Activated platelets retain and protect most of their factor XIII-A cargo from proteolytic activation and degradation.

ABSTRACT: Platelet factor XIII-A (FXIII-A) is a major cytoplasmic protein (∼3% of total), representing ∼50% of total circulating FXIII. However, mobilization of FXIII-A during platelet activation is not well defined. To determine mechanisms mediating the retention vs release of platelet FXIII-A, platelets from healthy humans and mice (F13a1-/-, Fga-/-, Plg-/-, Stim1fl/flPf4-Cre, and respective controls) were stimulated with thrombin, convulxin plus thrombin, or calcium ionophore (A23187), in the absence or presence of inhibitors of transglutaminase activity, messenger RNA (mRNA) translation, microtubule rearrangement, calpain, and Rho GTPase. Platelet releasates and pellets were separated by (ultra)centrifugation. FXIII-A was detected by immunoblotting and immunofluorescence microscopy. Even after strong dual agonist (convulxin plus thrombin) stimulation of human platelets, >80% platelet FXIII-A remained associated with the platelet pellet. In contrast, essentially all tissue factor pathway inhibitor, another cytoplasmic protein in platelets, was released to the supernatant. Pellet-associated FXIII-A was not due to de novo synthesis via platelet F13A1 mRNA. The proportion of platelet FXIII-A retained by vs released from activated platelets was partly dependent on STIM1 signaling, microtubule rearrangement, calpain, and RhoA activation but did not depend on the presence of fibrinogen or plasminogen. Immunofluorescence microscopy confirmed the presence of considerable FXIII-A within the activated platelets. Although released FXIII-A was cleaved to FXIII-A∗ and could be degraded by plasmin, platelet-associated FXIII-A remained uncleaved. Retention of substantial platelet-derived FXIII-A by activated platelets and its reduced susceptibility to thrombin- and plasmin-mediated proteolysis suggest platelet FXIII-A is a protected pool with biological role(s) that differs from plasma FXIII.

Integration of clotting and fibrinolysis: central role of platelets and factor XIIIa.

PURPOSE: The aim of the present study was to establish the role of platelets and activated factor XIIIa (FXIIIa) in the structuring of the fibrin network as well as to clarify the effect of network compaction on clot lysis. METHODS: Turbidimetry was used for the one-stage clotting test where platelet-free plasma (PFP) is regarded as single factor-deficient plasma (platelets as lacking factor) and autologous platelet-rich plasma (PRP) as deficiency corrected plasma. Structural features of the developed and subsequently lysed fibrin network, formed under static and flow conditions, were visualized by confocal microscopy. RESULTS: Thrombin-initiated plasma clotting revealed changes in the shape of the absorption curve, more pronounced in the presence of platelets. These changes correlate with the transformation of the fibrin scaffold during clot maturing. With the combined action of platelets, thrombin and Ca2+, plasma clotting passes through two phases: initial formation of a platelet-fibrin network (first peak in the polymerization curve), and then the compaction of fibrin, driven by FXIIIa (the second peak) which can be further modulate by the contractile action of platelets. These structural changes, mediated by platelets and FXIIIa, have been shown to determine subsequent clot lysis. CONCLUSIONS: Platelet aggregates serve as organizing centers that determine the distribution of fibrin in clot volume. The openwork structure of the platelet-transformed fibrin provides the necessary prerequisites for its timely lysis. The revealed aspects of the interaction of platelets and FXIIIa, which accompanies the maturation of a fibrin clot, may lead to new approaches in the pharmacological correction of disorders associated with both thrombotic episodes and bleeding tendency.

Linking inflammation and angiogenesis with fibrogenesis: Expression of FXIIIA, MMP-9, and VEGF in oral submucous fibrosis / Vinculación de la inflamación y la angiogénesis con la fibrogénesis: expresión de FXIIIA, MMP-9 y VEGF en la fibrosis submucosa oral

