentrated DD‐FG was unaffected by thrombin treatment, and light‐scattering, at lower concentration, showed no intensity and hydrodynamic radius changes. Compared with hp‐FG, both WT‐FG and DD‐FG showed no FpA cleavage difference, while ~50% FpB was not recovered. Correspondingly, SDS‐PAGE/Western‐blots revealed partial Bβ‐chain N‐terminal and Aα‐chain C‐terminal degradation. Nevertheless, ~70% DD‐FG molecules bearing (A)αC‐regions potentially able to associate were available. Higher‐concentration, nearly intact hp‐FG with 500‐fold molar excess GPRP‐NH2/GHRP‐NH2 knobs‐mimics experiments confirmed these no‐association findings. Conclusions(A)αC‐regions interactions appear too weak to assist native fibrin polymerization, at least without knobs engagement. Their role in all stages should be carefully reconsidered.

Intrinsically Unfolded Alpha-C Region Of Fibrinogen is Major Contributor to Mechanical Strength of Fibrin Fibers

Background. Fibrinogen is the key mechanical protein in blood coagulation since it is the building block of fibrin fibers, and these 100 nm thick fibers provide mechanical and structural stability to blood clots as they stem the flow of blood. Fibrinogen has two large, intrinsically unfolded regions, termed alphaC regions, which comprise 27% of its molecular weight. The role of these unfolded regions has long puzzled scientists. We hypothesize that they contribute significantly to the mechanical properties of fibrin fibers, which, in turn, determine the stability of blood clots. Methods. We used a nano-mechanical manipulation method, based on a combined atomic force and optical microscope, to determine the mechanical properties of native fibrin fibers, and of fibers formed from a variant in which the alphaC region was truncated. Results. Compared to native fibrin fibers, fibers formed from the truncated variant showed dramatically different mechanical properties. The extensibility (fracture strain) was reduced by a factor of 1.6 (from 1.96 to 1.26); the fracture stress was reduced by a factor of 22 (from 13.7 MPa to 0.61 MPa); and the modulus (stiffness) decreased by a factor of 5.8 (from 6.11 MPa to 1.05 MPa). Conclusion. The large, intrinsically unfolded alphaC region of fibrinogen is a major contributor to the mechanical strength of fibrin fibers, and thus to the strength of blood clots. These findings have significant implications for haemostasis and thrombosis.

Digging deeper: structural background of PEGylated fibrin gels in cell migration and lumenogenesis.

Fibrin is a well-known tool in tissue engineering, but the structure of its modifications created to improve its properties remains undiscussed despite its importance, e.g. in designing biomaterials that ensure cell migration and lumenogenesis. We sought to uncover the structural aspects of PEGylated fibrin hydrogels shown to contribute to angiogenesis. The analysis of the small-angle X-ray scattering (SAXS) data and ab initio modeling revealed that the PEGylation of fibrinogen led to the formation of oligomeric species, which are larger at a higher PEG : fibrinogen molar ratio. The improvement of optical properties was provided by the decrease in aggregates' sizes and also by retaining the bound water. Compared to the native fibrin, the structure of the 5 : 1 PEGylated fibrin gel consisted of homogenously distributed flexible fibrils with a smaller space between them. Moreover, as arginylglycylaspartic acid (RGD) sites may be partly bound to PEG-NHS or masked because of the oligomerization, the number of adhesion sites may be slightly reduced that may provide the better cell migration and formation of continuous capillary-like structures.

Aptamer-Functionalized Fibrin Hydrogel Improves Vascular Endothelial Growth Factor Release Kinetics and Enhances Angiogenesis and Osteogenesis in Critically Sized Cranial Defects.

An aging population, decreased activity levels and increased combat injuries, have led to an increase in critical sized bone defects. As more defects are treated using allografts, which is the current standard of care, the deficiencies of allografts are becoming more evident. Allografts lack the angiogenic potential to induce sufficient vasculogenesis to counteract the hypoxic environment associated with critical sized bone defects. In this study, aptamer-functionalized fibrin hydrogels (AFH), engineered to release vascular endothelial growth factor (VEGF), were evaluated for their material properties, growth factor release kinetics, and angiogenic and osteogenic potential in vivo. Aptamer functionalization to native fibrin did not result in significant changes in biocompatibility or hydrogel gelation. However, aptamer functionalization of fibrin did improve the release kinetics of VEGF from AFH and, when compared to FH, reduced the diffusivity and extended the release of VEGF several days longer. VEGF released from AFH, in vivo, increased vascularization to a greater degree, relative to VEGF released from FH, in a murine critical-sized cranial defect. Defects treated with AFH loaded with VEGF, relative to nonhydrogel loaded controls, showed a nominal increase in osteogenesis. Together, these data suggest that AFH more efficiently incorporates and retains VEGF in vitro and in vivo, which then enhances angiogenesis and osteogenesis to a greater extent in vivo than FH.

