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Human Leukocyte Antigen (HLA)-DQ2 and HLA-DQ8 are genetic risk factors for Type 1 Diabetes Mellitus (T1DM) and Celiac disease (CD) in Caucasians, but their association with Taiwanese Han population is unknown. We screened 532 Taiwanese T1DM patients for CD biomarkers including anti-tissue transglutaminase (TGM2), anti-gliadin and anti-neoepitope antibodies (Abs), sequencing DQB1 genotypes, and characterized the TGM2 Abs. We report that 3.76% of Taiwanese patients had TGM2-Abs and all had no CD's symptoms. In contrast to Caucasian's CD patients, DQ2/DQ8 only constituted ~4/5 of TGM2-Abs positive patients, while the other ~1/5 patients belonged to different HLA genotypes. Either anti-gliadin or anti-neoepitope Abs coexisted with ~3/4 of TGM2-Abs positive patients that were likely due to gluten-ingestion, while the cause of TGM2-Abs production for other ~1/4 of patients was unknown. Purified anti-TGM2 IgA (TGA) and anti-TGM2 IgG (TGG) could bind on endothelial cells surface, recognized native better than denatured forms of TGM2, and TGA inhibited TGM2's transamidation activity by up to 80% but TGG had no effects. Epitope mapping of all TGM2-Abs positive sera demonstrated that TGM2-Abs had heterogeneity in specificities. This is the first study on the differences between Taiwanese Han group and Caucasian in HLA genotypes and properties of TGM2-Abs.

Mitochondria are the cell's power plant to satisfy the energy demands. However, dysfunctional mitochondria can cause overproduction of reactive oxygen species (ROS), oxidative stress, and alteration of calcium homeostasis, which are the hallmarks of mitochondrial diseases. Under prolong oxidative stress, repeated cytosolic calcium elevations even only transiently, can lead to activation of some enzymes. One calcium-activated enzyme with demonstrated pathophysiological important in mitochondrial disease is tissue transglutaminase (TG2). TG2 is known as a post-translational modification (PTM) enzyme that is induced by oxidative stress. Compared to other types of PTMs, the physiological significance of TG2 mediated PTM is just beginning to be understood. Once activated, TG2 can modulate transcription, inactivate metabolic enzymes, and cause aggregation of critical proteins. Recent data indicate that TG2's activity not only can modulate the assembly of respiratory chain complexes but can also modulate the transcription of critical genes including PGC-1alpha and cytochrome C that are important for function and biogenesis of mitochondria. Here, we summarize dysfunctional mitochondria in diseases such as in neurodegenerative disorders can modulate TG2's activity and function. TG2 is also important for normal function of mitochondria.

Until recently, 10% of hepatocellular adenomas (HCAs) remained unclassified (UHCA). Among the UHCAs, the sonic hedgehog HCA (shHCA) was defined by focal deletions that fuse the promoter of Inhibin beta E chain with GLI1. Prostaglandin D2 synthase was proposed as immunomarker. In parallel, our previous work using proteomic analysis showed that most UHCAs constitute a homogeneous subtype associated with overexpression of argininosuccinate synthase (ASS1). To clarify the use of ASS1 in the HCA classification and avoid misinterpretations of the immunohistochemical staining, the aims of this work were to study (1) the link between shHCA and ASS1 overexpression and (2) the clinical relevance of ASS1 overexpression for diagnosis. Molecular, proteomic, and immunohistochemical analyses were performed in UHCA cases of the Bordeaux series. The clinico‐pathological features, including ASS1 immunohistochemical labeling, were analyzed on a large international series of 67 cases. ASS1 overexpression and the shHCA subgroup were superimposed in 15 cases studied by molecular analysis, establishing ASS1 overexpression as a hallmark of shHCA. Moreover, the ASS1 immunomarker was better than prostaglandin D2 synthase and only found positive in 7 of 22 shHCAs. Of the 67 UHCA cases, 58 (85.3%) overexpressed ASS1, four cases were ASS1 negative, and in five cases ASS1 was noncontributory. Proteomic analysis performed in the case of doubtful interpretation of ASS1 overexpression, especially on biopsies, can be a support to interpret such cases. ASS1 overexpression is a specific hallmark of shHCA known to be at high risk of bleeding. Therefore, ASS1 is an additional tool for HCA classification and clinical diagnosis., ShHCA is a new HCA subgroup with a high risk of bleeding with PTGDS and ASS1 proposed as immunomarkers, with conflicting results and interpretations in the literature. By molecular, proteomic, and immunohistochemistry analyses, we established that ASS1 overexpression was a specific hallmark of shHCA. Having shown that PTGDS was not a good marker, we demonstrated, using a large cohort of UHCAs, the sensitivity of ASS1 immunomarker and its clinical relevance. Therefore, ASS1 is an additional tool for HCA classification, clinical diagnosis of shHCA, and appropriate management.