Objectives Interplay of Factor XIIIa (FXIIIa), a transglutaminase, responsible for cross-linking of matrix proteins, Matrix Metalloproteinase-9 (MMP-9), a gelatinase, and Vascular Endothelial Growth Factor (VEGF), an angiogenic inducer, were studied in relation to fibrogenesis and disease progression in oral submucous fibrosis (OSMF). Material and methods Immunohistochemical expression of markers was studied in 60 formalin-fixed paraffin-embedded tissue blocks of OSMF and 20 normal oral mucosal tissues. FXIIIa was studied quantitatively while MMP-9 and VEGF were assessed semi-quantitatively. Expression was compared with histopathological grades of OSMF. Results FXIIIa expression significantly increased in OSMF (p-value 0.000). However, expression decreased and cells became quiescent with increasing grades (p-value 0.000). MMP-9 (p-value epithelium 0.011, p-value connective tissue 0.000) and VEGF expression (p-value epithelium 0.000, connective tissue 0.000) increased in OSMF. A negative correlation between FXIIIa and MMP-9 (−0.653) in early grade (p-value of 0.021) and a positive correlation between FXIIIa and VEGF (0.595) (p-value of 0.032) was found in the moderate grade OSMF. Regression analysis showed a significant association (p<0.01) of FXIIIa in OSMF and with increasing grades of OSMF. Conclusion FXIIIa may play a crucial role in initiation of fibrosis in OSMF. MMP-9 may have a diverse role to play in OSMF as a regulator of fibrosis. VEGF may show an angio-fibrotic switch and contribute to fibrosis in OSMF. These cytokines may show altered function and can contribute to fibrosis and chronicity of disease due to changes in the microenvironment. Tissue stiffness in OSMF itself creates an environment that enhances the chronicity of the disease. (AU)

Objetivos Se estudió la interacción del factor XIIIa (FXIIIa), una transglutaminasa responsable de los entrecruzamientos de las proteínas de la matriz, la metaloproteinasa de matriz-9 (MMP-9), una gelatinasa y el factor de crecimiento endotelial vascular (VEGF), un inductor angiogénico, en relación con la fibrogénesis y la progresión de la enfermedad en la fibrosis submucosa oral (OSMF). Material y métodos Se estudió la expresión inmunohistoquímica de marcadores en 60 bloques de tejido de OSMF fijados con formalina e incluidos en parafina y 20 tejidos de mucosa oral normales. FXIIIa se estudió cuantitativamente mientras que MMP-9 y VEGF se evaluaron semicuantitativamente. La expresión se comparó con los grados histopatológicos de OSMF. Resultados La expresión de FXIIIa aumentó significativamente en OSMF (valor de p 0,000). Sin embargo, la expresión disminuyó y las células se volvieron inactivas a medida que aumentaban los grados (valor de p 0,000). MMP-9 (valor de p epitelio 0,011, tejido conectivo valor de p 0,000) y expresión de VEGF (valor de p epitelio 0,000, tejido conectivo 0,000) aumentaron en OSMF. Se encontró una correlación negativa entre FXIIIa y MMP-9 (-0,653) en grado temprano (valor de p de 0,021) y una correlación positiva entre FXIIIa y VEGF (0,595) (valor de p de 0,032) en OSMF de grado moderado. El análisis de regresión mostró una asociación significativa (p<0,01) de FXIIIa en OSMF y con grados crecientes de OSMF. Conclusión FXIIIa puede desempeñar un papel crucial en el inicio de la fibrosis en OSMF. MMP-9 puede desempeñar un papel diverso en OSMF como regulador de la fibrosis. VEGF puede mostrar un interruptor angiofibrótico y contribuir a la fibrosis en OSMF. Estas citocinas pueden mostrar una función alterada y pueden contribuir a la fibrosis y la cronicidad de la enfermedad debido a cambios en el microambiente. La rigidez del tejido en el propio OSMF crea un entorno que mejora la cronicidad de la enfermedad. (AU)

Linking inflammation and angiogenesis with fibrogenesis: Expression of FXIIIA, MMP-9, and VEGF in oral submucous fibrosis / Vinculación de la inflamación y la angiogénesis con la fibrogénesis: expresión de FXIIIA, MMP-9 y VEGF en la fibrosis submucosa oral

