Macrophages-derived Factor XIII links coagulation to inflammation in COPD.

Background: The local, extravascular, activation of the coagulation system in response to injury is a key factor mediating the resulting inflammatory response. Coagulation Factor XIIIA (FXIIIA) found in alveolar macrophages (AM) and dendritic cells (DC), by influencing fibrin stability, might be an inflammatory modifier in COPD. Aims: To study the expression of FXIIIA in AM and Langerin+DC (DC-1) and their relation to the inflammatory response and disease progression in COPD. Methods: In 47 surgical lungs, 36 from smokers (22 COPD and 14 no-COPD) and 11 from non-smokers we quantified by immunohistochemistry FXIIIA expression in AM and DC-1 along with numbers of CD8+Tcells and CXCR3 expression in lung parenchyma and airways. Lung function was measured prior to surgery. Results: The percentage of AM expressing FXIII (%FXIII+AM) was higher in COPD than no-COPD and non-smokers. DC-1 expressed FXIIIA and their numbers were higher in COPD than no-COPD and non-smokers. DC-1 positively correlated with %FXIII+AM (r=0.43; p<0.018). CD8+Tcells, which were higher in COPD than in no-COPD, were correlated with DC-1 (p<0.01) and %FXIII+AM. CXCR3+ cells were increased in COPD and correlated with %FXIII+AM (p<0.05). Both %FXIII+AM (r=-0.6; p=0.001) and DC-1 (r=-0.7; p=0.001) correlated inversely with FEV1. Conclusion: FXIIIA, an important link between the extravascular coagulation cascade and inflammatory response, is significantly expressed in alveolar macrophages and dendritic cells of smokers with COPD, suggesting that it could play an important role in the adaptive inflammatory reaction characteristic of the disease.

Antibodies against Noncatalytic B Subunit of Factor XIII Inhibit Activation of Factor XIII and Fibrin Crosslinking.

BACKGROUND: Coagulation factor XIII (FXIII) is a proenzyme of plasma transglutaminase. It comprises two catalytic A subunits (FXIII-A) and two carrier B subunits (FXIII-B). We previously reported that alloantibodies against FXIII-B could promote FXIII clearance in a patient with congenital FXIII-B deficiency who had received infusions of plasma-derived human FXIII (A2B2 heterotetramer). OBJECTIVES: We aimed to investigate whether anti-FXIII-B antibodies affect the catalytic function of FXIII. METHODS: FXIII activation and fibrin crosslinking were examined in the presence of patient plasma, isolated patient IgG, or rat anti-FXIII-B monoclonal antibodies. RESULTS: Alloantibody levels were increased by repeated infusions of plasma-derived A2B2 heterotetramer, which enhanced binding to the functionally important FXIII-B sushi domains. The patient plasma strongly inhibited cleavage of the FXIII-A activation peptide, amine incorporation, and fibrin crosslinking in normal plasma. Furthermore, anti-FXIII-B alloantibodies blocked the formation of the complex of FXIII-B with FXIII-A, and fibrinogen. Rat monoclonal antibodies against the 10th sushi domain of FXIII-B inhibited the incorporation of FXIII-B to fibrin, FXIII activation (i.e., cleavage of FXIII-A activation peptide), and ultimately fibrin crosslinking in normal plasma, independent of their effect on heterotetramer assembly with FXIII-A. Alloantibody binding to the A2B2 heterotetramer blocked the access of thrombin to the FXIII-A cleavage site, as indicated by the reaction of the alloantibodies to the A2B2 heterotetramer and FXIII-B, but not to FXIII-A. CONCLUSION: Anti-FXIII-B antibodies binding to the A2B2 heterotetramer and FXIII-B inhibited FXIII activation and its crosslinking function despite being directed against its noncatalytic subunit (FXIII-B).

Peptidic Inhibitors and a Fluorescent Probe for the Selective Inhibition and Labelling of Factor XIIIa Transglutaminase.