Nitric oxide (NO) produced by endothelial cells in response to cytokines displays anti-inflammatory activity by preventing the adherence, migration and activation of neutrophils. The molecular mechanism by which NO operates at the blood-endothelium interface to exert anti-inflammatory properties is largely unknown. Here we show that on endothelial surfaces, NO is associated with the sulfhydryl-rich protein tissue transglutaminase (TG2), thereby endowing the membrane surfaces with anti-inflammatory properties. We find that tumor necrosis factor-α-stimulated neutrophil adherence is opposed by TG2 molecules that are bound to the endothelial surface. Alkylation of cysteine residues in TG2 or inhibition of endothelial NO synthesis renders the surface-bound TG2 inactive, whereas specific, high affinity binding of S-nitrosylated TG2 (SNO-TG2) to endothelial surfaces restores the anti-inflammatory properties of the endothelium, and reconstitutes the activity of endothelial-derived NO. We also show that SNO-TG2 is present in healthy tissues and that it forms on the membranes of shear-activated endothelial cells. Thus, the anti-inflammatory mechanism that prevents neutrophils from adhering to endothelial cells is identified with TG2 S-nitrosylation at the endothelial cell-blood interface.

Renal anemia is a common complication in patients with advanced chronic kidney disease.

Blood coagulation factor XIIIa is a calcium-dependent enzyme that covalently ligates fibrin molecules during blood coagulation. X-ray crystallography studies identified a major calcium-binding site involving Asp(438), Ala(457), Glu(485), and Glu(490). We mutated two glutamic acid residues (Glu(485) and Glu(490)) and three aspartic acid residues (Asp(472), Asp(476), and Asp(479)) that are in close proximity. Alanine substitution mutants of these residues were constructed, expressed, and purified from Escherichia coli. The K(act) values for calcium ions increased by 3-, 8-, and 21-fold for E485A, E490A, and E485A,E490A, respectively. In addition, susceptibility to proteolysis was increased by 4-, 9-, and 10-fold for E485A, E490A, and E485A,E490A, respectively. Aspartic acids 472, 476, and 479 are not involved directly in calcium binding since the K(act) values were not changed by mutagenesis. However, Asp(476) and Asp(479) are involved in regulating the conformation for exposure of the secondary thrombin cleavage site. This study provides biochemical evidence that Glu(485) and Glu(490) are Ca(2+)-binding ligands that regulate catalysis. The binding of calcium ion to this site protects the molecule from proteolysis. Furthermore, Asp(476) and Asp(479) play a role in modulating calcium-dependent conformational changes that cause factor XIIIa to switch from a protease-sensitive to a protease-resistant molecule.

