Citrullination is a post-translational protein modification, which is associated with inflammation in general and is thought to play an important pathogenic role in rheumatoid arthritis (RA). Here a mass spectrometry-based proteomics approach was applied to identify citrullination sites in synovial fluid fibrinogen from four RA patients. In general, high disease activity correlated with increased number of identified citrullination sites and higher relative citrulline occupancy. Altogether, 23 sites were identified, of which 9 have not been previously reported to be citrullinated in vivo. Citrullination at site α84, α123, α129, α547, α573, α591, β334 and γ134 was identified in more than one patient, and these positions were therefore regarded as hotspots. Following citrullination of fibrinogen in vitro using human recombinant peptidylarginine deiminase 2 (PAD2), a total of 46 citrullination sites were identified, including 6 hitherto unreported in vitro citrullination sites. Twenty-two out of the 23 citrullination sites identified in vivo were also detected in vitro, supporting the validity of the identifications. SIGNIFICANCE: This work provides information about previously uncharacterized citrullination sites in synovial fluid fibrinogen from rheumatoid arthritis patients. Detection of these novel citrullination sites may prove to have diagnostic or prognostic value in RA and enhance our understanding of the immune pathogenesis.

Increased vascular permeability (VP) has been indicated to play an important role in the pathogenesis of inflammatory bowel disease (IBD). However, the pathological causes of increased intestinal VP in IBD remain largely unknown.

How cancer cells evade the therapeutic effects of immune checkpoint blockade is largely unknown. Here, we report that fibrinogen-like protein 1 (FGL1), a newly identified immune checkpoint ligand, was modified by acetylation at Lys 98 in hepatocellular carcinoma (HCC), which targeted it for proteasomal degradation. Sirtuin 2 (SIRT2) deacetylated and stabilized FGL1, thus promoting immune evasion. Notably, the SIRT2 inhibitor 2-Cyano-3-[5-(2,5-dichlorophenyl)-2-furanyl]-N-5-quinolinyl-2-propenamide (AGK2) enhanced acetylation of FGL1 and reduced FGL1 protein levels in vitro. The combination of AGK2 and programmed death ligand 1 (PD-L1) blockade effectively suppressed tumor growth and improved overall survival of mice. Furthermore, aspirin, an old drug, could directly acetylate FGL1 at Lys 98 and promote its degradation in vitro. Aspirin enhanced the immunotherapeutic efficacy, induced tumor regression, and extended the lifespan of tumor-bearing mice. Furthermore, the SIRT2/FGL1 axis was expressed in HCC specimens. Collectively, these findings unveil an acetylation-mediated regulation of FGL1, identify a potential target for HCC immunotherapy, and provide therapeutic strategies for the clinical treatment of HCC.

Fibrin formation and structure may be affected by a plethora of factors, including both genetic and posttranslational modifications, such as glycation, nitration or acetylation.

Angiogenesis is a prerequisite of tumor growth and metastasis and, thus, anti-angiogenesis treatment has become an important part of cancer therapy. A 15-amino acid peptide of the fibrinogen α chain, fibrinostatin, was previously found in serum samples of gastric cancer patients. Herein we demonstrated that fibrinostatin has anti-angiogenesis activity in several angiogenesis models and it reduces tumor growth in mouse xenografts and allografts. Increased tumor necrosis and reduced microvessel density in tumors were observed in mouse xenograft models. Fibrinostatin inhibited proliferation and induced apoptosis in HUVEC, but not in cancer cells. In addition, fibrinostatin specifically entered HUVEC. Fibrinostatin also prevented migration, adhesion and tubule formation of HUVEC in vitro. A single-dose acute toxicity testing and a repeated-dose chronic toxicity study in the mouse, rat and monkey indicated that fibrinostatin had a wide margin of safety. Taken together, fibrinostatin shows promise as a potential anti-angiogenesis therapeutic agent.