Objectives Interplay of Factor XIIIa (FXIIIa), a transglutaminase, responsible for cross-linking of matrix proteins, Matrix Metalloproteinase-9 (MMP-9), a gelatinase, and Vascular Endothelial Growth Factor (VEGF), an angiogenic inducer, were studied in relation to fibrogenesis and disease progression in oral submucous fibrosis (OSMF). Material and methods Immunohistochemical expression of markers was studied in 60 formalin-fixed paraffin-embedded tissue blocks of OSMF and 20 normal oral mucosal tissues. FXIIIa was studied quantitatively while MMP-9 and VEGF were assessed semi-quantitatively. Expression was compared with histopathological grades of OSMF. Results FXIIIa expression significantly increased in OSMF (p-value 0.000). However, expression decreased and cells became quiescent with increasing grades (p-value 0.000). MMP-9 (p-value epithelium 0.011, p-value connective tissue 0.000) and VEGF expression (p-value epithelium 0.000, connective tissue 0.000) increased in OSMF. A negative correlation between FXIIIa and MMP-9 (−0.653) in early grade (p-value of 0.021) and a positive correlation between FXIIIa and VEGF (0.595) (p-value of 0.032) was found in the moderate grade OSMF. Regression analysis showed a significant association (p<0.01) of FXIIIa in OSMF and with increasing grades of OSMF. Conclusion FXIIIa may play a crucial role in initiation of fibrosis in OSMF. MMP-9 may have a diverse role to play in OSMF as a regulator of fibrosis. VEGF may show an angio-fibrotic switch and contribute to fibrosis in OSMF. These cytokines may show altered function and can contribute to fibrosis and chronicity of disease due to changes in the microenvironment. Tissue stiffness in OSMF itself creates an environment that enhances the chronicity of the disease. (AU)

Objetivos Se estudió la interacción del factor XIIIa (FXIIIa), una transglutaminasa responsable de los entrecruzamientos de las proteínas de la matriz, la metaloproteinasa de matriz-9 (MMP-9), una gelatinasa y el factor de crecimiento endotelial vascular (VEGF), un inductor angiogénico, en relación con la fibrogénesis y la progresión de la enfermedad en la fibrosis submucosa oral (OSMF). Material y métodos Se estudió la expresión inmunohistoquímica de marcadores en 60 bloques de tejido de OSMF fijados con formalina e incluidos en parafina y 20 tejidos de mucosa oral normales. FXIIIa se estudió cuantitativamente mientras que MMP-9 y VEGF se evaluaron semicuantitativamente. La expresión se comparó con los grados histopatológicos de OSMF. Resultados La expresión de FXIIIa aumentó significativamente en OSMF (valor de p 0,000). Sin embargo, la expresión disminuyó y las células se volvieron inactivas a medida que aumentaban los grados (valor de p 0,000). MMP-9 (valor de p epitelio 0,011, tejido conectivo valor de p 0,000) y expresión de VEGF (valor de p epitelio 0,000, tejido conectivo 0,000) aumentaron en OSMF. Se encontró una correlación negativa entre FXIIIa y MMP-9 (-0,653) en grado temprano (valor de p de 0,021) y una correlación positiva entre FXIIIa y VEGF (0,595) (valor de p de 0,032) en OSMF de grado moderado. El análisis de regresión mostró una asociación significativa (p<0,01) de FXIIIa en OSMF y con grados crecientes de OSMF. Conclusión FXIIIa puede desempeñar un papel crucial en el inicio de la fibrosis en OSMF. MMP-9 puede desempeñar un papel diverso en OSMF como regulador de la fibrosis. VEGF puede mostrar un interruptor angiofibrótico y contribuir a la fibrosis en OSMF. Estas citocinas pueden mostrar una función alterada y pueden contribuir a la fibrosis y la cronicidad de la enfermedad debido a cambios en el microambiente. La rigidez del tejido en el propio OSMF crea un entorno que mejora la cronicidad de la enfermedad. (AU)

Immunohistochemical analysis of chronic and recurrent dermatophytosis.

BACKGROUND: Dermatophytosis has assumed epidemic proportions with rising resistance, recalcitrance and recurrence, especially in tropical regions. While various factors contribute to high prevalence worldwide, yet little is known about the interactions between host defence mechanisms and dermatophytes, particularly in chronic and recalcitrant dermatophytosis. OBJECTIVES: We aimed to compare the population of various immune cells in specimens of chronic recurrent dermatophytosis and those with acute superficial dermatophytosis. METHODS: We investigated the density of various immune cells-Langerhans cells (CD1a+), macrophages (CD68+), dermal dendrocytes (Factor XIIIa+) in the skin of chronic dermatophytosis patients and those with successfully resolved lesions (controls). RESULTS: Langerhans cells were significantly decreased in the epidermis of patients, both in affected and unaffected areas in comparison with controls. In the dermis, however, no differences in the density of immune cells (macrophages and fibroblasts) were observed. LIMITATIONS: The limited sample size and immune cells evaluated could be expanded further in future research. CONCLUSION: These results indicate that the decreased number of Langerhans cells could be a potential risk factor for the development of chronic and recurrent dermatophytosis.