Plasma fibrinogen plays an important role in hemostasis and inflammation. Fibrinogen is converted to fibrin to impede blood loss and serves as the provisional matrix that aids wound healing. Fibrinogen also binds to cytokine activated endothelial cells and promotes the binding and migration of leukocytes into tissues during inflammation. Tissue transglutaminase (TGM-2) released from injured cells could cross-link fibrinogen to form multivalent complexes that could promote adhesion of platelets and vascular cells to endothelium. Histamine released by mast cells is a potent biogenic amine that promotes inflammation. The covalent attachment of histamine to proteins (histaminylation) by TGM-2 could modify local inflammatory reactions. We investigated TGM-2 crosslinking of several biogenic amines (serotonin, histamine, dopamine and noradrenaline) to fibrinogen. We identified histaminylation of fibrinogen by TGM-2 as a preferred reaction in solid and solution phase transglutaminase assays. Histamine caused a concentration-dependent inhibition of fibrinogen cross-linking by TGM-2. Fibrinogen that was not TGM-2 crosslinked bound to unactivated endothelial cells with low affinity. However, the binding was increased by sevenfold when fibrinogen was cross-linked by TGM-2. Histaminylation of fibrinogen also inhibited TGM-2 crosslinking of fibrinogen and the binding to un-activated HUVEC cells by 75–90 %. In summary, the histaminylation of fibrinogen by TGM-2 could play a role in modifying inflammation by sequestering free histamine and by inhibiting TGM-2 crosslinking of fibrinogen.

Tissue transglutaminase (tTG) exhibits a magnesium-dependent GTP/ATPase activity that is involved in the regulation of the cell cycle and cell receptor signaling. The portion of the molecule involved in GTP/ATP hydrolysis is unknown. We expressed and purified a series of C-terminal truncation mutants of human tTG as glutathione S-transferase fusion proteins (DeltaS538, DeltaE447, DeltaP345, DeltaC290, DeltaV228, and DeltaF185) to determine the effect on GTP/ATPase activity. The truncation of the C terminus did not change significantly the apparent Km value for either GTP or ATP. In contrast, the Kcat value for GTP was increased by 4.6- and 3-fold for the DeltaS538 and DeltaE447 mutants, respectively. The DeltaP345 mutant had the highest hydrolysis activity with a 34-fold increase. The hydrolysis activity then declined to 8.1-, 8.7-, and 1. 9-fold for the DeltaC290, DeltaV228, and DeltaF185 mutants, respectively. The Kcat for ATP changed in parallel with the GTPase results. Thin layer chromatography analysis of the hydrolysis reaction products revealed that ATP was rapidly converted to ADP followed by a much slower conversion of ADP to AMP when incubated with wild type tTG or the DeltaP345 mutant. There was a substantial decrease in the calcium-dependent TGase activity when the last 149 amino acid residues were deleted from the C terminus. Less than 5% of the TGase activity was detected for the DeltaS538 and DeltaE447 mutants. In conclusion, we have located the ATP and GTP hydrolytic domain to amino acid residues 1-185. The C terminus functions to inhibit the expression of endogenous GTP/ATPase activity of tTG, and the potential role of the C terminus in modulating this activity is discussed.

Human tissue transglutaminase (TGM2) is implicated in the pathogenesis of several neurodegenerative disorders including Alzheimer's, Parkinson's and expanded polyglutamine (polyQ) diseases. TGM2 promotes formation of soluble and insoluble high molecular weight aggregates by catalyzing a covalent linkage between peptide-bound Q residues in polyQ proteins and a peptide-bound Lys residue. Therapeutic approaches to modulate the activity of TGM2 are needed to proceed with studies to test the efficacy of TGM2 inhibition in disease processes. We investigated whether acetylation of Lys-residues by sulfosuccinimidyl acetate (SNA) or aspirin (ASA) would alter the crosslinking activity of TGM2. Acetylation by either SNA and/or ASA resulted in a loss of90% of crosslinking activity. The Lys residues that were critical for inhibition were identified by mass spectrometry as Lys(444), Lys(468), and Lys(663). Hence, acetylation of Lys-residues may modulate the enzymatic function of TGM2 in vivo and offer a novel approach to treatment of TGM2 mediated disorders.

