Skip to main content
NCBI home page
BBA Advances logoLink to BBA Advances
. 2023 Jan 5;3:100072. doi: 10.1016/j.bbadva.2023.100072

Functional properties of individual sub-domains of the fibrin(ogen) αC-domains

YM Stohnii a, TA Yatsenko b, VV Nikulina a, YP Kucheriavyi a, OO Hrabovskyi a, OYu Slominskyi a, KS Savchenko a,c, LV Garmanchuk c, LD Varbanets d, AO Tykhomyrov b, VO Chernyshenko a,⁎
PMCID: PMC10074951  PMID: 37082262

Highlights

  • •

    The use of specific proteases allowed us to obtain truncated forms of fibrin(ogen) lacking its C-terminal sub-domains or both N- and C-terminal sub-domains of their αC-domains. These fibrin(ogen) forms have been used for studying the role of the corresponding sub-domains in several fibrin(ogen)-dependent processes.

  • •

    It was shown that the N- and C-terminal sub-domains both play an important role in fibrin polymerization. These two sub-domains also contributed to platelet aggregation with the N-terminal sub-domains playing a more significant role in this process.

  • •

    It was also shown that the C-terminal sub-domains make the major contribution to the plasminogen activation process, endothelial cell viability, and migration of cancer cells.

Keywords: Fibrin(ogen), Fibrinogen αC-domain, Fibrin polymerization, Fibrinolysis, Platelet aggregation, Fibrin(ogen)-cell interactions

Abstract

Background

Fibrinogen is a large polyfunctional plasma protein consisting of a number of structural and functional domains. Among them, two αC-domains, each formed by the amino acid residues Аα392–610, are involved in fibrin polymerization, activation of fibrinolysis, platelet aggregation, and interaction with different cell types. Previous study revealed that each fibrinogen αC-domain consists of the N-terminal and C-terminal sub-domains. The major objections of the present study were to test functional role of these sub-domains in the above mentioned processes.

Methods

To achieve these objections, we used specific proteases to prepare two truncated forms of fibrinogen, fibrinogen desAα505–610 and fibrinogen desAα414–610, missing their N-terminal and both N- and C-terminal sub-domains, respectively.

Results

Our study with these truncated forms using turbidity measurements and electron microscopy revealed that the N- and C-terminal subdomains both contribute to protofibril formation and their lateral aggregation into fibers during fibrin polymerization process. These two sub-domains also contributed to platelet aggregation with the N-terminal sub-domains playing a more significant role in this process. At the same time, the C-terminal sub-domains make the major contribution to the plasminogen activation process. Further, our experiments revealed that the C-terminal sub-domains are involved in endothelial cell viability and migration of cancer cells.

Conclusions

Thus, the results obtained establish the functional role of individual sub-domains of the αC-domains in fibrin polymerization, activation of fibrinolytic system, platelet aggregation, and cellular interactions.

General significance

The present study expands our understanding of the functional role of individual fibrinogen domains and their specific portions in various fibrin(ogen)-dependent processes.

1. Introduction

Fibrinogen is a large glycoprotein consisting of three pairs of polypeptide chains, Aα, Bβ, and γ [1]. Blood vessel injury initiates the blood clotting cascade resulting in conversion of fibrinogen to fibrin which spontaneously polymerizes to form the core of blood clots that prevent blood loss [1,2]. Besides of its blood clotting function, fibrinogen as a multifunctional protein involved in various processes including platelet aggregation [3], [4], [5], migration and proliferation of endothelial cells [6,7], leucocyte migration [8], and regulation of vessel tonus [9], and endothelial permeability [10].

Structurally, fibrinogen consists of the central E-region, two peripheral D regions, and two αС regions formed by the 221–610 portions of the Аα chains. Each αC region includes the unordered αC-connector (amino acid residues Аα221–391) and the compact αC-domain formed by the Аα392–610 residues [11]. During fibrin polymerization, the αC regions mainly participate in lateral association of protofibrils [12,13,2] and subsequently in activation of fibrinolysis [14]. This portion of the molecule also contains the RGD motif (amino acid residues Аα572–574) which is known to bind integrin receptors on platelets and endothelial cells [15,16]. Such reactivity and various interactions of the αС regions suggest that distinct portions of these regions contain different binding sites that participate in protein-protein and protein-cellular interactions. Thus, it is important to identify which portions of the αC region are responsible for its specific functions.

In the present study, we focused on the role of different portions of fibrin(ogen) αС-domains in fibrin polymerization, activation of fibrinolytic system, platelet aggregation, endothelial cells proliferation, and cancer cells migration. In our experiments, we utilized two highly selective proteases prepared from the cultural liquid of Bacillus thuringiensis var. israelensis IMV B-7465 and from the venom of snake Gloydius halys halys. These proteases allowed us to obtain truncated forms of fibrin(ogen) lacking the Aα414-610 and Aα505-610 portions of their αC regions. Comparison of the properties of fibrin(ogen) desAα505-610 and desAα414-601 with those of the parent molecule allowed us to elucidate the role of the Aα414-504 and Aα505-610 portions of the αC-domains in the above mentioned processes.

2. Materials and methods

2.1. Materials

2.1.1. Chemicals

Molecular weight markers were from Thermo Fisher Scientific (Waltham, USA). Superdex G 75, SDS, and β-mercaptoethnol were from Bio-Rad Laboratories (Hercules, USA). Acrylamide, bis-acrylamide, ТЕМЕD, Thrombin (50 NIH units/ml), ADP, carboxypeptidase В, 4-Nitrophenyl phosphate disodium salt, Blue-Sepharose, NaCl, HEPES, KCl, MgCl2, NaH2PO4, BSA, SDS, PBS tablets, CaCl2, modified Dulbecco's medium, fetal bovine serum (FBS), gentamicin, 3-(4, 5-diethylthiazoly-2-yl)-2,5-diphenyltetrazolium bromide (MTT reagent), dimethyl sulfoxide (DMSO) were from Sigma-Aldrich (St. Louis, USA). Recombinant tPA ‘Actilyse’ was from Boehringer Ingelheim (Ingelheim am Rhein, Germany) and chromogenic substrate S2251 (Val-Leu-Lys-p-nitroanilide) was from Siemens (Munich, Germany).

2.1.2. Proteases

Proteolytic enzyme from the venom of G. halys halys was purified by the method developed previously [17]. This serine protease was described as the fibrinogen-specific enzyme that preferentially cleaves the Аα413-414 peptide bond. After 90 min of incubation of fibrinogen (10 mg/ml) with this enzyme (0.02 mg/ml) at 37 °С, most of the Аα chains were hydrolyzed while the Bβ and γ chains remained intact, as reported previously [18].

Proteolytic enzyme from the cultural liquid of Bacillus thuringiensis was purified by the method developed previously [19]. This serine protease is specific towards peptide bond Аα504-505. After 90 min of incubation of fibrinogen (10 mg/ml) with this enzyme (0.02 mg/ml) at 37 °С, most of the Аα chains were hydrolyzed while the Bβ and γ chains remained intact [20].

2.1.3. Antibodies

Mouse monoclonal antibody ІІ-5С (anti-Aα20-78) and 2D-2A (anti-Вβ12-25) used in the present study were prepared as described earlier [21,22]. Polyclonal rabbit antibody against human Glu-plasminogen and against recombinant tissue-type plasminogen activator were also prepared as described earlier [23,24]. All antibodies were produced in immunology department as well as in protein structure and functions department of Palladin Instutute of biochemistry of NAS of Ukraine and were used in 1:1000 dilution. Anti-Rabbit IgG (whole molecule) Alkaline Phosphatase conjugated antibody produced in goat was purchased from Sigma-Aldrich (St. Louis, USA).