The two coagulases, von Willebrand factor binding protein (vWbp) and Coagulase (Coa), are critical virulence factors in several animal models of invasive Staphylococcus aureus (S. aureus) infections. These proteins are part of an intricate system of proteins that S. aureus uses to assemble a fibrinogen (Fg)/fibrin protective shield surrounding itself. This shield allows the microorganism to evade clearance by the host phagocytic cells. The coagulases can non-proteolytically activate the zymogen prothrombin to convert Fg to fibrin and promote the Fg/fibrin shield formation. The coagulases also bind directly to Fg and the interaction between Coa and Fg has been previously characterized in some detail. However, the mechanism(s) by which vWbp interacts with Fg remains unclear. Here, we show that vWbp and Coa have distinct interactions with Fg, despite being structurally similar. Coa binds with a significantly higher affinity to soluble Fg than to Fg coated on a plastic surface, whereas vWbp demonstrates no preference between the two forms of Fg. The two coagulases appear to target different sites on Fg, as they do not compete with each other in binding to Fg. Similar to Coa, both the N- and C-terminal halves of vWbp (vWbp-N, vWbp-C, respectively) harbor Fg-binding activities. The higher affinity Fg-binding activity resides in vWbp-N; whereas, the C-terminal region of Coa encompasses the major Fg-binding activity. Peptides constituting the previously identified Coa/Efb1 Fg-binding motif fail to inhibit vWbp-C from binding to Fg, indicating that vWbp-C lacks a functional homolog to this motif. Interestingly, the N-terminal prothrombin-binding domains of both coagulases recognize the Fg β-chain, but they appear to interact with different sequence motifs in the host protein. Collectively, our data provide insight into the complex interactions between Fg and the S. aureus coagulases.

Fibrinogen is one of the plasma proteins most susceptible to oxidative modification. It has been suggested that modification of fibrinogen may cause thrombotic/bleeding complications associated with many pathophysiological states of organism. We exposed fibrinogen molecules to three different modification reagents-malondialdehyde, sodium hypochlorite, and peroxynitrite-that are presented to various degrees in different stages of oxidative stress. We studied the changes in fibrin network formation and platelet interactions with modified fibrinogens under flow conditions. The fastest modification of fibrinogen was caused by hypochlorite. Fibers from fibrinogen modified with either reagent were thinner in comparison with control fibers. We found that platelet dynamic adhesion was significantly lower on fibrinogen modified with malondialdehyde and significantly higher on fibrinogen modified either with hypochlorite or peroxynitrite reflecting different prothrombotic/antithrombotic properties of oxidatively modified fibrinogens. It seems that, in the complex reactions ongoing in living organisms at conditions of oxidation stress, hypochlorite modifies proteins (e.g., fibrinogen) faster and more preferentially than malondialdehyde. It suggests that the prothrombotic effects of prior fibrinogen modifications may outweigh the antithrombotic effect of malondialdehyde-modified fibrinogen in real living systems.

Fibrinogen, a protein involved in blood coagulation, is very susceptible to oxidation. Oxidation alters its function and usually makes it more thrombogenic. Bilirubin, an end-product of the haem degradation in vertebrates, is known for its antioxidant properties. The present paper describes interaction between fibrinogen and bilirubin, and the influence of bilirubin on the formation of fibrin and protection against oxidation. The binding constant of 4.5 × 104 M-1 was determined for the fibrinogen/bilirubin complex at 37 °C. There is no change in secondary and tertiary structure of fibrinogen or its thermal stability upon bilirubin binding. The binding site of fibrinogen is not stereospecific for bilirubin and is able to accommodate both bilirubin conformers. A change in absorption maximum of bilirubin occurs upon its interaction with fibrinogen, suggesting an alteration in the conformation of bilirubin to the more cyclic one. Bilirubin exerts antioxidant effect on fibrinogen, preventing its carbonylation and aggregation. The presence of bilirubin induces the formation of fibrin with thicker fibres, as assessed by the coagulation assay. Fibrinogen and bilirubin interact at physiological concentrations, bilirubin may act as an antioxidant for fibrinogen and may modulate an important event in haemostasis, which altogether suggests possible physiological relevance of this interaction.