The epithelial to mesenchymal transition (EMT), which converts epithelial cells into an elongated, motile, and invasive phenotype, is considered to be a critical step in the dissemination of tumor cells. During EMT, the loss of E-cadherin (ECad) and upregulation of N-cadherin (NCad) or fibronectin (FN) have been most frequently described. FN binds integrin Alpha5 and is known for its upregulation in aggressive tumor cells. In addition, FN binds tissue transglutaminase (TGM-2) with high affinity (∼in nM) and plays a role in TGM-2 secretion. TGM-2, a multifunctional enzyme with transamidation or crosslinking, and GTP/ATP binding activities, is up-regulated in various malignant diseases, including lung cancer. In breast, cervical and ovarian cancers, TGM-2 has been shown to play a role in chemotherapy resistance and tumor metastasis during induction of EMT. TGM-2 overexpression results in the loss of epithelial features, up-regulation of mesenchymal markers, increased migration and invasion, and up-regulation of transcriptional repressors (e.g., ZEB1/2, Snail) known to mediate the EMT process. However, most of these studies were performed in aggressive tumor cells. In this study, we investigated the role of TGM-2 in TGFbeta-induced EMT process using NSCLC H358 cells. H358 cells express high levels of epithelial genes and are relatively resistant to TGFbeta induced EMT when compared to A549 cells. Particularly, H358 expresses barely detectable TGM-2 and FN. To investigate the role of TGM-2 during TGFbeta induced EMT, we constructed three isogenic cell lines with doxycycline (Dox)-inducible TGM-2 or C277A (an inactive mutant of TGM-2 with no transamidating activity) using InVitrogen's Flp-in TRex system. We found continuing treatment of 10 ng/ml TGFbeta for 6 days inducing H358 flp-in (vector control) cells to mescenchymal phenotype as demonstrated by the disappearance of Ecad, and the increased expression of NCad and FN. Under the same condition, Dox inducible TGM-2 and C277A facilitate the disappearance of Ecad, and Occludin and the appearance of Ncad. However, the expression of FN was undetectable in H358/TGM-2 and H358/C277A cells. As a result, H358/TGM-2 and H358/C277A remained localized and failed to migrate as demonstrated by a scratch wound healing assay. Seahorse mitochondria stress assay also demonstrated H358/TGM-2 and H358/C277A utilize more oxidative phosphorylation pathway than glycolytic pathway when compared with H358/flp-in, an indication of shifting cells to more normal phenotype. In summary, overexpression of TGM-2 (or inactive C277A mutant) inhibited the expression of FN and resulted in a less migratory H358 cells. Our results demonstrated that the role of TGM-2 during EMT in less aggressive epithelial cells such as H358 is different from its role in promoting EMT in other aggressive tumor cell; the suppression of FN might be a critical factor. Note: This abstract was not presented at the meeting. Citation Format: Thung S. Lai, Pin-Tsen Lin, Charles S. Greenberg, Harry A. Drabkin, Robert Gemmill. TGM-2 mediated downregulation of fibronectin during TGFb-induced epithelial-mesenchymal transition in lung cancer NCI-H358 cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5192. doi:10.1158/1538-7445.AM2015-5192

Tissue transglutaminase (TG) is an enzyme that stabilizes the structure of tissues by covalently ligating extracellular matrix molecules. Expression and localization of TG are not well established during wound healing. We performed punch biopsy wounds on anesthetized rats and monitored the wound healing process by histological and immunohistochemical methods. The TG antigen and activity are expressed at sites of neovascularization in the provisional fibrin matrix within 24 h of wounding. Endothelial cells, macrophages, and skeletal muscle cells expressed TG throughout the healing process. The TG antigen within the wound was active in vivo based on the detection of isopeptide bonds. The TG antigen increased four- to fivefold by day 3 postwounding and was proteolytically degraded. TG expression occurred in association with TGF-beta, TNF-alpha, IL-6, and VEGF production in the wound. Recombinant TG increased vessel length density (a measure of angiogenesis) when applied topically in rat dorsal skin flap window chambers. We have established that TG is an important tissue stabilizing enzyme that is active during wound healing and can function to promote angiogenesis.