Factor XIIIa (FXIIIa) is a transglutaminase of major therapeutic interest for the development of anticoagulants due to its essential role in the blood coagulation cascade. While numerous FXIIIa inhibitors have been reported, they failed to reach clinical evaluation due to their lack of metabolic stability and low selectivity over transglutaminase 2 (TG2). Furthermore, the chemical tools available for the study of FXIIIa activity and localization are extremely limited. To combat these shortcomings, we designed, synthesised, and evaluated a library of 21 novel FXIIIa inhibitors. Electrophilic warheads, linker lengths, and hydrophobic units were varied on small molecule and peptidic scaffolds to optimize isozyme selectivity and potency. A previously reported FXIIIa inhibitor was then adapted for the design of a probe bearing a rhodamine B moiety, producing the innovative KM93 as the first known fluorescent probe designed to selectively label active FXIIIa with high efficiency (kinact/KI = 127,300 M-1 min-1) and 6.5-fold selectivity over TG2. The probe KM93 facilitated fluorescent microscopy studies within bone marrow macrophages, labelling FXIIIa with high efficiency and selectivity in cell culture. The structure-activity trends with these novel inhibitors and probes will help in the future study of the activity, inhibition, and localization of FXIIIa.

Transglutaminase Activities of Blood Coagulant Factor XIII Are Dependent on the Activation Pathways and on the Substrates.

Factor XIII (FXIII) catalyzes formation of γ-glutamyl-ε-lysyl crosslinks between reactive glutamines (Q) and lysines (K). In plasma, FXIII is activated proteolytically (FXIII-A*) by the concerted action of thrombin and Ca2+. Cellular FXIII is activated nonproteolytically (FXIII-A°) by elevation of physiological Ca2+ concentrations. FXIII-A targets plasmatic and cellular substrates, but questions remain on correlating FXIII activation, resultant conformational changes, and crosslinking function to different physiological substrates. To address these issues, the characteristics of FXIII-A* versus FXIII-A° that contribute to transglutaminase activity and substrate specificities were investigated. Crosslinking of lysine mimics into a series of Q-containing substrates were measured using in-gel fluorescence, mass spectrometry, and UV-Vis spectroscopy. Covalent incorporation of fluorescent monodansylcadaverine revealed that FXIII-A* exhibits greater activity than FXIII-A° toward Q residues within Fbg αC (233-425 WT, Q328P Seoul II, and Q328PQ366N) and actin. FXIII-A* and FXIII-A° displayed similar activities toward α2-antiplasmin (α2AP), fibronectin, and Fbg αC (233-388, missing FXIII-binding site αC 389-402). Furthermore, the N-terminal α2AP peptide (1-15) exhibited similar kinetic properties for FXIII-A* and FXIII-A°. MALDI-TOF mass spectrometry assays with glycine ethyl ester and Fbg αC (233-425 WT, αC E396A, and truncated αC (233-388) further documented that FXIII-A* exerts greater benefit from the αC 389-402 binding site than FXIII-A°. Conformational properties of FXIII-A* versus A° are proposed to help promote transglutaminase function toward different substrates. A combination of protein substrate disorder and secondary FXIII-binding site exposure are utilized to control activity and specificity. From these studies, greater understandings of how FXIII-A targets different substrates are achieved.

The Effect of Activated FXIII, a Transglutaminase, on Vascular Smooth Muscle Cells.

Plasma factor XIII (pFXIII) is a heterotetramer of FXIII-A and FXIII-B subunits. The cellular form (cFXIII), a dimer of FXIII-A, is present in a number of cell types. Activated FXIII (FXIIIa), a transglutaminase, plays an important role in clot stabilization, wound healing, angiogenesis and maintenance of pregnancy. It has a direct effect on vascular endothelial cells and fibroblasts, which have been implicated in the development of atherosclerotic plaques. Our aim was to explore the effect of FXIIIa on human aortic smooth muscle cells (HAoSMCs), another major cell type in the atherosclerotic plaque. Osteoblastic transformation induced by Pi and Ca2+ failed to elicit the expression of cFXIII in HAoSMCs. EZ4U, CCK-8 and CytoSelect Wound Healing assays were used to investigate cell proliferation and migration. The Sircol Collagen Assay Kit was used to monitor collagen secretion. Thrombospondin-1 (TSP-1) levels were measured by ELISA. Cell-associated TSP-1 was detected by the immunofluorescence technique. The TSP-1 mRNA level was estimated by RT-qPCR. Activated recombinant cFXIII (rFXIIIa) increased cell proliferation and collagen secretion. In parallel, a 67% decrease in TSP-1 concentration in the medium and a 2.5-fold increase in cells were observed. TSP-1 mRNA did not change significantly. These effects of FXIIIa might contribute to the pathogenesis of atherosclerotic plaques.