Fabrication of 3D-nanofibrous fibrinogen scaffolds using salt-induced self assembly

Fibrinogen has become highly attractive for tissue engineering scaffolds since it is a naturally occurring blood protein, which contains important binding sites to facilitate cell adhesion. Here, we introduce a novel biofabrication technique to prepare three-dimensional, nanofibrous fibrinogen scaffolds by salt-induced self assembly. For the first time, we were able to fabricate either free-standing or immobilized fibrinogen scaffolds on demand by tailoring the underlying substrate material and adding a fixation and washing procedure after the fiber assembly. Using scanning electron microscopy we observed that different buffers including phosphate buffered saline and sodium phosphate reproducibly yielded dense fiber networks on bare and silanized glass surfaces, gold as well as polystyrene upon drying. Fibrillogenesis could be induced with a fibrinogen concentration of at least 2 mg ml−1 in a pH regime of 7–9. Fiber diameters ranged from 100 to 300 nm, thus resembling native fibrin and ECM protein fibers. By adjusting the salt concentration we could prepare fibrinogen scaffolds with overall dimensions in the centimeter range and a thickness of 3 to 5 μm. Using FTIR analysis we observed peak shifts of the amide bands for fibrinogen nanofibers in comparison to planar fibrinogen, which indicates changes in the secondary structure. Since fibrillogenesis was only induced upon drying when salt ions were present we assume that protein molecules were locally oriented in the respective buffers, which—in combination with the observed conformational changes—led to the assembly of individual molecules into fibers. In summary, our novel self assembly process offers a simple and well controllable method to prepare large scale 3D-scaffolds of fibrinogen nanofibers under physiological conditions. The unique possibility to chose between free-standing and immobilized scaffolds makes our novel biofabrication process highly attractive for the preparation of versatile tissue engineering scaffolds.

Prompt peripheral nerve regeneration induced by a hierarchically aligned fibrin nanofiber hydrogel.

In peripheral nervous system defect repair, a wide variety of strategies have been proposed for preparing functionalized nerve guidance conduits (NGC) with more complex configurations to obtain optimal repair effects. Longitudinally oriented fibrin cables were reported to form spontaneously during the initial stages of peripheral nerve regeneration in an empty NGC, which can direct the migration and proliferation of Schwann cells and promote axonal regrowth. Therefore, based on the biomimetic idea, we prepared a three-dimensional hierarchically aligned fibrin nanofiber hydrogel (AFG) through electrospinning and molecular self-assembly, resembling the architecture and biological function of the native fibrin cable and serving as an intraluminal filling to accelerate axon regeneration. We found that the AFG was a beneficial microenvironment to support SCs cable formation and accelerate axonal regrowth with improved motor functional recovery.

Integration of colloids into a semi-flexible network of fibrin.

Typical colloid-polymer composites have particle diameters much larger than the polymer mesh size, but successful integration of smaller colloids into a large-mesh network could allow for the realization of new colloidal states of spatial organization and faster colloid motion which can allow the possibility of switchable re-configuration of colloids or more dramatic stimuli-responsive property changes. Experimental realization of such composites requires solving non-trivial materials selection and fabrication challenges; key questions include composition regime maps of successful composites, the resulting structure and colloidal contact network, and the mechanical properties, in particular the ability to form a network and retain strain stiffening in the presence of colloids. Here, we study these fundamental questions by formulating composites with fluorescent (though not stimuli-responsive) carboxylate modified polystyrene/latex (CML) colloidal particles (diameters 200 nm and 1000 nm) in bovine fibrin networks (a semi-flexible biopolymer network with mesh size 1-5 μm). We describe and characterize two methods of composite preparation: adding colloids before fibrinogen polymerization (Method I), and electrophoretically driving colloids into a network already formed by fibrinogen polymerization (Method II). We directly image the morphology of colloidal and fibrous components with two-color fluorescent confocal microscopy under wet conditions and SEM of fixed dry samples. Mechanical properties are studied with shear and extensional rheology. Both fabrication methods are successful, though with trade-offs. Method I retains the nonlinear strain-stiffening and extensibility of the native fibrin network, but some colloid clustering is observed and fibrin network integrity is lost above a critical colloid concentration that depends on fibrinogen and thrombin concentration. Larger colloids can be included at higher volume fractions before massive aggregation occurs, indicating surface interactions as a limiting factor. Method II results in a loss of measurable strain-stiffening, but colloids are well dispersed and template along the fibrous scaffold. The results here, with insight into both structure and rheology, form a foundational understanding for the integration of other colloids, e.g. with stimuli-responsive functionalities, into semi-flexible networks.