Tissue transglutaminase (tTG) catalyzes a Ca2+-dependent transglutaminase reaction resulting in the formation of gamma-glutamyl-epsilon-lysine bonds and is activated during apoptosis to catalyze the formation of apoptotic body. We investigate whether lipids that are membrane components and involved in cell signaling could modify the Ca2+-dependent activation of tTG. We found that sphingosylphosphocholine (lyso-SM) was the only lipid to activate transglutaminase at low Ca2+ concentrations. In the presence of lyso-SM (125 microM), transglutaminase was detectable at 10 microM Ca2+, whereas in the absence of lyso-SM, similar activity was obtained at 160 microM Ca2+. Furthermore, in the presence of lipid vesicles lyso-SM retained the ability to enhance the Ca2+-dependent activation of tTG. Lyso-SM did not significantly change the Km for the glutamyl and primary amine substrates. However, the Kact for Ca2+ was reduced from 300 microM to 90 microM. Structure-function studies of lyso-SM analogs indicate that phosphocholine group on C1, the free amino group at C2 and a C4-C5 double bond are critical for the activation of transglutaminase activity. This is the first demonstration that a specific sphingolipid could enhance the activity of tTG and could play a role in vivo in activation of the tTG at physiologic Ca2+ levels.

Alternative splicing is an important mechanism for modulating gene function that accounts for a considerable proportion of proteomic complexity in higher eukaryotes. Alternative splicing is often tightly regulated in a cell-type- or developmental-stage- specific manner and can cause a single gene to have multiple functions. Human Tissue transglutaminase (TGM2) is a multifunctional enzyme with transglutaminase crosslinking (TGase), G protein signaling and kinase activities that are postulated to play a role in many disease states. TGM2 mRNA is regulated by alternative splicing, producing C-terminal truncated forms of TGM2 that are predicted to have distinct biochemical properties and biological functions. In this review, we will discuss how alternatively spliced forms of TGM2 could modulate its roles in cancer, neurodegeneration, inflammation and wound healing.

Abstract 3304 Recent studies have demonstrated that a wide variety of biogenic amines can be covalently cross-linked to intracellular and extracellular proteins. This “aminylation” reaction regulates several important reactions in vascular biology. Serotinylation of small GTPases recently was shown to regulate cell signaling events, platelet activation and promote vascular smooth muscle cell proliferation. The role of extracellular crosslinking of biogenic amines to plasma and extracellular matrix proteins is not well established and could play a role in altering transglutaminase reactions in vascular tissue. Tissue translutaminase (TGM-2) crosslinking of the a-C domain of fibrinogen has been shown to promote clustering of RGD domains, enhance binding of the cross-linked polymers to endothelial cells and promote cell adhesion. While Factor XIIIa prefers to crosslink fibrin, the TGM-2 molecule which is abundantly expressed by erythrocytes, endothelial and vascular smooth muscle cells does not show any preference for fibrin compared to fibrinogen. TGM-2 is a sulfhydryl rich calcium-dependent enzyme that could cross-link a variety of biogenic amines to fibrinogen. We investigated the preference of TGM-2 crosslinking of biogenic amines to fibrinogen in vitro and determined whether the crosslinking would modify the transglutaminase-dependent binding of fibrinogen to endothelial cells. We found histamine was the most effective primary amine inhibitor of the TGM2-mediated cross-linking reaction (Histamine > putrescine >>> serotonin, dopamine, noradrenaline). 1.25 mM histamine inhibited > 75% TGM2-mediated fibrinogen and fibrin cross-linking. The ability of TGM-2 crosslinked fibrinogen complexes to bind to confluent human umbilical vascular endothelial cell (HUVEC) was also studied. Free [125I]-fibrinogen bound to endothelial cells with low affinity, however the binding was increased ∼7 fold when fibrinogen was cross-linked by TGM2. The increase in crosslinked fibrinogen binding was dependent on TGM2 and Ca+2 concentration. Unlabeled crosslinked fibrinogen inhibited the binding by more than 85%. In contrast, unlabeled fibrinogen actually enhanced the binding 1.7 fold suggesting that fibrinogen and cross-linked fibrinogen formed multivalent complexes with cross-linked fibrinogens on the endothelial cell surface. When bound complex were eluted from HUVEC cells after binding experiments, >95% of bound materials were extensively crosslinked and could not enter a 5–15% polyacrylamide gel. In contrast, no high molecular weight material was eluted when non-crosslinked fibrinogen was used in the binding experiments. When fibrinogen was cross-linked in the presence of 8 to 500 micromolar of histamine, the binding was inhibited by ∼75 to 90 %, respectively. In summary, TGM-2 cross-linked fibrinogen showed enhanced binding to endothelial cells as previously reported for purified aC domains of fibrinogen. The binding of fibrinogen to endothelial cells is known to enhance endothelial cell adhesion and leukocyte transmigration, reactions that are involved in wound healing, angiogenesis and inflammation. The TGM-2 crosslinked fibrinogen/fibrinogen complexes may serve as proinflammatory, prothrombotic and proangiogenic factors in vivo. Histaminylation of fibrinogen by TGM-2 could provide a mechanism to regulate these vascular events. Fibrinogen could also serve to control local histamine function as only free histamine can bind to histamine receptors. Transglutaminase mediated histaminylation of fibrinogen could have multiple effects on acute and chronic inflammatory reactions. Disclosures: No relevant conflicts of interest to declare.