2.1.4. Proteins

Fibrinogen was purified from the human blood plasma according to the method described earlier [25]. Glu-Plasminogen was obtained from the human blood plasma by affinity chromatography on Lys-Sepharose as described previously [26]. Thrombin-like enzyme Ancistron, which removes from fibrinogen only fibrinopeptides A, was purified from the venom of G. halys halys using Blue-Sepharose (Sigma-Aldrich, St. Louis, USA) according to the previously described method [27].

2.1.5. Platelets

Venous blood of healthy volunteers (n = 5) who had not taken any medication for 7 days was collected by venipuncture of basilic vein using 19 G sterile needle. Blood was collected into sterile plastic 10 ml tubes. Blood was spinned down at 160 g for 30 min at 25 °C. Platelet rich plasma was collected and spinned down again at 300 g for 15 min. Pellet of platelets was re-suspended in 0.004 M HEPES, pH 7.4, 0.137 M NaCl, 0.0027 M KCl, 0.001 M MgCl2, 0.0056 mM glucose, 0.003 M NaH2PO4, and 0.35 mg/ml BSA to obtain the homogenous suspension of washed platelets, as recommended earlier [28]. Patients signed informed consent prior to blood sampling. This study was approved by the Ethics Committee of the Palladin Institute of Biochemistry (09.09.2021, N12).

2.1.6. Cell lines

HeLa (human cervix epitheloid carcinoma, ECACC 93021013) were obtained from European Collection of Authenticated Cell Cultures.

Murine aortic endothelial cell line (MAEC) [29] were purchased by National Bank of Cell Lines and Tumor Strains of R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology of National Academy of Sciences of Ukraine (IEPOR, NAS of Ukraine).

2.2. Methods

2.2.1. Preparation of fibrinogen desAα505-610 and fibrinogen desAα414-610

Fibrinogen hydrolysis was performed by incubation with proteases from the cultural liquid of Bacillus thuringiensis var. israelensis IMV B-7465 and from the venom of snake Gloydius halys halys for 60 and 30 min, respectively, at enzyme:substrate ratio of 1:300 for both enzymes. The reaction was performed in 0.05 M tris-HCl buffer, pH 7.4, containing 0.13 M NaCl, at 37 °С, and was terminated by adding benzamidine solution to a final concentration of 0.0016 М. The incubation mixture after hydrolysis was fractionated on Superdex 75 column using FPLC-system Akta Prime equilibrated with the same buffer. Truncated forms of fibrinogen, fibrinogen desAα414-610 and fibrinogen desAα505-610, were then characterized using SDS-PAGE and Western blotting. For the MALDI-TOF analysis, fractions eluted from Superdex 75, which contained cleaved peptides, were concentrated using the centrifuge microconcentrators Amicon Ultra 3 K (MilliporeSigma, Burlington, USA).

Both truncated forms of fibrinogen were used for preparation of monomeric fibrins desAB-desAα414–610 and desAB-desAα505–610. These forms were incubated with 0.15 NIH units of thrombin per 1 mg of fibrinogen to convert fibrinogen to fibrin. After 30 min of incubation at 37 °С, fibrin clots were removed, washed with 0.15 M NaCl solution, and then dissolved in 0.125% acetic acid as described in [30]. All samples of truncated fibrinogen and fibrin were frozen in liquid nitrogen and lyophilized using LyoQuest (Telstar, Terrassa, Spain). The lyophilized samples of fibrinogen and fibrin were stored at +4 °С and dissolved before experiments in 0.05 М tris-HCl buffer, pH 7.4, or in 0.125% acetic acid, respectively.

2.2.2. SDS-PAGE and Western blotting

The purity of truncated fibrin(ogen) samples and their molecular masses were determined by SDS-PAGE using 10% or 12% gels according to Laemmli. The products of hydrolysis of fibrinogen and fibrin by fibrinogenase were also analyzed by SDS-PAGE under reducing conditions. The purified proteins were further transferred to a nitrocellulose membrane to specify the bands by immunoprobing. The membrane was blocked with 5% milk in PBS for 1 h, incubated with a mouse monoclonal antibody ІІ−5С (for the Aα chain detection) or antibody 2D-2A (for the Вβ chain detection) for another hour and then developed with a secondary HRP-labelled goat anti-mouse antibody. The bands were visualized using 0.001 M 4-chloro-1-naphtol solution in 0.05 M Tris, pH 7.5, and 0.3% H2O2.

2.2.3. MALDI-TOF analysis

Mass spectrometry analysis was performed on a MALDI-TOF spectrometer Voyager DE PRO (Applied Biosystems, USA). H+-matrix ionization was performed by laser irradiation. The concentration of Sinapinic acid (Sigma, USA) in the matrix reagent was 1 mg/ml. The reagent was dissolved in a solution containing equal volumes of acetonitrile (Sigma, USA) and 1% aqueous trifluoroacetic acid (Sigma, USA). The spectra obtained were processed by Data Explorer 4.0.0.0 (Applied Biosystems) [31].

2.2.4. Turbidity measurements

The process of fibrin polymerization was monitored by measuring the change in turbidity at 350 nm during thrombin-induced polymerization of fibrin using spectrophotometer POP (Optizen, Daejeon, Korea) [32]. The samples of native or truncated fibrinogens were dissolved in 0.05 M Tris buffer, pH 7.5, containing 0.13 M NaCl, and 10−3 М CaCl2 to final concentration of 0.1 mg/ml). Polymerization was initiated by addition of thrombin (0.25 NIH units/ml).

2.2.5. Surface plasmon resonance

Studies were performed using ‘Plasmon 6′ device developed at the V.E. Lashkarev Institute of Semiconductor Physics (Kyiv, Ukraine). The gold surface of a chip was covered by the self-assembled layer formed by 11-mercaptoundecanoic acid and 6-mercapto-1-hexanol [33]. Human fibrinogen was immobilized covalently onto the functionalized chip using standard amino group coupling chemistry. Native or truncated forms of fibrinogen at 0.01 mg/ml were applied to the chip in 0.05 M Tris buffer, pH 7.5, with 0.13 M NaCl. Protein-protein interactions were registered as the change in Response.

2.2.6. Electron microscopy

Polymerization of native fibrin and its truncated forms was studied by transmission electron microscopy of negatively contrasted samples using H-600 Transmission Electron Microscope (Hitachi, Tokyo, Japan) at 75 kV; 1% water solution of uranyl acetate (Merck, Germany) was used as a negative contrast. For sample preparation, 0.32 mg/ml fibrinogen in 0.05 M ammonium formiate buffer, pH 7.9, with 0.025 M CaCl2 was placed in sterile glass tubes. Fibrin polymerization was initiated by adding thrombin to a final concentration of 0.25 NIH units/ml. After 180 s, aliquots were taken from the polymerization mixture. Each aliquot was diluted to a final fibrinogen concentration of 0.07 mg/ml, and 0.01 ml probes of fibrinogen solution were transferred to a carbon lattice which was treated with a 1% uranyl acetate solution after 2 min. Electron microscopic images were obtained at magnification of 20,000–50,000 as described before [34].