Linking inflammation and angiogenesis with fibrogenesis: Expression of FXIIIA, MMP-9, and VEGF in oral submucous fibrosis.

OBJECTIVES: Interplay of Factor XIIIa (FXIIIa), a transglutaminase, responsible for cross-linking of matrix proteins, Matrix Metalloproteinase-9 (MMP-9), a gelatinase, and Vascular Endothelial Growth Factor (VEGF), an angiogenic inducer, were studied in relation to fibrogenesis and disease progression in oral submucous fibrosis (OSMF). MATERIAL AND METHODS: Immunohistochemical expression of markers was studied in 60 formalin-fixed paraffin-embedded tissue blocks of OSMF and 20 normal oral mucosal tissues. FXIIIa was studied quantitatively while MMP-9 and VEGF were assessed semi-quantitatively. Expression was compared with histopathological grades of OSMF. RESULTS: FXIIIa expression significantly increased in OSMF (p-value 0.000). However, expression decreased and cells became quiescent with increasing grades (p-value 0.000). MMP-9 (p-value epithelium 0.011, p-value connective tissue 0.000) and VEGF expression (p-value epithelium 0.000, connective tissue 0.000) increased in OSMF. A negative correlation between FXIIIa and MMP-9 (-0.653) in early grade (p-value of 0.021) and a positive correlation between FXIIIa and VEGF (0.595) (p-value of 0.032) was found in the moderate grade OSMF. Regression analysis showed a significant association (p<0.01) of FXIIIa in OSMF and with increasing grades of OSMF. CONCLUSION: FXIIIa may play a crucial role in initiation of fibrosis in OSMF. MMP-9 may have a diverse role to play in OSMF as a regulator of fibrosis. VEGF may show an angio-fibrotic switch and contribute to fibrosis in OSMF. These cytokines may show altered function and can contribute to fibrosis and chronicity of disease due to changes in the microenvironment. Tissue stiffness in OSMF itself creates an environment that enhances the chronicity of the disease.

Reciprocal stabilization of coagulation factor XIII-A and -B subunits is a determinant of plasma FXIII concentration.

ABSTRACT: Transglutaminase factor XIII (FXIII) is essential for hemostasis, wound healing, and pregnancy maintenance. Plasma FXIII is composed of A and B subunit dimers synthesized in cells of hematopoietic origin and hepatocytes, respectively. The subunits associate tightly in circulation as FXIII-A2B2. FXIII-B2 stabilizes the (pro)active site-containing FXIII-A subunits. Interestingly, people with genetic FXIII-A deficiency have decreased FXIII-B2, and therapeutic infusion of recombinant FXIII-A2 (rFXIII-A2) increases FXIII-B2, suggesting FXIII-A regulates FXIII-B secretion, production, and/or clearance. We analyzed humans and mice with genetic FXIII-A deficiency and developed a mouse model of rFXIII-A2 infusion to define mechanisms mediating plasma FXIII-B levels. Like humans with FXIII-A deficiency, mice with genetic FXIII-A deficiency had reduced circulating FXIII-B2, and infusion of FXIII-A2 increased FXIII-B2. FXIII-A-deficient mice had normal hepatic function and did not store FXIII-B in liver, indicating FXIII-A does not mediate FXIII-B secretion. Transcriptional analysis and polysome profiling indicated similar F13b levels and ribosome occupancy in FXIII-A-sufficient and -deficient mice and in FXIII-A-deficient mice infused with rFXIII-A2, indicating FXIII-A does not induce de novo FXIII-B synthesis. Unexpectedly, pharmacokinetic/pharmacodynamic modeling of FXIII-B antigen after rFXIII-A2 infusion in humans and mice suggested FXIII-A2 slows FXIII-B2 loss from plasma. Accordingly, comparison of free FXIII-B2 vs FXIII-A2-complexed FXIII-B2 (FXIII-A2B2) infused into mice revealed faster clearance of free FXIII-B2. These data show FXIII-A2 prevents FXIII-B2 loss from circulation and establish the mechanism underlying FXIII-B2 behavior in FXIII-A deficiency and during rFXIII-A2 therapy. Our findings reveal a unique, reciprocal relationship between independently synthesized subunits that mediate an essential hemostatic protein in circulation. This trial was registered at www.ClinicalTrials.com as #NCT00978380.