Abstract 3055 Poster Board II-1031 Fibrin deposition triggers an injury response that involves the migration of inflammatory cells, formation of new blood vessels and the synthesis of extracellular matrix (ECM). Tissue transglutaminase (TGM2) is a calcium dependent enzyme that covalently crosslinks a wide variety of ECM proteins producing a protease resistant matrix. TGM2 is secreted by inflammatory and endothelial cells, involved in activating transforming growth factor beta-1 (TGFbeta-1) and expressed during tissue injury. In this study, we investigated how TGM2 modulated fibrin-dependent wound healing and the associated angiogenic response. We used an animal model consisting of fibrin Z-chambers (F-ZC, dual porous plexiglass chambers containing fibrin) implanted into the subcutaneous tissue of rats and harvested subsequently for quantitative assessment of granulation tissue formation (wound healing) and microvessel density (angiogenesis). We found that local administration of recombinant TGM2 into F-ZC resulted in a dose-dependent, 2-fold increase in granulation tissue thickness by day 6 of wound healing (p We then investigated whether Aspirin (Acetylsalicylic Acid, ASA) a potent anti-inflammatory agent would inhibit TGM2. ASA and another chemical acetylating agent, sulfosuccinimidyl acetate (SNA), were used to investigate whether acetylation would alter the crosslinking activity of TGM2. We found acetylation by either SNA or ASA resulted in a loss of >90% of crosslinking activity. The Lys residues that were critical for inhibition were identified by mass spectrometry as Lys468 and Lys663. Molecular modeling indicates that these Lys residues play an important role in the conformation change that occurs in TGM2 from a closed-to-open shape, i.e. inactive-to-active, transitions. In conclusion, we show that TGM2-fibrin crosslinking accelerates angiogenesis and promotes ECM deposition. This suggests that TGM2-fibrin interactions mediates outside-in signaling events that aides wound healing. Furthermore aspirin can acetylate and inhibit critical residues in TGM2 that regulate TGM-2 function. Disclosures No relevant conflicts of interest to declare.