2.2.7. Plasminogen activation assay

Activation of Glu-plasminogen by tissue-type plasminogen activator was studied on native fibrin, as well as on desA-desAα414–504 and desA-desAα505–610 truncated forms of fibrin. To prepare these forms, we used thrombin-like enzyme Ancistron. Incubation mixture contained fibrinogen (0.2175 µМ), Ancistron (0.01 mg/ml), Glu-plasminogen (0–0.11 µМ), tissue-type plasminogen activator (3 IU/ml), and chromogenic substrate S2251 (0.3 mM). The reaction was performed in 0.05 М tris-НСl buffer, pH 7.4, containing 0.15 М NaCl at 37 °С. The resulting plasmin activity was monitored using chromogenic substrate S2251. Generation of p-nitroaniline was monitored using Multiscan FC (ThermoFisher, Waltham, USA). Native fibrinogen and its truncated forms were incubated with carboxypeptidase B for 15 min at ambient temperature at 1:50 mass ratio for the removal of the C-terminal Lysine residues.

2.2.8. Aggregometry

Platelet aggregation measurements were based on the changes in the turbidity of human PRP [35]. The measurements were performed on Aggregometer Solar AP2110 (Belorussia) according to manufacturer's recommendations. Washed platelets (0.2 ml) were added to the sample tube, mixed with 0.05 ml of fibrinogen (9 mg/ml) and were activated by the addition of 0.025 ml CaCl2 (0.025 M) and 0.025 ml of platelet agonist ADP (25 µM). Aggregation was registered for 5 min at 37 °C.

2.2.9. Study of endothelial cells viability

Immortalized line of endothelial cells MAEC were cultivated in Iscove's modified Dulbecco's medium (Sigma-Aldrich, St. Louis, USA) supplemented with 10% FBS and gentamicin (50 μg/ml). Cells were incubated at 37 °C in 5% CO2. Cell viability was measured by 3-(4, 5-diethylthiazoly-2-yl)−2,5-diphenyltetrazolium bromide (MTT) assay. Cells were plated on 96-well plates and were incubated with 100 μl of complete medium containing 1 mg/ml MTT (3-(4,5-Dimethylthiazol-2-yl)−2,5-Diphenyltetrazolium Bromide (Sigma) at 37 °C for 4 h followed by solubilization with dimethyl sulfoxide (Sigma). The absorbance at 540 nm was measured with Multiscan FC (ThermoFisher, Waltham, USA) [36].

2.2.10. Scratch test

The scratch wound-healing assay was applied as described previously [37]. This assay is widely used for estimating cell migration and is especially useful for the study of migration of cancer cells [38]. Briefly, 4 × 105 cells were seeded onto 24-well cell culture plates and allowed to grow to form a confluent monolayer (24 h). Next day, the cells were serum-starved in RPMI containing 0.5% FBS for 24 h prior to treatment. The monolayer was gently scratched with a sterile pipette tip to create a 1 cm long “scratch”. The wells were washed 2 times with 1 ml of the growth media to remove the debris and smooth the edge of the scratch. Lyophilized fibrinogen was reconstituted in 50 mM Tris, pH 7.5, with 130 mM NaCl prior to treatment. Fresh media containing 50 µg/ml of control or truncated fibrinogen was added to cells and cells were incubated in fibrinogen-containing media for 2 h. After 2 h, media was replaced with RPMI + 0.5% FBS and cells were incubated for up to 24 h. The images of scratches were acquired at 0, 12, and 24 h post treatment. Wound area was measured, and number of migrated cells were counted using ImageJ software (NIH). The number of cells migrated into a zone of scratching was normalized to vehicle-treated wells (no fibrinogen).

2.2.11. Molecular modeling of αC-domain structure

The structure of the human fibrinogen αC-domain was obtained from https://faculty.uml.edu//vbarsegov/research/fibrin.html#the-structures-of-fibrin-oligomers-and-protofibrils. The structure of the αC-domain (amino acid residues Aα392-610) was constructed by the authors with the Modeller software using sequence homology with the structure of the bovine αC-domain (PDB code: 2JOR). The αC-domain models were used as initial structures in all-atom MD simulations. Energy minimization of the αC-domain was performed using the steepest descent algorithm, heated αC-domains to 300 K, and equilibrated the obtained structures for 1 µsec. Then the structures of the αC-domains were randomly selected from the last 500-ns portion of equilibrium simulations and incorporated into the structure of protofibrils. In coarse-grained modeling, the structures of the αC-domains were stabilized by native contacts identified with Modeller [39].

2.2.12. Statistics

One-Way ANOVA was used to compare the means of independent groups in order to determine significant difference. Results are presented as means ± standard deviation calculated using Microsoft Excel (Microsoft, Redmond, USA). Data were considered significant when P < 0.05.

3. Results

3.1. Characterization of truncated forms of fibrinogen

The truncated forms of fibrinogen were obtained using proteases from the venom of G. halys halys and from the culture medium of B. thuringiensis. They were characterized by SDS-PAGE and Western blotting. SDS-PAGE analysis revealed that both forms had truncated Аα chains with the molecular masses of 54 and 43 kDa for fibrinogen desАα505-610 and fibrinogen desАα414-610, respectively (Fig. 1A and B). Western blot analysis using monoclonal antibody (mAb) ІІ-5С specific to the fibrinogen Аα chain residues 20–78 [21] revealed that Aα chain truncation occurs at its C-terminal part (Fig. 1C and D). In addition, Western blot analysis with mAb 2D-2A specific to the fibrinogen Вβ chain residues 12–25 [22] confirmed that this chain was intact in both forms of fibrinogen (Fig. 1E).

Fig. 1.

Fig. 1

Open in a new tab

SDS-PAGE and Western blot analyses of the truncated forms of fibrinogen. [A and B] SDS-PAGE analysis of fibrinogen desАα505–610 and fibrinogen desАα414–610, respectively. [C and D] Western blot analysis of fibrinogen desАα505–610 and fibrinogen desАα414–610, respectively, using mAb II-5C specific to the Аα chain residues 20–78. [E] Western-blot analysis of fibrinogen desАα505–610 and fibrinogen desАα414–610 using mAb 2D-2A specific to the Вβ chain residues 12–25. 1 – native fibrinogen; 2 - fibrinogen desАα505–610; 3 – fibrinogen desАα414–610; M – molecular mass markers.

Further, MALDI-TOF analysis of the purified products of fibrinogen's hydrolysis by the snake venom protease yielded a peak with M/Z ratio of 21,109. According to the Peptide Mass Calculator, this product of hydrolysis corresponds to the Аα414-610 peptide. MALDI-TOF analysis of the purified products of fibrinogen's hydrolysis by the protease from the bacterial culture medium revealed a peak with M/Z ratio of 11,441 which corresponds to the Аα505-610 peptide. Thus, this analysis further confirmed that the above mentioned proteases allowed us to obtain truncated forms of fibrinogen missing the Аα414-610 and Аα505-610 portions, i.e., fibrinogen desАα414-610 and fibrinogen desАα505-610, respectively [18,20]. In one of these forms, desАα414-610, practically the entire αC-domains were missing while the other form, desАα505-610, contained only about half of each αC-domain. These fibrinogen forms have been used in our subsequent study.

3.2. Effect of the fibrinogen Aα chain truncations on fibrin polymerization

To evaluate the role of the αС-domains in fibrin polymerization process, we measured changes in turbidity of fibrinogen solutions during thrombin-induced conversion of fibrinogen to fibrin. This method allowed us to evaluate the time which is required for protofibril formation (lag-stage), the rate of lateral association of protofibrils into fibers (exponential phase), and the final turbidity of the clot which characterizes the structure of polymerized fibrin. Both truncated fibrins, desAB-desАα505-610 and desAB-desАα414-610, demonstrated longer lag-stage in comparison to the native fibrin desAB tested in the same conditions (Fig. 2). Despite of this prominent delay in lateral aggregation of protofibrils, the rate of polymerization and the final turbidity of fibrin clots formed by the truncated fibrinogen forms were higher than those observed for native fibrin.