Juvenile Xanthogranuloma: A Comparative Immunohistochemical Study of Factor XIIIa, CD11c, and CD4.

ABSTRACT: Juvenile xanthogranuloma is a group C and L non-Langerhans cell histiocytosis, and its cell of origin is still debatable. The expression of CD11c, a more recently described macrophage marker, and CD4 have not been studied comprehensively. This study aimed to expand immunophenotypic profile and hence our understanding of the origin of these lesions. The surgical pathology archive was searched for the cases with the pathologic diagnosis of "xanthogranuloma" from 1995 to 2019. Immunohistochemical (IHC) stains were performed for factor XIIIa, CD11c, and CD4. Morphologically, each lesion was classified into early classic, classic, or transitional subtypes. Seventy-seven cases were included with the median age of 7.8 years (male:female 1.3:1). Uniform positivity was noticed for CD4 (n = 77), CD68 (n = 37), CD163 (n = 5), and vimentin (n = 4) stains. Other stains included CD11c 75/77 (97.4%), factor XIIIa 71/76 (93.4%), S-100 protein 4/23 (17.4%), and CD1a 0/18 (0%). Despite insignificant association between morphologic subtype and main studied IHC stains, factor XIIIa reactivity was highest in transitional lesions and CD11c showed higher reactivity in early classic lesions. CD11c and CD4 are sensitive markers and showed promising results in the diagnosis of juvenile xanthogranuloma compared with factor XIIIa. Despite different reactivity of factor XIIIa and CD11c in various morphologic subtypes, such association was statistically insignificant.

Multiple myxoid cellular neurothekeomas in a patient with systemic lupus erythematosus.

Cellular neurothekeoma is a cutaneous tumor with a distinctive histopathologic appearance characterized by a dermal-based multinodular proliferation of epithelioid to spindled cells. Although the tumor may show varying amounts of myxoid stroma, extensive myxoid change is uncommon. The tumor typically presents as a solitary nodule with a predilection for the head and neck and upper limbs; examples of multiple cellular neurothekeomas are decidedly rare. The present report describes a unique case of multiple myxoid cellular neurothekeomas arising in a 60-year-old female with systemic lupus erythematosus. Two papular lesions were identified involving the skin inferior to the umbilicus and the left inguinal crease. Both lesions were histopathologically similar, forming a nodular mass composed of epithelioid cells in a prominent myxoid stroma. By immunohistochemistry the lesional cells expressed NKI/C3, microphthalmia transcription factor (MiTF), and CD68, with focal staining for PGP9.5, factor XIIIa, and CD10 also observed. The tumors were negative for S-100, SOX-10, epithelial membrane antigen, desmin, smooth muscle actin, glial fibrillary acid protein, and CD34. The present case confirms that cellular neurothekeoma can present clinically as multiple lesions and can have a predominantly myxoid appearance, potentially mimicking other cutaneous myxoid lesions.

Pleomorphic Fibroma: A Clinicopathologic Case Series With the Review of the Literature.

BACKGROUND: Pleomorphic fibromas are rare flesh-colored benign neoplasms first described in 1989. Their histopathology is notable for nuclear pleomorphism of spindle cells and multinucleate giant cells but lacking mitoses. The cellular origin of these tumors is unknown. This case series describes an additional 18 lesions with discussion of histopathology and immunohistochemistry. METHODS: This case series of 18 pleomorphic fibromas uses immunohistochemical staining for CD34, CD68, factor XIIIa, and S-100 and general histopathologic examination under light microscopy to describe the lesions. RESULTS: Immunohistochemical stains for CD34 showed nearly universal positivity of the pleomorphic spindle cells, although some more focally. The pleomorphic cells were negative for CD68, variably positive for factor XIIIa, and universally negative for S-100. All the lesions showed characteristic nuclear pleomorphism with absent mitoses. Collagen thickening was variable, mucin was absent, and perivascular inflammation was present in all lesions. CONCLUSIONS: Pleomorphic fibromas are fibrous lesions with benign clinical course and histopathologic findings including nuclear pleomorphism. Immunohistochemical staining characteristics of the lesion, along with unique spindle cells and multinucleate giant cells help to differentiate this from other tumors.