Critical illness leads to rapid fibrinogen consumption, hyperfibrinolysis, and coagulopathy that exacerbates bleeding and increases mortality. Immune cell activation and inflammation are associated with coagulopathy after injury but play an undetermined role. We performed high dimensional immunophenotyping and single-cell imaging flow cytometry to investigate for a pathophysiological mechanism governing the effects of leukocyte-associated inflammation on fibrinogen function. Fibrinogen was oxidized early, followed by its degradation after 3 hours of lipopolysaccharides (LPS)-induced sterile inflammation in a rat model in vivo. Fibrinogen incubated with human leukocytes activated by TNFα was similarly oxidized, and later proteolyzed after 3 hours in vitro. TNFα induced mitochondrial superoxide generation from neutrophils and monocytes, myeloperoxidase (MPO)-derived reactive oxygen species (ROS) from neutrophils, and nitric oxide from lymphocytes and monocytes. Inhibition of mitochondrial superoxide prevented oxidative modification and proteolysis of fibrinogen, whereas inhibition of MPO attenuated only fibrinogen proteolysis. Quenching of both mitochondrial superoxide and MPO-derived ROS prevented coagulopathy better than tranexamic acid. Collectively, these findings indicate that neutrophil and monocyte mitochondrial superoxide generation can rapidly oxidize fibrinogen as a priming step for fibrinogen proteolysis and coagulopathy during inflammation.

Platelet Mapping can measure both the degree of platelet inhibition and fibrinogen activation, was not originally designed to measure fibrinogen concentration. Traditional laboratory fibrinogen concentration testing requires around 60 minutes; however, fibrinogen activation only takes 10 minutes, and is indicated as maximum amplitude of activator f. If Platelet Mapping can predict fibrinogen concentration during cardiopulmonary bypass, this could facilitate rapid hemostasis management. The aim of this study was to verify whether fibrinogen concentration could be predicted using Platelet Mapping results. Thus, a pilot study was conducted to evaluate this concept during cardiopulmonary bypass. This prospective, observational pilot study investigated 15- to 90-year-old patients who underwent cardiac or aortic surgery from August 2019 to September 2019. Twenty-one patients enrolled in this study, and 43 blood samples were obtained for both fibrinogen activation measurements using Platelet Mapping and traditional laboratory-based tests, respectively. Correlations between results were analyzed using linear regression and the receiver operating characteristic curve. Correlation by Pearson's correlation analysis indicates a significant relationship (correlation coefficient of r = 0.91), and a receiver operating characteristic curve indicated that sensitivity, specificity, and receiver operating characteristic area were 100% (95% confidence interval, 75.3-100%), 93.8% (79.2-99.2%), and 0.995 (0.984-1.00), respectively. Our results indicate a strong correlation between fibrinogen activation and serum fibrinogen concentration. The maximum amplitude of activator f can estimate low fibrinogen concentration faster than traditional methods; this method quickly provides important information for anesthesia and hemostatic management in cardiac surgery.

There is very limited of evidence linking fibrinogen and bone mineral density (BMD) in postmenopausal women. Therefore, this study intended to examine the relationship between fibrinogen and total BMD in postmenopausal women.

Fibrinogen Caracas II is an abnormal fibrinogen involving the mutation of A alpha serine 434 to N-glycosylated asparagine. Some effects of this mutation on the ultrastructure of fibrinogen Caracas II molecules, fibers, and clots were investigated by electron microscopy. Electron microscopy of rotary shadowed individual molecules indicated that most of the alphaC domains of fibrinogen Caracas II do not interact with each other or with the central domain, in contrast to control fibrinogen. Negatively contrasted Caracas II fibers were thinner and less ordered than control fibers, and many free fiber ends were observed. Scanning electron microscopy of whole clots revealed the presence of large pores bounded by local fiber networks made up of thin fibers. Permeation experiments also indicated that the average pore diameter was larger than that of control clots. The viscoelastic properties of the Caracas II clot, as measured by a torsion pendulum, were similar to those of control clots. Both the normal stiffness and increased permeability of the Caracas II clots are consistent with the observation that subjects with this dysfibrinogenemia are asymptomatic.