Fig. 2.

Fig. 2

Open in a new tab

The change in turbidity during thrombin-induced polymerization of native and truncated forms of fibrin. [A] Turbidity curves showing polymerization of fibrin desАВ-desАα505–610 vs native fibrin desAB. [B] Turbidity curve showing polymerization of fibrin desАВ-desАα414–610 vs native fibrin desAB. Data are representative of typical experiments (n = 5). 1 – native fibrin; 2 - fibrin desAB-desАα505–610; 3 – fibrin desAB-desАα414–610.

To further evaluate the effects observed with the truncated fibrin forms, we analyzed the structure of fibrin polymers using electron microscopy. Samples for electron microscopy were collected 300 s after addition of thrombin to native fibrinogen and its truncated forms in conditions used for turbidity measurements. As shown in Fig. 3A–C, both truncated fibrinogens formed thinner fibers than native fibrinogen. However, their fibrin clots were denser (Fig. 3 D–F). This may explain higher final turbidity of fibrin clots formed by truncated forms of fibrinogen observed in the turbidity study.

Fig. 3.

Fig. 3

Open in a new tab

Electron microscopy of fibrin clots formed by thrombin-induced polymerization of native and truncated forms of fibrin obtained at the different magnification. [A and D] Polymerized native fibrin desAB. [B and E] Polymerized fibrin desАВ-desАα505–610. [C and F] Polymerized fibrin desАВ-desАα414–610. Data represent typical experiments (n = 5). Scale bars: 0.2 µm in panels A-C and 2 µm in panels d-F.

To evaluate the role of the αС regions in the intermolecular interactions, we selected fibrinogen instead of fibrin. While polymerization sites A and B are not available in the fibrinogen molecule, its αС regions can be involved in fibrinogen-to-fibrinogen interactions [40]. To study αС-αС interactions, we immobilized native fibrinogen on the surface of a sensor chip and tested its interaction with added native fibrinogen (control) and fibrinogens desАα505-610 and desАα414-610 using SPR (Fig. 4). The experiments revealed that the removal of the Аα505-610 portion from the αC regions decreased the ability of fibrinogen to bind to immobilized fibrinogen through αС-αС interactions by about 30% when compared to control. The loss of the Аα414–504 portion further decreased such ability by approximately 70% compared to control. These findings further confirm the importance of the αС-domains in the intermolecular fibrinogen-fibrinogen interactions that was reported earlier [41].

Fig. 4.

Fig. 4

Open in a new tab

Surface Plasmon resonance-detected interaction of immobilized native fibrinogen with fibrinogen desАα505–610 and fibrinogen desАα414–610. Data are representative of typical experiments (n = 5). 1 – native fibrinogen; 2 - fibrinogen desАα505–610; 3 – fibrinogen desАα414–610.

3.3. Effect of the fibrinogen Aα chain truncations on plasminogen activation

It is known that in fibrinolytic process fibrin is required for efficient activation of plasminogen by its activator, tissue-type plasminogen activator (tPA). To evaluate the involvement of the Аα414-504 and Аα505-610 portions of the αC-domain in fibrinolysis, we tested generation of active plasmin by tPA in the presence of truncated forms of fibrinogen. The Aα chains of fibrinogen desAα505-610 do not contain C-terminal Lys residues, which can support plasminogen activation [42], while the Aα chains of fibrinogen desAα414-610 have such residues. To remove these Lys residues, fibrinogen desАα414-610 was treated with carboxypeptidase B. Fibrin desA and its truncated forms, desA-desAα414-610 and desA-desAα505-610, were generated ex tempore using thrombin-like enzyme Ancistron. Generation of active plasmin was monitored using specific chromogenic substrate S2251. Both truncated forms of fibrin exhibited lower ability to stimulate activation of plasminogen (0.044 µM) by tPA (3 IU/ml) compared to native fibrin desA (Fig. 5A). In the range of plasminogen concentrations close to saturation, this difference reached 20% (Fig. 5B). Importantly, the rates of plasmin generation on both truncated forms of fibrin were very close. This observation suggests that plasminogen- and tPA-binding sites involved in activation of fibrinolysis are located within the Aα505-610 portion of the αС-domain.

Fig. 5.

Fig. 5

Open in a new tab

Generation of plasmin from Glu-plasminogen by tissue-type plasminogen activator in the presence of fibrin desA (curves 1), fibrin desA-desАα414–610 (curves 2), and fibrin desА-desAα505–610 (curves 3). [A] Typical kinetic curves of plasminogen activation in the presence of fibrin and its truncated forms. Data are representative of typical experiments (n = 5). [В] The rate of hydrolysis of chromogenic plasmin-specific substrate S2251 at different concentrations of plasminogen (0-0.11 µM) and constant concentration of tPA (3 IU/ml) in the presence of fibrin and its truncated forms.

3.4. Platelet aggregation in the presence of fibrinogens desАα505-610 and desАα414-610

Since interaction of fibrinogen with platelet receptor αIIbβ3 plays an important role in platelet aggregation [43], we studied ADP-induced aggregation of washed human platelets in the presence of native and truncated forms of fibrinogen. In a typical experiment, we added fibrinogen or its truncated forms (final concentration of 1.5 mg/ml) to suspension of washed platelets in HEPES buffer. After 2 min of incubation under stirring, platelets were activated by the addition of ADP to a final concentration of 12.5 µM. The experiments revealed that platelet aggregation rate in the presence of fibrinogen desАα505-610 was decreased by 15 ± 5% compared to that in the presence of native fibrinogen; the speed of aggregation was practically the same for both fibrinogens (Fig. 6A). At the same time, the loss of the Аα414-504 portions in fibrinogen desАα414-610 resulted in a significant reduction of its ability to support platelet aggregation. As shown in Fig. 6B, the rate of platelet aggregation decreased by 50 ± 9% and the speed of the process declined by 35 ± 7% compared to control fibrinogen. These findings suggest that the loss of Аα414-504 and Аα505-610 portions of the αC-domains in truncated fibrinogens reduced their ability to support platelet aggregation with the Аα414-504 portion to be more critical than the Аα505-610 one.

Fig. 6.

Fig. 6

Open in a new tab

Typical aggregation curves of washed platelets activated by ADP in the presence of native and truncated forms of fibrinogen. [А] Aggregation of washed platelets activated by ADP (12.5 µM) in the presence of native fibrinogen vs fibrinogen desАα505–610 (1.5 mg/ml). [В] Aggregation of washed platelets activated by ADP (12.5 µM) in the presence of native fibrinogen vs fibrinogen desАα414–610 (1.5 mg/ml). Data are representative of typical experiments (n = 5). 1 – native fibrinogen; 2 - fibrinogen desАα505–610; 3 – fibrinogen desАα414–610.