Interplay of fibrinogen αEC globular domains and factor XIIIa cross-linking dictates the extensibility and strain stiffening of fibrin networks.

BACKGROUND: Fibrinogen is a plasma protein forming the fibrin scaffold of blood clots. Its mechanical properties therefore affect the risk of bleeding as well as thrombosis. There has been much recent interest in the biophysical mechanisms controlling fibrin mechanics; however, the role of molecular heterogeneity of the circulating fibrinogen in determining clot mechanical function remains poorly characterized. OBJECTIVES: By comparing 2 fibrinogen variants where the only difference is the Aα-chain length, with one variant having a globular domain at its C-terminus, this study aimed to reveal how the molecular structure impacts the structure and mechanics of fibrin networks. METHODS: We characterized the mechanical response to large shear for networks formed from 2 recombinant fibrinogen variants: the most prevalent variant in circulation with a molecular weight of 340 kDa (recombinant human fibrinogen [rFib] 340) and a minor variant with a molecular weight of 420 kDa (rFib420). RESULTS: We show that the elastic properties of the 2 variants are identical when fibrin is cross-linked with factor XIIIa but differ strongly in its absence. Uncross-linked rFib420 networks are softer and up to 3-fold more extensible than rFib340 networks. Electron microscopy imaging showed that the 2 variants formed networks with a comparable structure, except at 4 mg/mL, where rFib420 formed denser networks. CONCLUSION: We propose that the αEC domains of rFib420 increase the extensibility of uncross-linked fibrin networks by promoting protofibril sliding, which is blocked by FXIIIa cross-linking. Our findings can help explain the functional role of different circulating fibrinogen variants in blood clot mechanics and tissue repair.

von Willebrand factor-binding protein (vWbp)-activated factor XIII and transglutaminase 2 (TG2) promote cross-linking between FnBPA from Staphylococcus aureus and fibrinogen.

The secreted von Willebrand factor-binding protein (vWbp) from Staphylococcus aureus interacts with the coagulation factors prothrombin and fibrinogen (Fbg), leading to the non-proteolytic transglutaminase activation of Factor XIII (FXIII). In this study we found that vWbp-activated FXIII catalyses the incorporation of amino-donor dansylcadaverine into region A of fibronectin-binding protein A (FnBPA). Incubation of Fbg with recombinant region A of S. aureus Fbg-binding proteins FnBPA, FnBPB, ClfA or ClfB in presence of vWbp-activated FXIII resulted in the formation of high molecular heteropolymers with FnBPA only, suggesting a specificity of the cross-linking reaction between fibrin(ogen) and the staphylococcal surface. As previously observed, cross-linking sites were mapped to the α-chain and the N1 subdomain of fibrin(ogen) and region A of FnBPA, respectively. Comparable results were obtained when tissue tranglutaminase-2 (TG2) was tested for cross-linking of FnBPA and Fbg. Of note, FnBPA-mediated covalent cross-linking promoted by vWbp-activated FXIII was also observed when bacteria were allowed to attach to fibrin(ogen). Together these findings suggest a novel pathogenetic mechanism by which the transglutaminase action of FXIII and/or TG2 contributes to entrapment and persistence of S. aureus in blood and host tissues.

Fbg αC 389-402 Enhances Factor XIII Cross-Linking in the Fibrinogen αC Region Via Electrostatic and Hydrophobic Interactions.