Platelets and megakaryocytes are critical players in immune responses. Recent reports suggest infection and inflammation alter the megakaryocyte and platelet transcriptome to induce altered platelet reactivity. We determined whether nonviral sepsis induces differential platelet gene expression and reactivity. Nonviral sepsis upregulated IFN-induced transmembrane protein 3 (IFITM3), an IFN-responsive gene that restricts viral replication. As IFITM3 has been linked to clathrin-mediated endocytosis, we determined whether IFITM3 promoted endocytosis of α-granule proteins. IFN stimulation enhanced fibrinogen endocytosis in megakaryocytes and platelets from Ifitm+/+ mice, but not Ifitm-/- mice. IFITM3 overexpression or deletion in megakaryocytes demonstrated IFITM3 was necessary and sufficient to regulate fibrinogen endocytosis. Mechanistically, IFITM3 interacted with clathrin and αIIb and altered their plasma membrane localization into lipid rafts. In vivo IFN administration increased fibrinogen endocytosis, platelet reactivity, and thrombosis in an IFITM-dependent manner. In contrast, Ifitm-/- mice were completely rescued from IFN-induced platelet hyperreactivity and thrombosis. During murine sepsis, platelets from Ifitm+/+ mice demonstrated increased fibrinogen content and platelet reactivity, which was dependent on IFN-α and IFITMs. Platelets from patients with nonviral sepsis had increases in platelet IFITM3 expression, fibrinogen content, and hyperreactivity. These data identify IFITM3 as a regulator of platelet endocytosis, hyperreactivity, and thrombosis during inflammatory stress.

Cardiomyocytes exposed to inflammatory processes express intracellular adhesion molecule-1 (ICAM-1). We investigated whether fibrinogen and fibrinogen degradation products, including D-dimer, could alter cardiomyocyte contractile function through interaction with ICAM-1 found on inflamed cardiomyocytes.

Protein micropatterning allows proteins to be precisely deposited onto a substrate of choice and is now routinely used in cell biology and in vitro reconstitution. However, drawbacks of current technology are that micropatterning efficiency can be variable between proteins and that proteins may lose activity on the micropatterns. Here, we describe a general method to enable micropatterning of virtually any protein at high specificity and homogeneity while maintaining its activity. Our method is based on an anchor that micropatterns well, fibrinogen, which we functionalized to bind to common purification tags. This enhances micropatterning on various substrates, facilitates multiplexed micropatterning, and dramatically improves the on-pattern activity of fragile proteins like molecular motors. Furthermore, it enhances the micropatterning of hard-to-micropattern cells. Last, this method enables subcellular micropatterning, whereby complex micropatterns simultaneously control cell shape and the distribution of transmembrane receptors within that cell. Altogether, these results open new avenues for cell biology.

Hereditary fibrinogen Aα-chain (AFib) amyloidosis is a relatively uncommon renal disease associated with a small number of pathogenic fibrinogen Aα (FibA) variants; wild-type FibA normally does not result in amyloid deposition. Proteomics is now routinely used to identify the amyloid type in clinical samples, and we report here our algorithm for identification of FibA in amyloid.

Fibrin is considered a highly promising biomaterial for manifold medical applications. Although it is a well-established material in this field, the required enzyme thrombin bears some striking downsides such as high costs and health risks. Current research discovers more and more ways to use fibrin's precursor fibrinogen as a substitute. Fibrinogen's full potential is, however, only retained when using it as fibrous gel, as it is the case for fibrin. In our previous work, we introduced such a kind of material for the first time. This material, called pseudo-fibrin, shows striking similarities to fibrin regarding its supramolecular structure and is created in a facile salt-induced process, which we further improved in this study. In particular, we shine light on the role of Ca2+ in pseudo-fibrin buildup, which turned out to drastically improve the outcome. Never before has it been observed that Ca2+ can induce fibrillogenesis and the gelation of native, enzyme-free fibrinogen. Enzyme catalysis was ruled out by the addition of thrombin and factor XIII inhibitors. Even more striking, Ca2+ induces gelation even under physiological conditions, leading again to stable and fibrous hydrogels. Although this latter approach is possibly co-induced by residual factor XIII, the resulting gels are for the first time recognized as promising materials and not discounted as unwanted side effects. The finding that these gels again consist of fibers especially renders a new perspective on the role of factor XIII and fibrinogen's well-known Ca2+ binding sites. In this study, we aim to provide first insights into this highly feasible material and its characteristics.