3.5. Viability of endothelial cells grown on scaffolds of truncated variants of fibrin

The involvement of fibrinogen and fibrin in the migration and proliferation of endothelial cells, as well as in the vascularization process [44,45], prompted us to compare the viability of endothelial cells grown on scaffolds of truncated forms of fibrin with that of native fibrin. Different forms of fibrin, fibrin desAB, fibrin desАВ-desАα505–610, and fibrin desАВ-desАα414–610, were obtained from fibrinogen and its truncated forms by incubation with thrombin with subsequent dissolution of fibrin clots in 0.125% acetic acid as described in Materials and methods. These forms of fibrin were adsorbed to the wells of 96-well cultural plates at the final amount of 40 µg/cm2. After aspiration and washing wells with PBS, cell suspension was applied to the wells and cultivated as described in Methods section. Mouse aorta endothelial cells (MAEC) were chosen for these experiments. MTT-test was used for the analysis of cell viability after their cultivation on scaffolds formed by the truncated forms of fibrin in comparison to native fibrin. The experiments revealed that the loss of both C-terminal portions of the fibrinogen Aα chains substantially decreased the viability of MAEC grown on fibrin scaffolds (Fig. 7). No difference between fibrin desАВ-desАα505–610 and fibrin desАВ-desАα414–610 was found. These results suggest that interaction fibrin with endothelial cells is mediated mainly by the Аα505–610 portions of its αC-domains.

Fig. 7.

Fig. 7

Open in a new tab

Viability of mouse aorta endothelial cells (MAEC) cultivated on fibrin scaffolds according to the results of MTT-test. Cells were cultivated on fibrin desAB (1), desАВ-desАα505–610 (2), and fibrin desАВ-desАα414–610 (3). *P ≤ 0.05 (n = 8).

3.6. Migration of HeLa cells on truncated variants of fibrinogen

Having the sites for interaction with cancer cells and being the component of intracellular matrix, fibrinogen supports tumor growth and invasion [46]. To investigate the role of the Аα414–610 and Аα505–610 portions missing in the truncated forms of fibrinogen in the process of cancer cells migration, we utilized scratch-test. After forming the monolayer of HeLa cancer cells, we made a scratch with pipette tip and added to the culture media different forms of fibrinogen at the final concentration of 0.05 mg/ml. Cell migration to the wounded zone was observed at 12 and 24 h (Fig. 8). For the quantitative analysis of series of images (n = 5), we used ImageJ software. We calculated the number of cells migrated to the scratch-zone at 24 h after scratch. The results of quantification are presented in Fig. 9. It was found that truncated forms of fibrinogen supported cell migration less effectively compared to native fibrinogen. The calculated 10–15% impairment of the migration was statistically significant and correlated well with the data of MAEC viability impairment. As in the viability test on endothelial cells, we detected no difference between cell migration on fibrinogens lacking the Аα505-610 or Аα414-610 portions. These results suggest the greater importance of the Аα505-610 portions of the αC-domains for the interactions with endothelial cells.

Fig. 8.

Fig. 8

Open in a new tab

Migration of HeLa cells to the scratch zone in the presence of native fibrinogen or its truncated forms after 0, 12, and 24 h of incubation. Magnification 1 × 40. 1 – native fibrinogen; 2 - fibrinogen desАα505–610; 3 – fibrinogen desАα414–610. Data are representative of typical experiments (n = 5).

Fig. 9.

Fig. 9

Open in a new tab

Number of HeLa cells migrated to the scratch zone in wound-healing test in the presence of native fibrinogen or its truncated forms after 0, 12, and 24 h of incubation. Calculations were performed using ImageJ software. 1 – native fibrinogen; 2 - fibrinogen desАα505–610; 3 – fibrinogen desАα414–610. *P ≤ 0.05 (n = 8).

4. Discussion

The use of proteases from the cultural liquid of Bacillus thuringiensis and from the venom of G. halys halys allowed us to obtain two truncated forms of fibrinogen, desАα505–610 and desАα414–610. It was suggested earlier that fibrinogen αC-domain (amino acid residues Aα392–610) consists of two compact sub-domains, N-terminal sub-domain and C-terminal one, formed by the N- and C-terminal halves of this domain, respectively [47]. While the structure of the N-terminal sub-domain has been solved [47,48] that of the N-terminal sub-domain was predicted to contain β-sheet structures [47]. We have constructed the structure of the complete αC-domain using approach described in Methods section. The structure revealed two distinct sub-domains with the cleavage site for the above mentioned proteases at the beginning of the N-terminal sub-domain and approximately in between the N- and C-terminal sub-domains (Fig. 10). Accordingly, fibrinogen desАα505–610 contained the N-terminal sub-domains while in fibrinogen desАα414–610 both sub-domains of the αC-domains were missing. Thus, these truncated forms of fibrinogen represent convenient models for studying functional properties of individual sub-domains of the αC-domains in fibrin(ogen)-mediated processes.

Fig. 10.

Fig. 10

Open in a new tab

Structure of the fibrinogen αC-domain constructed as described in the Methods section. Red arrows indicate cleavage sites by proteases from the cultural liquid of Bacillus thuringiensis (Аα504–505) and from the venom of snake Gloydius halys (Аα413–414). Potential sites of platelet receptors binding (Lys-Gly-Asp, Arg-Gly-Asp, and Asp-Gly-His) are shown by white arrows; β-sheets and α-helix are shown by blue and red ribbons, respectively.

First, we tested the role of the N- and C-terminal sub-domains in fibrin polymerization process. Turbidity measurements revealed a significant delay in lag-stage for both truncated fibrinogens compared to the native one indicating that both sub-domains of the αC-domain contribute significantly to the process of protofibril formation at the early stage of fibrin polymerization. Further, electron microscopy study revealed that both truncated forms had thinner fibers compared to native fibrin highlighting a significant role of the αC-domains in lateral aggregation of protofibrils at the stage of fibrin fiber formation. These results are in a good agreement with the previous observations with other truncated variants of fibrinogen that have been recently reviewed [40].

Our SPR experiments revealed that native fibrinogen and its truncated forms bound to immobilized fibrinogen although binding of the truncated fibrinogens was less pronounced (Fig. 4). As was shown earlier, fibrinogen in solution can bind to adsorbed fibrinogen through the intermolecular interaction between their αC-domains [39]. It was also shown that formation of αC polymers in fibrin occurs mainly through intermolecular interactions between the N-terminal sub-domains of the αC-domains and between their C-terminal sub-domains and the αC-connectors [40,49]. Thus, the fact that fibrinogen desАα505–610 containing the N-terminal sub-domains binds to immobilized fibrinogen can be easily explained by the involvement of these sub-domains in the αC-αC interaction. As to fibrinogen desАα414–610, its interaction with immobilized fibrinogen was less pronounced than that of fibrinogen desАα505–610. Taking into account that this truncated fibrinogen devoid of both αC-domains but still contains the αC-connectors, its binding to fibrinogen most probably occurs through the interaction of its αC-connectors with the C-terminal sub-domains of immobilized fibrinogen. Thus this finding provides further experimental evidences for the proposed molecular mechanism of αC polymer formation in fibrin [40,48].

It was previously shown that the plasmin- and tPA-binding sites are cryptic in fibrinogen and are exposed in fibrin due to conformational changes during formation of fibrin polymers [50], [51], [52]. It is also known that the major binding sites for plasminogen and tPA are located in the D regions of fibrin [51] and the additional binding sites for these components of fibrinolytic system are located in the αC-domains [51,53]. To evaluate the involvement of individual sub-domains of the αC-domains in the activation of fibrinolytic system, we studied generation of plasmin activity in the activation complex formed by plasminogen, tPA, and different forms of fibrin. Since both truncated forms of fibrin contained the major tPA- and plasminogen-binding sites, they both stimulated generation of plasmin although to the lesser extent than native fibrin due to the absence of their additional binding sites located in the αC-domains. Importantly, the stimulating effects of both truncated forms were very similar in spite of the absence of the N-terminal sub-domains in fibrinogen desАα414–610. This finding indicates that the tPA- and plasminogen-binding sites are located within the C-terminal sub-domains of the αC-domains.