Factor XIII-A in Diseases: Role Beyond Blood Coagulation.

Multidisciplinary research from the last few decades has revealed that Factor XIII subunit A (FXIII-A) is not only involved in blood coagulation, but may have roles in various diseases. Here, we aim to summarize data from studies involving patients with mutations in the F13A1 gene, performed in FXIII-A knock-out mice models, clinical and histological studies assessing correlations between diseases severity and FXIII-A levels, as well as from in vitro experiments. By providing a complex overview on its possible role in wound healing, chronic inflammatory bowel diseases, athe-rosclerosis, rheumatoid arthritis, chronic inflammatory lung diseases, chronic rhinosinusitis, solid tumors, hematological malignancies, and obesity, we also demonstrate how the field evolved from using FXIII-A as a marker to accept and understand its active role in inflammatory and malignant diseases.

NMR-Based Structural Characterization of a Two-Disulfide-Bonded Analogue of the FXIIIa Inhibitor Tridegin: New Insights into Structure-Activity Relationships.

The saliva of blood-sucking leeches contains a plethora of anticoagulant substances. One of these compounds derived from Haementeria ghilianii, the 66mer three-disulfide-bonded peptide tridegin, specifically inhibits the blood coagulation factor FXIIIa. Tridegin represents a potential tool for antithrombotic and thrombolytic therapy. We recently synthesized two-disulfide-bonded tridegin variants, which retained their inhibitory potential. For further lead optimization, however, structure information is required. We thus analyzed the structure of a two-disulfide-bonded tridegin isomer by solution 2D NMR spectroscopy in a combinatory approach with subsequent MD simulations. The isomer was studied using two fragments, i.e., the disulfide-bonded N-terminal (Lys1-Cys37) and the flexible C-terminal part (Arg38-Glu66), which allowed for a simplified, label-free NMR-structure elucidation of the 66mer peptide. The structural information was subsequently used in molecular modeling and docking studies to provide insights into the structure-activity relationships. The present study will prospectively support the development of anticoagulant-therapy-relevant compounds targeting FXIIIa.

Self-Amplifying Nanotherapeutic Drugs Homing to Tumors in a Manner of Chain Reaction.

Active tumor-targeting drug delivery has great potency in cancer therapy. However, the targeting efficiency of traditional active tumor-targeting nanotherapeutic drugs is limited by the scarcity of their accessible targets/receptors in tumors. Here, a novel self-amplifying tumor-targeting strategy with a chain reaction mechanism is developed. A coagulation targeting peptide (GNQEQVSPLTLLKXC, termed A15)-decorated poly(L-glutamic acid)-graft-maleimide poly(ethylene glycol)/combretastatin A4 conjugate (A15-PLG-CA4) is prepared to obtain a self-amplifying nanotherapeutic platform homing to tumors. After administration to tumor-bearing mice, A15-PLG-CA4 starts a chain reaction cycle consisting of intratumoral hemorrhage, target FXIIIa amplification, blood clot binding, and CA4 release in tumors. In this way, A15-PLG-CA4 increases the level of its accessible targets (FXIIIa) in a manner of chain reaction. The FXIIIa activity at 8 h is 4.1-fold more than the one at 0 h in the C26 tumors treated with A15-PLG-CA4. The total CA4 concentration at 24 h is 2.9-fold more than the control. A15-PLG-CA4 shows a significantly higher antitumor effect against large C26 tumors (≈500 mm3 ) thanks to the remarkable tumor-targeting ability compared with the control. Therefore, this report highlights the potential of the self-amplifying tumor-targeting strategy in the development of next generation active tumor-targeting nanotherapeutic drugs for tumor therapy.

F13A1 transglutaminase expression in human adipose tissue increases in acquired excess weight and associates with inflammatory status of adipocytes.