Computational imaging analysis of fibrin matrices with the inclusion of erythrocytes from homozygous SS blood reveals agglomerated and amorphous structures.

Sickle cell disease is a single point mutation disease that is known to alter the coagulation system, leading to hypercoagulable plasma conditions. These hypercoagulable conditions can lead to complications in the vasculature, caused by fibrin clots that form undesirably. There is a need to understand the morphology and structure of fibrin clots from patients with sickle cell disease, as this could lead to further discovery of treatments and life-saving therapies. In this work, a computational imaging analysis method is presented to evaluate fibrin agglomeration in the presence of erythrocytes (RBCs) homozygous for the sickle cell mutation (SS). Numerical algorithms were used to determine agglomeration of fibrin fibers within a matrix with SS RBCs to test the hypothesis that fibrin matrices with the inclusion of SS RBCs possess a more agglomerated structure than native fibrin matrices with AA RBCs. The numerical results showed that fibrin structures with SS RBCs displayed an overall higher degree of agglomeration as compared to native fibrin structures. The computational algorithm was also used to evaluate fibrin fiber overlap (aggregation) and anisotropy (orientation) in normal fibrin matrices compared to fibrin matrices polymerized around SS RBCs; however, there was no statistical difference. Ultrasound measurements of stiffness revealed rigid RBCs in the case of samples derived from homozygous SS blood, and densely evolving matrices, when compared to normal fibrin with the inclusion of AA RBCs. An agglomeration model is suggested to quantify the fibrin aggregation/clustering near RBCs for both normal fibrin matrices and for the altered structures. The results of this work are important in the sense that the understanding of aggregation and morphology in fibrin clots with incorporation of RBCs from persons living with sickle cell anemia may elucidate the complexities of comorbidities and other disease complications.

Fibrin(ogen)olytic and platelet modulating activity of a novel protease from the Echis multisquamatis snake venom.

The variety of enzymes including serine proteases that possess fibrin(ogen)olytic and platelet modulating activity have been discovered in different snake venoms. In our work the fibrin(ogen)olytic and platelet modulating activity of a new protease from Echis multisquamatis snake venom was studied. It was shown that purified enzyme cleaved the ВβR42-A43 bond of fibrinogen during first contact with the substrate following much slower hydrolysis of C-terminus of fibrinogen Aα-chain. Protease hydrolysed fibrin clot too, but at much slower rate and cleaved both C-terminus of Aα-chain and ВβR42-A43 bond of Bβ-chain simultaneously. Preincubation of fibrinogen with protease dramatically elongated thrombin clotting time and the clot formed from a mixture of native fibrinogen and fibrinogen desВβ(1-42)2 digested by plasmin much faster than a native fibrin clot. The protease did not activate platelets nor cause changes in their shape and granularity, but it reduced platelets aggregation induced by ADP.

Ozone-induced oxidative modification of fibrinogen molecules

Ozone-induced oxidation of fibrinogen has been investigated. The conversion of oxidized fibrinogen to fibrin catalyzed either by thrombin or by a reptilase-like enzyme, ancistron, in both cases is accompanied by production of gels characterized by a higher weight/length ratio of fibrils in comparison with the native fibrin gels. IR spectra of the D and E fragments isolated from unoxidized and oxidized fibrinogen suggest a noticeable transformation of functional groups by oxidation. A decrease in content of N-H groups in the peptide backbone and in the number of C-H bonds in aromatic structures, as well as a decrease in the intensity of the C-H valence vibrations in aliphatic fragments CH2 and CH3 were found. The appearance in the differential spectra of the D fragments of rather intense peaks in the interval of 1200-800 cm(-1) clearly indicates the interaction of ozone with amino acid residues of methionine, tryptophan, histidine, and phenylalanine. Comparison of the differential spectra for the D and E fragments suggests that fibrinogen fragment D is more sensitive to the oxidant action than fragment E. Using EPR spectroscopy, differences are found in the spectra of spin labels bound with degradation products of oxidized and unoxidized fibrinogen, the D and E fragments, caused by structural and dynamical modifications of the protein molecules in the areas of localization of the spin labels. The relationship between the molecular mechanism of oxidation of fibrinogen and its three-dimensional structure is discussed.