Another process that strongly depends on fibrinogen is platelet aggregation. Previous studies revealed that fibrinogen γ chain residues γ400–411 and the Aα chain residues Аα95–97 and Аα572–574 containing Arg-Gly-Asp motif play an important role in platelet aggregation [1]. In our study, proteolytic removal of the Aα505–610 portions of the αС-domains, i. e. their C-terminal sub-domains, slightly suppressed the ability of fibrinogen desАα505–610 to support platelet aggregation (Fig. 6A), most likely due to the loss of the Аα572–574 Arg-Gly-Asp motif located in these sub-domains (Fig. 10). At the same time, we found that the ability of fibrinogen desАα414–610 to support platelet aggregation was significantly reduced (Fig. 6B). The most probable explanation for this finding is the following. It is known that Lys-Gly-Asp motif, which is located in the N-terminal sub-domains (Fig. 10), can interact with platelet receptor αIIbβ3 [54]; therefore, it is most likely that the absence of these sub-domains in fibrinogen desАα414–610 results in the significant reduction of platelet aggregation. Altogether, our experiments indicate that both sub-domains of the αC-domains are involved in platelet aggregation and the N-terminal sub-domains play a more significant role in this process.

Previous studies revealed that fibrin(ogen) interacts with endothelial cells through various receptors including the VLDL receptor, VE-cadherin, integrin receptors αvβ3 and α5β1, etc., and such interactions play an important role in fibrin-induced inflammation, angiogenesis, and other important physiological and pathological processes [40,[55], [56], [57]].. Our study revealed a significant decrease in viability of endothelial cells (MAEC) grown on both truncated forms of fibrin. Namely, the removal of the C-terminal domains from fibrin resulted in a significant decrease of the viability (Fig. 7). Importantly, further removal of the N-terminal sub-domains did not change this effect suggesting that interaction of fibrin with endothelial cells is mediated mainly by the C-terminal domains, most probably through their Arg-Gly-Asp-motif (Fig. 10). Similarly, the C-terminal sub-domains of the αC-domains play the major role in HeLa cell migration observed in our scratch test.

Despite the numerous intermolecular interactions of different sub-domains of αC-domains that were demonstrated, their exact role in vivo remains to be addressed [41]. It is important to underline that findings presented in the current work can contribute the understanding of the role of αC-domains in various (patho)physiological conditions accompanied by the disturbance of fibrin clot structure including clotting abnormalities or amyloid deposits [58].

5. Conclusion

In the present study, in which we utilized the truncated fibrin(ogen) forms lacking either its C-terminal sub-domains of the αC-domains or both the N- and C-terminal sub-domains, we distinguished the involvement of these sub-domains in several fibrin(ogen)-dependent processes. Specifically, the results obtained revealed that the N- and C-terminal sub-domains both play an important role in fibrin polymerization. These sub-domains also play an important role in platelet aggregation with the N-terminal sub-domains playing a more significant role in this process. At the same time, the C-terminal sub-domains make the major contribution to the plasminogen activation process, endothelial cell viability, and migration of cancer cells. These findings may contribute to the development of molecular effectors able to regulate such important physiological and pathological processes as blood coagulation, wound healing, and tumorigenesis.

CRediT authorship contribution statement

Y.M.S: manuscript preparation, methodology, preparation of fibrinogen forms, turbidity measurements and aggregometry; T.A.Y: fibrinolysis study; V.V.N: cell cultivation; Y.P.K: SPR study; O.O.H: visualization and data analysis; O.Yu.S: chromatography, protein purification; K.S.S: MTT test, cell migration test; L.V.G: endothelial cells cultivation; L.D.V: preparation of the proteases; A.O.T: fibrinolysis study; V.O.C: methodology, concept of the article, review and editing, supervision.

Funding

This study was supported by the research program of National Academy of Sciences of Ukraine for research laboratories/groups of young scientists of the National Academy of Sciences of Ukraine for conducting research in the priority directions of the development of science and technology in 2022–2023. ‘Identification of sites of intermolecular interactions of fibrin(ogen) molecule #0122U002132.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

  • No data was used for the research described in the article.