OBJECTIVE: F13A1/FXIII-A transglutaminase has been linked to adipogenesis in cells and to obesity in humans and mice, however, its role and associated molecular pathways in human acquired excess weight have not been explored. METHODS: We examined F13A1 expression and association to human weight gain in weight-discordant monozygotic twins (Heavy-Lean difference (ΔWeight, 16.8 kg ± 7.16 for n = 12). The twin pairs were examined for body composition (by dual-energy X-ray absorptiometry), abdominal body fat distribution (by magnetic resonance imaging), liver fat content (by magnetic resonance spectroscopy), circulating adipocytokines, leptin and adiponectin, as well as serum lipids. Affymetrix full transcriptome mRNA analysis was performed from adipose tissue and adipocyte-enriched fractions from subcutaneous abdominal adipose tissue biopsies. F13A1 differential expression between the heavy and lean co-twins was examined and its correlation transcriptome changes between co-twins were performed. RESULTS: F13A1 mRNA showed significant increase in adipose tissue (p < 0.0001) and an adipocyte-enriched fraction (p = 0.0012) of the heavier co-twin. F13A1 differential expression in adipose tissue (Heavy-Lean ΔF13A1) showed significant negative correlation with circulating adiponectin (p = 0.0195) and a positive correlation with ΔWeight (p = 0.034), ΔBodyFat (0.044) and ΔAdipocyte size (volume, p = 0.012;) in adipocyte-enriched fraction. A whole transcriptome-wide association study (TWAS) on ΔF13A1 vs weight-correlated ΔTranscriptome identified 182 F13A1-associated genes (r > 0.7, p = 0.05) with functions in several biological pathways including cell stress, inflammatory response, activation of cells/leukocytes, angiogenesis and extracellular matrix remodeling. F13A1 did not associate with liver fat accumulation. CONCLUSIONS: F13A1 levels in adipose tissue increase with acquired excess weight and associate with pro-inflammatory, cell stress and tissue remodeling pathways. This supports its role in expansion and inflammation of adipose tissue in obesity.

Distinct 3-disulfide-bonded isomers of tridegin differentially inhibit coagulation factor XIIIa: The influence of structural stability on bioactivity.

Tridegin is a 66mer cysteine-rich coagulation factor XIIIa (FXI-IIa) inhibitor from the giant amazon leech Haementeria ghilianii of yet unknown disulfide connectivity. This study covers the structural and functional characterization of five different 3-disulfide-bonded tridegin isomers. In addition to three previously identified isomers, one isomer containing the inhibitory cystine knot (ICK, knottin) motif, and one isomer with the leech antihemostatic protein (LAP) motif were synthesized in a regioselective manner. A fluorogenic enzyme activity assay revealed a positive correlation between the constriction of conformational flexibility in the N-terminal part of the peptide and the inhibitory potential towards FXI-IIa with clear differences between the isomers. This observation was supported by molecular dynamics (MD) simulations and subsequent molecular docking studies. The presented results provide detailed structure-activity relationship studies of different tridegin disulfide isomers towards FXI-IIa and reveal insights into the possibly existing native linkage compared to non-native disulfide tridegin species.

Factor XIIIa inhibitors as potential novel drugs for venous thromboembolism.

Human factor XIIIa (FXIIIa) is a multifunctional transglutaminase with a significant role in hemostasis. FXIIIa catalyzes the last step in the coagulation process. It stabilizes the blood clot by cross-linking the α- and γ-chains of fibrin. It also protects the newly formed clot from plasmin-mediated fibrinolysis, primarily by cross-linking α2-antiplasmin to fibrin. Furthermore, FXIIIa is a major determinant of clot size and clot's red blood cells content. Therefore, inhibitors targeting FXIIIa have been considered to develop a new generation of anticoagulants to prevent and/or treat venous thromboembolism. Several inhibitors of FXIIIa have been discovered or designed including active site and allosteric site small molecule inhibitors as well as natural and modified polypeptides. This work reviews the structural, biochemical, and pharmacological aspects of FXIIIa inhibitors so as to advance their molecular design to become more clinically relevant.

Uncommon cutaneous non-Langerhans cell histiocytosis arising from dermal dendritic cells in adults: a clinicopathological study of five cases.