Three-dimensional bioprinting has emerged as an attractive technology due to its ability to mimic native tissue architecture using different cell types and biomaterials. Nowadays, cell-laden bioink development or skin tissue equivalents are still at an early stage. The aim of the study is to propose a bioink to be used in skin bioprinting based on a blend of fibrinogen and alginate to form a hydrogel by enzymatic polymerization with thrombin and by ionic crosslinking with divalent calcium ions. The biomaterial ink formulation, composed of 30 mg/mL of fibrinogen, 6% of alginate, and 25 mM of CaCl2, was characterized in terms of homogeneity, rheological properties, printability, mechanical properties, degradation rate, water uptake, and biocompatibility by the indirect method using L929 mouse fibroblasts. The proposed bioink is a homogeneous blend with a shear thinning behavior, excellent printability, adequate mechanical stiffness, porosity, biodegradability, and water uptake, and it is in vitro biocompatible. The fibrinogen-based bioink was used for the 3D bioprinting of the dermal layer of the skin equivalent. Three different normal human dermal fibroblast (NHDF) densities were tested, and better results in terms of viability, spreading, and proliferation were obtained with 4 × 106 cell/mL. The skin equivalent was bioprinted, adding human keratinocytes (HaCaT) through bioprinting on the top surface of the dermal layer. A skin equivalent stained by live/dead and histological analysis immediately after printing and at days 7 and 14 of culture showed a tissuelike structure with two distinct layers characterized by the presence of viable and proliferating cells. This bioprinted skin equivalent showed a similar native skin architecture, paving the way for its use as a skin substitute for wound healing applications.

Thrombosis is a leading cause of morbidity and mortality. Fibrinogen, the soluble substrate for fibrin-based clotting, has a central role in haemostasis and thrombosis and its plasma concentration correlates with cardiovascular disease event risk and a prothrombotic state in experimental models. We aimed to identify chemical entities capable of changing fibrinogen production and test their impact on experimental thrombosis. A total of 1,280 bioactive compounds were screened for their ability to alter fibrinogen production by hepatocyte-derived cancer cells and a selected panel was tested in zebrafish larvae. Anthralin and all-trans retinoic acid (RA) were identified as fibrinogen-lowering and fibrinogen-increasing moieties, respectively. In zebrafish larvae, anthralin prolonged laser-induced venous- occlusion times and reduced thrombocyte accumulation at injury sites. RA had opposite effects. Treatment with RA, a nuclear receptor ligand, increased fibrinogen mRNA levels. Using an antisense morpholino oligonucleotide to deplete zebrafish fibrinogen, we correlated a shortening of laser-induced venous thrombosis times with RA treatment and fibrinogen protein levels. Anthralin had little effect on fibrinogen mRNA in zebrafish larvae, despite leading to lower detectable fibrinogen. Therefore, we made a proteomic scan of anthralin-treated cells and larvae. A reduced representation of proteins linked to the canonical secretory pathway was detected, suggesting that anthralin affects protein secretion. In summary, we found that chemical modulation of fibrinogen levels correlates with measured effects on experimental venous thrombosis and could be investigated as a therapeutic avenue for thrombosis prevention.

Non-genetic MMP-2 insufficiency is a relatively unexplored condition which could be induced by pathological overexpression of endogenous MMP-2 inhibitors such as TIMPs and/or the acute phase reactant alpha-2-macroglobulin. Here, we investigate the hypothesis that human fibrinogen (FBG) - an acute phase reactant - inhibits human MMP-2. Following an unexpected observation where sera from human donors including arthritis patients with increased levels of serum FBG exhibited reduced binding of serum proMMP-2 to gelatin, we found that human FBG (0 to 3.6 mg/mL i.e., 0 to 10.6 μM) concentration-dependently inhibited human proMMP-2 and MMP2 from binding to gelatin. Moreover, at normal physiological concentrations, FBG (5.29-11.8 μM) concentration-dependently inhibited (40-70% inhibition) the cleavage of fluorescein-conjugated gelatin by MMP-2, but not MMP-9. Indicative of a mixed-type (combination of competitive and non-competitive) inhibition mechanism, FBG reduced the Vmax (24.9 ± 0.7 min-1 to 17.7 ± 0.9 min-1, P < 0.05) and increased the Michaelis-Menten constant KM (204 ± 6 nM to 478 ± 50 nM, P < 0.05) for the reaction of MMP-2 cleavage of fluorescein-conjugated gelatin. In silico analyses and studies of FBG neutralization with anti-FBG antibodies implicated the domains D and E of FBG in the inhibition of MMP-2. In conclusion, FBG is a natural selective MMP-2 inhibitor, whose pathological elevation could lead to MMP-2 insufficiency in humans.