References

  • 1.Mosesson M.W. Fibrinogen and fibrin structure and functions. J. Thromb Haemost. 2005;3:1894–1904. doi: 10.1111/j.1538-7836.2005.01365.x. [DOI] [PubMed] [Google Scholar]
  • 2.Weisel J.W., Litvinov R.I. Fibrin formation, structure and properties. Subcell. Biochem. 2017;82:405–456. doi: 10.1007/978-3-319-49674-0_13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Plow E.F., E.Ruoslahti M.H.Ginsberg, M.D.Pierschbacher G.Marguerie. Arginyl-Glycyl-Aspartic acid sequences and fibrinogen binding to platelets. Blood. 1987;70:110–115. [PubMed] [Google Scholar]
  • 4.Podolnikova N.P., Gorkun O.V., Loreth R.M., Yee V.C., Lord S.T., Ugarova T.P. A cluster of basic amino acid residues in the γ370-381 sequence of fibrinogen comprises a binding site for platelet integrin α(IIb)β3 (glycoprotein IIb/IIIa) Biochemistry. 2005;44:16920–16930. doi: 10.1021/bi051581d. [DOI] [PubMed] [Google Scholar]
  • 5.Rivera J., Lozano M.L., Navarro-Núñez L., Vicente V. Platelet receptors and signaling in the dynamics of thrombus formation. Haematologica. 2009;94:700–711. doi: 10.3324/haematol.2008.003178. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Sporn L.A., Bunce L.A., Francis C.W. Cell proliferation on fibrin: modulation by fibrinopeptide cleavage. Blood. 1995;86:1801–1810. [PubMed] [Google Scholar]
  • 7.Yakovlev S., Mikhailenko I., Tsurupa G., Belkin A.M., Medved L. Polymerization of fibrin αC-domains promotes endothelial cell migration and proliferation. Thromb. Hatmost. 2014;112:1244–1251. doi: 10.1160/TH14-01-0079. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Flick M.J., Du X., Witte D.P., Jirouskova M., Soloviev D.A., Busuttil S.J., Plow E.F., Degen J.L. Leukocyte engagement of fibrin(ogen) via the integrin receptor αMβ2/Mac-1 is critical for host inflammatory response in vivo. J. Clin. Investig. 2004;113:1596–1606. doi: 10.1172/JCI20741. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Sen U., Tyagi N., Patibandla P.K., Dean W.L., Tyagi S.C., Roberts A.M., Lominadze D. Fibrinogen-induced endothelin-1 production from endothelial cells. Am. J. Physiol. Cell Physiol. 2009;296:840–847. doi: 10.1152/ajpcell.00515.2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Tyagi N., Roberts A.M., Dean W.L., Tyagi S.C., Lominadze D. Fibrinogen induces endothelial cell permeability. Mol. Cell. Biochem. 2008;307:13–22. doi: 10.1007/s11010-007-9579-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Tsurupa G., Mahid A., Veklich Y., Weisel J.W., Medved L. Structure, stability, and interaction of fibrin αc-domain polymers. Biochemistry. 2011;50:8028–8037. doi: 10.1021/bi2008189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Weisel J.W., Medved L. The structure and function of the αC Domains of fibrinogen. Ann. N. Y. Acad. Sci. 2001;936:312–327. doi: 10.1111/j.1749-6632.2001.tb03517.x. [DOI] [PubMed] [Google Scholar]
  • 13.Litvinov R.I., Yakovlev S., Tsurupa G., Gorkun O.V., Medved L., Weisel J.W. Direct evidence for specific interactions of the fibrinogen αC-domains with the central e region and with each other. Biochemistry. 2007;46:9133–9142. doi: 10.1021/bi700944j. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Hudson N.E. Biophysical mechanisms mediating fibrin fiber lysis. BioMed Res. Int. 2017 doi: 10.1155/2017/2748340. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Plow E.F., Ruoslahti E., Ginsberg M.H., Pierschbacher M.D., Marguerie G. Arginyl-Glycyl-Aspartic acid sequences and fibrinogen binding to platelets. Blood. 1987;70:110–115. [PubMed] [Google Scholar]
  • 16.Suehiro Т.К., Mizuguchi J., Nishiyama K., Iwanaga S., Farrell D.H. Fibrinogen binds to integrin α5β1 via the carboxyl-terminal RGD site of the Aα-chain. J. Biochem. 2000;128:705–710. doi: 10.1093/oxfordjournals.jbchem.a022804. [DOI] [PubMed] [Google Scholar]
  • 17.Gornitskaia O.V., Rovinskaya I.N., Platonova T.N. Purification and characterization of the fibrinolytic enzyme from Agkistrodon halys halys venom. Ukr. Biokhim. Zh. 2002;74:42–49. (1999) [PubMed] [Google Scholar]
  • 18.Stohnii Ye.M., Rebriev A.V., Hornytska O.V., Slominskiy O.Yu., Kostiuchenko O.P., Klymenko K.P., Chernyshenko V.O. Directed proteolysis of fibrinogen by protease of Gloydius halys halys venom. Biotechnol. Acta. 2021;14:37–43. doi: 10.15407/biotech14.06.037. [DOI] [Google Scholar]
  • 19.Nidialkova N.А., Varbanets L.D., Chernyshenko V.O. Isolation and purification of Bacillus thuringiensis var. israelensis IМV В-7465 peptidase with specificity toward elastin and collagen. Ukr. Biochem. J. 2016;88:18–28. doi: 10.15407/ubj88.03.018. [DOI] [PubMed] [Google Scholar]
  • 20.Stohniі E.M., Chernyshenko V.O., Nidialkova N.A., Rebriev A.V., Varbanets L.D., Hadzhynova V.E., Chernyshenkо T.M., Kolesnikova I.M., Lugovskoy E.V. Mapping of residues of fibrinogen cleaved by protease II of Bacillus thuringiensis var. israelensis IMV B-7465. Ukr. Biochem. J. 2016;88:79–86. doi: 10.15407/ubj88.03.018. [DOI] [PubMed] [Google Scholar]
  • 21.Urvant L.P., Makogonenko Е.М., Pozniak Т.А., Pydiura N.А., Kolesnikova I.N., Tsap P.Y., Bereznitzkiy G.К., Lugovskoy E.V., Komisarenko S.V. Binding of mAb II-5c to Aα20–78 fragment of fibrinogen inhibits a neoantigenic determinant exposure within Bβ126–135 site of a molecule. Rep. NAS. 2014;5:149–156. doi: 10.15407/dopovidi2014.05.149. [DOI] [Google Scholar]
  • 22.Lugovskoy E.V., Chudnovets V.S., Makogonenko E.M., Derzskaia S.G., Gogolinskaia G.K., Kolesnikova I.N., Mikhalovskaia L.I., Komissarenko S.V. Study of the polymerization of fibrin using monoclonal antibodies 2D-2A and their Fab-fragments. Ukr. Biokhim. Zh. 1995;67:64–70. Russian. PMID: 8588256. [PubMed] [Google Scholar]
  • 23.Yatsenko T.A., Kharchenko S.M. Polyclonal antibodies against human plasminogen: purification, characterization and application. Biotechnol. Acta. 2020;13:50–57. doi: 10.15407/biotech13.06.050. [DOI] [Google Scholar]
  • 24.Yusova E.I., Grinenko T.V., Goncharenko T.O., Kapustyanenko L.G., Sobchenko A.A., Tykhomyrov A.A. Production and characterization of antibodies to tissue plasminogen activator: application for the platelet flow cytometry assay. Biotechnol. Acta. 2020;13:63–72. doi: 10.15407/biotech13.05.062. [DOI] [Google Scholar]
  • 25.Iskandarov E., Gryshchuk V., Platonov O., Kucheriavyi Y., Slominskyi O., Stohnii Y., Vartanov V., Chernyshenko V. Fractionation of Vipera berus berus snake venom and detection of bioactive compounds targeted to blood coagulation system. Southeast. Eur. Med. J. 2022;6:20–31. doi: 10.26332/seemedj.v6i2.256. [DOI] [Google Scholar]
  • 26.Summaria L., Spitz F., Arzadon L., Boreisha I.G., Robbins K.C. Isolation and characterization of the affinity chromatography forms of human Glu- and Lys-plasminogens and plasmins. J. Biol. Chem. 1976;251:3693–3699. PMID: 132440. [PubMed] [Google Scholar]
  • 27.Platonova T.N., Sushko O.O., Colovĭov D.O., Iena Ia.M. Determination of fibrinogen content in human blood plasma using thrombin-like enzyme of ancistron N and analysis of hemostasis under presence of blood coagulation inhibitors. Fiziol. Zh. 1993;39:15–19. [PubMed] [Google Scholar]
  • 28.Chernyshenko V., Shteinberg K., Lugovska N., Ryzhykova M., Platonova T., Korolova D., Lugovskoy E. Preparation of highly-concentrated autologous platelet-rich plasma for biomedical use. Ukr. Biochem. J. 2019;91:19–27. doi: 10.15407/ubj91.02.019. [DOI] [Google Scholar]
  • 29.Bastaki M., Nelli E.E., Dell'Era P., Rusnati M., Molinari-Tosatti M.P., Parolini S., Auerbach R., Ruco L.P., Possati L., Presta M. Basic fibroblast growth factor-induced angiogenic phenotype in mouse endothelium. A study of aortic and microvascular endothelial cell lines. Arterioscler. Thromb. Vasc. Biol. 1997;17:454–464. doi: 10.1161/01.atv.17.3.454. [DOI] [PubMed] [Google Scholar]