BACKGROUND: Non-Langerhans cell histiocytosis (non-LCH) is a collective term that encompasses a long list of rare "histiocytosis" that do not meet the criteria for Langerhans cell histiocytosis (LCH). Among cutaneous non-LCH, the xanthogranuloma (XG) family represents a distinct group of disorders derived from dermal dendritic cells (DDCs) at different stages of differentiation. OBJECTIVES: To investigate the clinicopathological characteristics of the XG family in adults and review the relevant literature. MATERIALS AND METHODS: We performed a retrospective clinicopathological study of five adult cases with a previous diagnosis of non-LCH. Clinicopathological features, immunophenotypes, genetic alterations and ultrastructural characteristics were analysed. RESULTS: Skin biopsies revealed that all five cases were characterized by diffuse infiltration of polymorphic cells, which were immunoreactive to factor XIIIa but negative for Langerin, CD1a, and S100. None of the cases harboured the BRAF V600E mutation. Electron microscopy of two cases exhibited abundant cytoplasmic processes with numerous lysosome-like dense bodies and electron-lucent vesicles in the cytoplasm and extracellular matrix. The overall features suggested that DDCs are the cellular origin, and these cases fulfilled the criteria for the XG family. CONCLUSION: The XG family represents a spectrum of rare diseases with different clinical presentations, a wide range of morphological appearances, and a shared common origin (DDCs). This group of disorders has been proposed as a unique entity with diagnostic challenges that should not be underestimated.

Melanogenic Properties and Expression Profiles of Melanogenic Paracrine Molecules in Riehl's Melanosis.

Riehl's melanosis is a hyperpigmentary disorder that occurs predominantly on the face and neck. To date, the pathogenesis of Riehl's melanosis with regards to the melanogenic properties and paracrine melanogenic molecules has not well been studied. This study was aimed to provide a novel perspective on the pathogenesis of Riehl's melanosis by identifying the relevant paracrine melanogenic molecules in Riehl's melanosis. Skin biopsies were performed on lesional and normal-appearing perilesional skin of 12 patients with Riehl's melanosis and 12 age- and sex-matched healthy controls. Histopathological and immunohistochemical staining for paracrine melanogenic molecules was analyzed. The major histopathological findings of Riehl's melanosis were basal hyperpigmentation, melanocyte proliferation, interface change, dermal pigmentary incontinence, vascular proliferation, and dermal inflammation. Dermal expression intensities of stem cell factor (SCF) and c-kit were increased in the lesional skin of Riehl's melanosis. In addition, increased expression of epidermal and dermal ET-1 was also observed in the lesional skin of Riehl's melanosis. Increased tissue expressions of SCF, c-kit, and ET-1 in Riehl's melanosis support the role of these paracrine melanogenic molecules in the pathogenesis of Riehl's melanosis. The findings from this study might present useful information on the pathogenetic mechanism of Riehl's melanosis.

A retrospective review of 93 cases of cellular dermatofibromas.

BACKGROUND: Cellular dermatofibromas (CDF) are an uncommon variant of benign fibrous histiocytomas with propensity to recur and rarely metastasize as well as demonstrate histologic similarities to more dangerous lesions. OBJECTIVES: The aim of this present study was to further describe the presentation and outcome of the cellular variant of benign fibrous histiocytomas so that it can be diagnosed and treated appropriately. METHODS: A retrospective chart review was performed on all patients seen in a single hospital system in Detroit, Michigan, from 2007 to 2017. CDF was confirmed by pathology. Baseline demographics, specialty service of diagnosis and treatment, treatment modality, and outcome were collected. RESULTS: Of the 93 qualifying patients, the average age at diagnosis was 42.65 years. The most common specialty service that diagnosed and treated patients was dermatology (38.71%). About 95.0% of CDF stained positive for Factor 13A (19/20), and 90.48% were CD34 negative (19/21). Of patients, 33.33% had recurrences of their CDF (9/27). Two patients had three or more recurrences. One patient's death was attributed to the CDF. CONCLUSION: CDF have a high local recurrence rate and similarities to more dangerous and malignant lesions. Patients with cellular dermatofibromas present to many subspecialty services for diagnosis and should be treated aggressively.

Assessment of expression and specific activities of transglutaminases TG1, TG2, and FXIII-A during osteoclastogenesis.