Coagulation Factor XIII (FXIII) stabilizes blood clots by cross-linking glutamines and lysines in fibrin and other proteins. FXIII activity in the fibrinogen αC region (Fbg αC 221-610) is critical for clot stability and growth. Fbg αC 389-402 is a binding site for thrombin-activated FXIII, (FXIII-A*), with αC E396 promoting FXIII-A* binding and activity in αC. The current study aimed to discover additional residues within Fbg αC 389-402 that accelerate transglutaminase activity toward αC. Electrostatic αC residues (E395, E396, and D390), hydrophobic αC residues (W391 and F394), and residues αC 328-425 were studied by mutations to recombinant Fbg αC 233-425. FXIII activity was monitored through MS-based glycine ethyl ester (GEE) cross-linking and gel-based fluorescence monodansylcadaverine (MDC) cross-linking assays. Truncation mutations 403 Stop (Fbg αC 233-402), 389 Stop (Fbg αC 233-388), and 328 Stop (Fbg αC 233-327) reduced Q237-GEE and MDC cross-linking compared to wild-type (WT). Comparable cross-linking between 389 Stop and 328 Stop showed that FXIII is mainly affected by the loss of Fbg αC 389-402. Substitution mutations E396A, D390A, W391A, and F394A decreased cross-linking relative to WT, whereas E395A, E395S, E395K, and E396D had no effect. Similar FXIII-A* activities were observed for double mutants (D390A, E396A) and (W391A, E396A), relative to D390A and W391A, respectively. In contrast, cross-linking was reduced in (F394A, E396A), relative to F394A. In conclusion, Fbg αC 389-402 boosts FXIII activity in Fbg αC, with D390, W391, and F394 identified as key contributors in enhancing αC cross-linking.

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.

Identification of a novel mutation in the factor XIII A subunit in a patient with inherited factor XIII deficiency.

Inherited factor XIII (FXIII) deficiency is an extremely rare and under-diagnosed autosomal recessive inherited coagulopathy, which is caused by genetic defects in the F13A1 or F13B gene. More than 200 genetic mutations have been identified since the first case of inherited FXIII deficiency was reported. This study aimed to identify underlying gene mutations in a patient with inherited FXIII deficiency who presented with recurrent intracerebral hemorrhage. Levels of plasma FXIII-A antigen were measured, F13A1 and F13B genes were sequenced, mutation information was analyzed, and the mutated protein structure was predicted using bioinformatics methods. Molecular genetic analysis identified four mutations of FXIII-related genes in the proband, including three previously reported mutations inherited from his parents (c.631G>A, p.Gly210Arg and c.1687G>A, p.Gly562Arg of F13A1 gene and c.344G>A, p.Arg115His of F13B gene) and a novel spontaneous mutation of F13A1 gene (c.2063C>G, p.Ser687Cys). Molecular structural modeling demonstrated that the novel Ser687Cys mutation may cause changes in the spatial structure of FXIII-A and increase its instability. In conclusion, we identified a novel and likely pathogenic mutation of the F13A1 gene, which enriched the gene mutation spectrum of inherited FXIII deficiency. The findings may provide promising targets for diagnosis and treatment of inherited FXIII deficiency.

Macrophages CD163+ and Factor XIIIa+ Provide a First-Line Defence against Proliferative Verrucous Leukoplakia Antigens.

This study aimed to evaluate the density of the dendritic cells (DCs) and macrophages in oral leukoplakia (OL) and proliferative verrucous leukoplakia (PVL) by immunohistochemical analysis. We analysed paraffined tissue samples of PVL (n = 27), OL (n = 20), and inflammatory fibrous hyperplasia (n = 20) as the control group using the immunomarkers for DCs (CD1a, CD207, CD83, CD208 and CD123) and macrophages (CD68, CD163, FXIIIa and CD209). A quantitative analysis of positive cells in the epithelial and subepithelial areas was determined. Our results showed a reduction in CD208+ cells in the subepithelial area of the OL and PVL compared to the control. Additionally, we found a higher density of FXIIIa+ and CD163+ cells in the subepithelial area in PVL compared to the OL and control. Four-way MANOVA revealed a relationship between increased CD123+ cell density in the subepithelial area of "high-risk" samples regardless of disease. Macrophages provide the first line of defence against PVL antigens, suggesting a distinct pattern of innate immune system activation in PVL compared to OL, which may contribute to the complexity and the high rate of malignant transformation in the PVL.

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.

Cloning of human anti-factor XIII monoclonal antibody dissects mechanisms of polyclonal antibodies in a single patient.