  • 30.Varetskaia V. Preparation of a fibrin monomer and studies on some of its properties. Ukr. Biokhim. Zh. 1965;37:194–206. [PubMed] [Google Scholar]
  • 31.Chaurand P., Luetzenkirchen F., Spengler B. Peptide and protein identification by matrix-assisted laser desorption ionization (MALDI) and MALDI-post-source decay time-of-flight mass spectrometry. J. Am. Soc. Mass Spectrom. 1999;10:91–103. doi: 10.1016/S1044-0305(98)00145-7. [DOI] [PubMed] [Google Scholar]
  • 32.Weisel J.W., Nagaswami C. Computer modeling of fibrin polymerization kinetics correlated with electron microscope and turbidity observations: clot structure and assembly are kinetically controlled. Biophys. J. 1992;63:111–128. doi: 10.1016/S0006-3495(92)81594-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Li X.M., Huskens J., Reinhoudt D.N. Reactive self-assembled monolayers on flat and nanoparticle surfaces, and their application in soft and scanning probe lithographic nanofabrication technologies. J. Mater. Chem. 2004;14:2954–2971. doi: 10.1039/B406037G. [DOI] [Google Scholar]
  • 34.Müller M.F., Ris H., Ferry J.D. Electron microscopy of fine fibrin clots and fine and coarse fibrin films. Observations of fibers in cross-section and in deformed states. J. Mol. Biol. 1984;174:369–384. doi: 10.1016/0022-2836(84)90343-7. [DOI] [PubMed] [Google Scholar]
  • 35.Cattaneo M., Cerletti C., Harrison P., Hayward C.P., Kenny D., Nugent D., Nurden P., Rao A.K., Schmaier A.H., Watson S.P., Lussana F., Pugliano M.T., Michelson A.D. Recommendations for the standardization of light transmission aggregometry: a consensus of the working party from the Platelet Physiology Subcommittee of SSC/ISTH. J. Thromb. Haemost. 2013;11:1183–1189. doi: 10.1111/jth.12231. [DOI] [PubMed] [Google Scholar]
  • 36.Gao W., Chen S.-.R., Li H. Methylprednisolone exerts neuroprotective effects by regulating autophagy and apoptosis. Neural Regen. Res. 2016;11:823–828. doi: 10.4103/1673-5374.182711. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Liang C., Park A., Guan J. In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nat. Protoc. 2007;2:329–333. doi: 10.1038/nprot.2007.30. [DOI] [PubMed] [Google Scholar]
  • 38.Mouritzen V.M., Jenssen H. Optimized scratch assay for In vitro testing of cell migration with an automated optical camera. J. Vis. Exp. 2018;138:57691. doi: 10.3791/57691. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Zhmurov A., Protopopova A.D., Litvinov R.I., Zhukov P., Weisel J.W., Barsegov V. Atomic structural models of fibrin oligomers. Structure. 2018;26:857–868. doi: 10.1016/j.str.2018.04.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Koo J., Rafailovich M.H., Medved L., Tsurupa G., Kudryk B.J., Liu Y., Galanakis D.K. Evaluation of fibrinogen self-assembly: role of its αC region. J. Thromb. Haemost. 2010;8:2727–2735. doi: 10.1111/j.1538-7836.2010.04072.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Medved L., Weisel J.W. The Story of the fibrin(ogen) αC-domains: evolution of our view on their structure and interactions. Thromb. Haemost. 2022;122:1265–1278. doi: 10.1055/a-1719-5584. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Miles L.A., Ny L., Wilczynska M., Shen Y., Ny T., Parmer R.J. Plasminogen receptors and fibrinolysis. Int. J. Mol. Sci. 2021;22:1712. doi: 10.3390/ijms22041712. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Xu X.R., Wang Y., Adili R., Ju L., Spring C.M., Jin J.W., Yang H., Neves M.A.D., Chen P., Yang Y., Lei X., Chen Y., Gallant R.C., Xu M., Zhang H., Song J., Ke P., Zhang D., Carrim N., Si-Yang Y., Zhu G., Yi-Min S., Cyr T., Fu W., Liu G., Connelly P.W., Rand M.L., Adeli K., Freedman J., Lee J.E., Tso P., Marchese P., Davidson W.S., Jackson S.P., Zhu C., Ruggeri Z.M., Ni H. Apolipoprotein A-IV binds αIIbβ3 integrin and inhibits thrombosis. Nat. Commun. 2018;9:3608. doi: 10.1038/s41467-018-05806-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Yakovlev S., Mikhailenko I., Tsurupa G., Belkin A.M., Medved L. Polymerization of fibrin αC-domains promotes endothelial cell migration and proliferation. Thromb. Hatmost. 2014;112:1244–1251. doi: 10.1160/TH14-01-0079. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Brown A.C., Barker T.H. Fibrin-based biomaterials: modulation of macroscopic properties through rational design at the molecular level. Acta Biomater. 2014;10:1502–1514. doi: 10.1016/j.actbio.2013.09.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Kwaan H.C., Lindholm P.F. Fibrin and fibrinolysis in cancer. Semin. Thromb. Hemost. 2019;45:413–422. doi: 10.1055/s-0039-1688495. [DOI] [PubMed] [Google Scholar]
  • 47.Tsurupa G., Hantgan R.R., Burton R.A., Pechik I., Tjandra N., Medved L. Structure, stability, and interaction of the fibrin(ogen) αC-Domains. Biochemistry. 2009;48:12191–12201. doi: 10.1021/bi901640e. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Burton R.A., Tsurupa G., Hantgan R.R., Tjandra N., Medved L. NMR solution structure, stability, and interaction of the recombinant bovine fibrinogen αC-Domain fragment. Biochemistry. 2007;46:8550–8560. doi: 10.1021/bi700606v. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Tsurupa G., Pechik I., Litvinov R.I., Hantgan R.R., Tjandra N., Weisel J.W., Medved L. On the mechanism of αC polymer formation in fibrin. Biochemistry. 2012;51:2526–2538. doi: 10.1021/bi2017848. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Yakovlev S., Makogonenko E., Kurochkina N., Nieuwenhuizen W., Ingham K., Medved L. Conversion of fibrinogen to fibrin: mechanism of exposure of tPA- and plasminogen-binding sites. Biochemistry. 2000;39:15730–15741. doi: 10.1021/bi001847a. [DOI] [PubMed] [Google Scholar]
  • 51.Medved L., Tsurupa G., Yakovlev S. Conformational changes upon conversion of fibrinogen into fibrin. Ann. N. Y. Acad. Sci. 2001;936:185–204. doi: 10.1111/j.1749-6632.2001.tb03505.x. [DOI] [PubMed] [Google Scholar]
  • 52.Medved L., Nieuwenhuizen W. Molecular mechanisms of initiation of fibrinolysis by fibrin. Thromb. Haemost. 2003;89:409–419. doi: 10.1055/s-0037-1613368. [DOI] [PubMed] [Google Scholar]
  • 53.Tsurupa G., Medved L. Identification and characterization of novel tPA- and plasminogen-binding sites within fibrin(ogen) alpha C-domains. Biochemistry. 2001;40:801–808. doi: 10.1021/bi001789t. [DOI] [PubMed] [Google Scholar]
  • 54.Reiss S., Sieber M., Oberle V., Wentzel A., Spangenberg P., Claus R., Kolmar H., Lösche W. Inhibition of platelet aggregation by grafting RGD and KGD sequences on the structural scaffold of small disulfide-rich proteins. Platelets. 2006;17:153–157. doi: 10.1080/09537100500436663. [DOI] [PubMed] [Google Scholar]
  • 55.Martinez J., Ferber A., Bach T.L., Yaen C.H. Interaction of Fibrin with VE-Cadherin. Ann. N. Y. Acad. Sci. 2001;936:386–405. doi: 10.1111/j.1749-6632.2001.tb03524.x. [DOI] [PubMed] [Google Scholar]
  • 56.Yakovlev S., Strickland D.K., Medved L. Current view on the molecular mechanisms underlying fibrin(ogen)-Dependent inflammation. Thromb. Haemost. 2022;122:1858–1868. doi: 10.1055/a-1910-4538. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 57.Lominadze D., Dean W.L., Tyagi S.C., Roberts A.M. Mechanisms of fibrinogen-induced microvascular dysfunction during cardiovascular disease. Acta Physiol. (Oxf). 2010;198:1–13. doi: 10.1111/j.1748-1716.2009.02037.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 58.Soria J., Mirshahi S., Mirshahi S.Q., Varin R., Pritchard L.L., Soria C., Mirshahi M. Fibrinogen αC domain: its importance in physiopathology. Res. Pract. Thromb. Haemost. 2019;3:173–183. doi: 10.1002/rth2.12183. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

  • No data was used for the research described in the article.

Articles from BBA Advances are provided here courtesy of Elsevier

RESOURCES