Osteoclasts are large multinucleated bone-resorbing cells derived from monocyte/macrophage lineage. Macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL) drive the multi-stage osteoclastogenesis. Transglutaminases (TGs) are Ca2+- and thiol-dependent acyl transferases and protein crosslinking enzymes. TG enzyme family contains eight catalytically active enzymes TG1-7 and Factor XIII-A (FXIII-A). Recent studies have shown that TG1, TG2, and FXIII-A are present in osteoclasts and that TG2 and FXIII-A regulate osteoclastogenesis. In this study, we examined gene and protein expression and specific activities of TG1, TG2, and FXIII-A during osteoclastogenesis using "Hitomi peptides" in a day-by-day manner. We report that TG activities are highest in the differentiation and early fusion phases and then decrease dramatically. TG activities were upregulated by M-CSF and downregulated by addition of RANKL. FXIII-A was dramatically downregulated by RANKL, suggesting its involvement in M-CSF-mediated precursor commitment phase. TG1 and TG2 proteins were present throughout osteoclastogenesis, suggesting that they may have functions in both differentiation and fusion. In summary, the three TGs likely exert distinct functions at different stages of osteoclastogenesis. Our work also demonstrates that the "Hitomi peptides" are highly specific tools for detection of distinct TGs in a system where multiple TGs are present.

Mass spectrometry-based molecular mapping of native FXIIIa cross-links in insoluble fibrin clots.

The roles of factor XIIIa-specific cross-links in thrombus formation, regression, or probability for embolization are largely unknown. A molecular understanding of fibrin architecture at the level of these cross-links could inform the development of therapeutic strategies to prevent the sequelae of thromboembolism. Here, we present an MS-based method to map native factor XIIIa cross-links in the insoluble matrix component of whole-blood or plasma-fibrin clots and in in vivo thrombi. Using a chaotrope-insoluble digestion method and quantitative cross-linking MS, we identified the previously mapped fibrinogen peptides that are responsible for covalent D-dimer association, as well as dozens of novel cross-links in the αC region of fibrinogen α. Our findings expand the known native cross-linked species from one to over 100 and suggest distinct antiparallel registries for interprotofibril association and covalent attachment of serpins that regulate clot dissolution.

Let's cross-link: diverse functions of the promiscuous cellular transglutaminase factor XIII-A.

Essentials Plasma Factor XIII, a heterodimer of A and B subunits FXIIIA2 B2 , is a transglutaminase enzyme with a well-established role in haemostasis. Cells of bone marrow and mesenchymal lineage express the FXIII-A gene (F13A1) that encodes the cellular form of the transglutaminase, a homodimer of the A subunits, FXIII-A. FXIII-A was presumed to function intracellularly, however, several lines of evidence now indicate that FXIII-A is externalised by an as yet unknown mechanism This review describes the mounting evidence that FXIII-A is a diverse transglutaminase with many intracellular and extracellular substrates that can participate in an array of biological processes SUMMARY: Factor XIII is a tranglutaminase enzyme that catalyzes the formation of ε-(γ-glutamyl)lysyl isopeptide bonds in protein substrates. The plasma form, FXIII-A2 B2 , has an established function in hemostasis, where its primary substrate is fibrin. A deficiency in FXIII manifests as a severe bleeding diathesis, underscoring its importance in this pathway. The cellular form of the enzyme, a homodimer of the A-subunits, denoted FXIII-A, has not been studied in as extensive detail. FXIII-A was generally perceived to remain intracellular, owing to the lack of a classical signal peptide for its release. In the last decade, emerging evidence has revealed that this diverse transglutaminase can be externalized from cells, by an as yet unknown mechanism, and can cross-link extracellular substrates and participate in a number of diverse pathways. The FXIII-A gene (F13A1) is expressed in cells of bone marrow and mesenchymal lineage, notably megakaryocytes, monocytes/macrophages, dendritic cells, chrondrocytes, osteoblasts, and preadipocytes. The biological processes that FXIII-A is coupled with, such as wound healing, phagocytosis, and bone and matrix remodeling, reflect its expression in these cell types. This review describes the mounting evidence that this cellular transglutaminase can be externalized, usually in response to stimuli, and participate in extracellular cross-linking reactions. A corollary of being involved in these biological pathways is the participation of FXIII-A in pathological processes. In conclusion, the functions of this transglutaminase extend far beyond its role in hemostasis, and our understanding of this enzyme in terms of its secretion, regulation and substrates is in its infancy.