BACKGROUND: Coagulation factor XIII (FXIII) consists of 2 A (FXIII-A) and 2 B (FXIII-B) subunits that cross-link and strengthen the hemostatic fibrin thrombus; thus, abnormal bleeding occurs when FXIII is significantly reduced. Autoimmune-acquired FXIII deficiency (AiF13D) is characterized by lethal bleeding secondary to the development of autoantibodies against FXIII. However, since anti-FXIII autoantibodies are polyclonal, the mechanism underlying FXIII dysfunction is unclear. OBJECTIVES: The objective of this study was to dissect the inhibitory mechanisms of polyclonal anti-FXIII autoantibodies. METHODS: In this study, we prepared the human monoclonal antibodies (hmAbs) from the peripheral blood of an 86-year-old man with AiF13D by using a new complementary DNA cloning method and analyzed the properties of each autoantibody. RESULTS: Seventeen clones obtained from hmAbs were divided into the following 3 groups: dissociation inhibitors of FXIII-A2B2 (6 clones), assembly inhibitors of FXIII-A2B2 (3 clones), and nonneutralizing/inhibitory hmAbs (8 clones). Dissociation inhibitors strongly inhibited fibrin cross-linking and amine incorporation. Assembly inhibitors extracted FXIII-A from FXIII-A2B2, strongly inhibited binding of FXIII-A to FXIII-B, and activation peptide cleavage. However, the patient's plasma presented a strong inhibition of A2B2 heterodimer assembly but only a slight inhibition of thrombin-Ca2+-dependent dissociation, suggesting that the assembly inhibitors concealed the effect of dissociation inhibitors in plasma. By contrast, nonneutralizing antibodies had little effect on the function of FXIII, suggesting that nonneutralizing hmAbs (and/or dissociation inhibitors and/or assembly inhibitors) promoted the clearance of FXIII-A from the blood. CONCLUSION: Cloning of anti-FXIII autoantibodies enabled us to not only elucidate the mechanism and pathophysiology of AiF13D but also develop a completely new type of anticoagulant.

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.

Coagulation Factor XIIIa and Activated Protein C Activate Platelets via GPVI and PAR1.

Platelet and coagulation activation are highly reciprocal processes driven by multi-molecular interactions. Activated platelets secrete several coagulation factors and expose phosphatidylserine, which supports the activation of coagulation factor proteins. On the other hand, the coagulation cascade generates known ligands for platelet receptors, such as thrombin and fibrin. Coagulation factor (F)Xa, (F)XIIIa and activated protein C (APC) can also bind to platelets, but the functional consequences are unclear. Here, we investigated the effects of the activated (anti)coagulation factors on platelets, other than thrombin. Multicolor flow cytometry and aggregation experiments revealed that the 'supernatant of (hirudin-treated) coagulated plasma' (SCP) enhanced CRP-XL-induced platelet responses, i.e., integrin αIIbß3 activation, P-selectin exposure and aggregate formation. We demonstrated that FXIIIa in combination with APC enhanced platelet activation in solution, and separately immobilized FXIIIa and APC resulted in platelet spreading. Platelet activation by FXIIIa was inhibited by molecular blockade of glycoprotein VI (GPVI) or Syk kinase. In contrast, platelet spreading on immobilized APC was inhibited by PAR1 blockade. Immobilized, but not soluble, FXIIIa and APC also enhanced in vitro adhesion and aggregation under flow. In conclusion, in coagulation, factors other than thrombin or fibrin can induce platelet activation via GPVI and PAR receptors.

Mast cell-derived factor XIIIA contributes to sexual dimorphic defense against group B streptococcal infections.

Invasive bacterial infections remain a major cause of human morbidity. Group B streptococcus (GBS) are Gram-positive bacteria that cause invasive infections in humans. Here, we show that factor XIIIA-deficient (FXIIIA-deficient) female mice exhibited significantly increased susceptibility to GBS infections. Additionally, female WT mice had increased levels of FXIIIA and were more resistant to GBS infection compared with isogenic male mice. We observed that administration of exogenous FXIIIA to male mice increased host resistance to GBS infection. Conversely, administration of a FXIIIA transglutaminase inhibitor to female mice decreased host resistance to GBS infection. Interestingly, male gonadectomized mice exhibited decreased sensitivity to GBS infection, suggesting a role for gonadal androgens in host susceptibility. FXIIIA promoted GBS entrapment within fibrin clots by crosslinking fibronectin with ScpB, a fibronectin-binding GBS surface protein. Thus, ScpB-deficient GBS exhibited decreased entrapment within fibrin clots in vitro and increased dissemination during systemic infections. Finally, using mice in which FXIIIA expression was depleted in mast cells, we observed that mast cell-derived FXIIIA contributes to host defense against GBS infection. Our studies provide insights into the effects of sexual dimorphism and mast cells on FXIIIA expression and its interactions with GBS adhesins that mediate bacterial dissemination and pathogenesis.