Novel Use of Scanning Electron Microscopy for Detection of Iron‐Induced Morphological Changes in Human Blood

Fibrinogen is key to the maintenance of hemostasis and is an acute phase protein that is part of the coagulation cascade of proteins. It plays a fundamental role in inflammation, particularly as indicator for a proinflammatory state and is a prominent marker for developing vascular inflammatory diseases. The ultrastructure of fibrin nets can be studied using scanning electron microscopy (SEM) with the addition of thrombin to plasma. In inflammatory conditions such as thromboembolic ischemic stroke and diabetes, the fibrin networks are changed to from dense matted fibrin deposits (DMDs) instead of typical netlike appearance. Similar DMDs can also be induced with the addition of FeCl(2) and FeCl(3). Importantly, the iron-induced DMDs look similar to those from patients with prothrombotic conditions. Excessive or misplaced tissue iron now is recognized to pose a substantial health risk. The current research therefore investigates the establishment of a laboratory fibrinogen model to study that might mimic fibrin fiber generation that is achieved using plasma from healthy and diseased individuals. Furthermore, to determine whether the addition of iron to purified fibrinogen will show DMDs and whether hydrophilic agents can prevent them. We conclude that SEM is a very effective tool for the visualization of circulatory consequences of the interaction of iron-induced hydroxyl radicals with human fibrinogen. Furthermore, this novel fibrinogen model provides a convenient method to study the interactions of the intramolecular and intermolecular hydrophobic forces responsible for the maintenance of the tertiary structure of native fibrin(ogen) and the prevention of iron-induced DMDs formation by hydrophilic agents.

Citrullination enhances the pro-inflammatory response to fibrin in rheumatoid arthritis synovial fibroblasts

ObjectiveFibrin deposits are characteristic of the synovial tissues in rheumatoid arthritis (RA). Once citrullinated, fibrin becomes an autoantigen and is thought to contribute in this way to perpetuate the disease....

Identification of Respective Lysine Donor and Glutamine Acceptor Sites Involved in Factor XIIIa-catalyzed Fibrin α Chain Cross-linking

Factor XIIIa-catalyzed ε-(γ-glutamyl)-lysyl bonds between glutamine and lysine residues on fibrin α and γ chains stabilize the fibrin clot and protect it from mechanical and proteolytic damage. The cross-linking of γ chains is known to involve the reciprocal linkages between Gln(398) and Lys(406). In α chains, however, the respective lysine and glutamine partners remain largely unknown. Traditional biochemical approaches have only identified the possible lysine donor and glutamine acceptor sites but have failed to define the respective relationships between them. Here, a differential mass spectrometry method was implemented to characterize cross-linked α chain peptides originating from native fibrin. Tryptic digests of fibrin that underwent differential cross-linking conditions were analyzed by high resolution Fourier transform mass spectrometry. Differential intensities associated with monoisotopic masses of cross-linked peptides were selected for further characterization. A fit-for-purpose algorithm was developed to assign cross-linked peptide pairs of fibrin α chains to the monoisotopic masses relying on accurate mass measurement as the primary criterion for identification. Equipped with hypothesized sequences, tandem mass spectrometry was then used to confirm the identities of the cross-linked peptides. In addition to the reciprocal cross-links between Gln(398) and Lys(406) on the γ chains of fibrin (the positive control of the study), nine specific cross-links (Gln(223)-Lys(508), Gln(223)-Lys(539), Gln(237)-Lys(418), Gln(237)-Lys(508), Gln(237)-Lys(539), Gln(237)-Lys(556), Gln(366)-Lys(539), Gln(563)-Lys(539), and Gln(563)-Lys(601)) on the α chains of fibrin were newly identified. These findings provide novel structural details with respect to the α chain cross-linking compared with earlier efforts.

Self-assembly of fibrin monomers and fibrinogen aggregation during ozone oxidation

The mechanism of self-assembly of fibrin monomers and fibrinogen aggregation during ozone oxidation has been studied by the methods of elastic and dynamic light-scattering and viscosimetry. Fibrin obtained from oxidized fibrinogen exhibits higher average fiber mass/length ratio compared with native fibrin. Fibrinogen ozonation sharply reduced the latent period preceding aggregation of protein molecules; however, the mechanism of self-assembly of ozonated and non-ozonated fibrinogen cluster was identical. In both cases flexible polymers are formed and reaching a certain critical length they form densely packed structures and aggregate. Using infrared spectroscopy, it has been shown that free radical oxidation of amino acid residues of fibrinogen polypeptide chains catalyzed by ozone results in formation of carbonyl, hydroxyl, and ether groups. It is concluded that fibrinogen peripheral D-domains are the most sensitive to ozonation, which causes local conformational changes in them. On one hand, these changes inhibit the reaction of longitudinal polymerization of monomeric fibrin molecules; on the other hand, they expose reaction centers responsible for self-assembly of fibrinogen clusters.