Tissue transglutaminase (TTG) is a multifunctional protein that plays a role in several different hematological processes. TTG is a unique member in transglutaminase gene family in that it exhibits multiple enzymatic properties including transglutaminase (TGase) and GTP/ATP hydrolysis activities. The Ca+2-dependent TGase activity catalyzes an isopeptide bond between a specific γ-glutamyl (Q) containing peptide and ε-amine group from a peptide-bound lysine (K) residue that functions to stabilize proteins and plays a role in wound healing, angiogenesis, cell proliferation, and apoptosis. In the presence of Mg+2, TTG hydrolyzes GTP to GDP, and functions as a G protein (Gαh). TTG can also hydrolyze ATP and functions as a kinase that phosphorylates histones and P53. In contrast to GTP, ATP binding does not inhibit TGase activity and involves distinct binding residues. Although normal activation of TTG is required for normal physiological process, aberrant activation of TGase function is reported to play a role in many inflammatory disorders and has been selected as target for therapeutic intervention to control fibrosis, angiogenesis, inflammation and apoptosis. In an effort to screen for specific small chemical inhibitors of TTG, we investigated a structurally diverse Lopac library (Sigma) containing 1280 pharmacologically active compounds that span a broad range of biological areas. This library contains marketed drugs, failed development candidates and “gold standards” that have well-characterized activities. Initial screening was performed using a solution-phase continuous fluorescent TGase assay performed at 6 μg/ml of purified recombinant human TTG at 37°C for 1 hour in the presence of 10 mM Ca+2 in a 96-well microtiter plate designed for high-throughput screening. In the absence of chemicals, the assay had a 3-fold increase in fluorescent intensity (λEx 340nm; λEm 520nm) when BOC-K-EDA-Dansyl (KXD; a K-substrate) was crosslinked to N, N′-dimethylcasein (a Q substrate). Initially, 20 hits were identified based on inhibition of ≥ 90% of TGase activity at 50 μM of chemicals excluding the well-characterized inhibitors cystamine and iodoacetamide, which target the active site Cys-SH of TTG. These hits were more potent than GTP as 50 μM of GTP only inhibited ≤ 25 % of TGase activity. These hits were subjected to a secondary screening using a colorimetric TGase assay that measured the covalent incorporation of biotinylated pentylamine (BP) into NMC coated microtiter plate. A total of six chemicals designated as LL1 (a c-Raf1 kinase inhibitor), LL2 (a JNK-3 kinase inhibitor), LL3 (a EGFR tyrosine kinase inhibitor), LL4 (protein kinase C and Calmodulin Kinase inhibitors), LL5 (cdc25 phosphatase inhibitor) and LL6 (DNA topoisomerase I inhibitor) were validated. All these inhibitors are known to target kinases and phosphatase by functioning as GTP and ATP analogs with guanine, adenine and quinone as backbone structures. The mechanism of inhibition of TGase activity is under investigation and might be similar to that of GTP. The fact that some kinase and phosphatase inhibitors also inhibit TGase activity further consolidating TTG as a member of kinase supergene family, but also raise a challenging task to develop a specific inhibitor to TTG. However, the chemical inhibitors discovered in current study can be used as a warhead to develop more specific inhibitor of TTG.

FactorXIIIa (FXIIIa) plays an important role in regulating hemostatic and thrombotic events. FXIIIa modifies the structure of fibrin making the insoluble fibrin gel resistant to plasmin degradation. Methods to detect FXIIIa crosslinking of soluble fibrin(ogen) complexes (SFCs) are not readily available. The purpose of this study was to 1) develop a sensitive assay to detect FXIIIa crosslinked (XL)-SFCs designated: cryptic D-dimer assay, 2) determine whether soluble XL-SFCs are formed prior to formation of an insoluble clot, 3) determine whether soluble XL-SFCs circulated in human plasma and could be incorporated into a fibrin clot, 4) determine whether levels of soluble XL-SFCs are elevated in patients suspected of having intravascular coagulation. We postulated that the cryptic D-dimer antigen would be created by FXIIIa crosslinking of SFCs and could be detected after plasmin digestion. We added purified recombinant FXIIIa (119 nM) to afibrinogenemic plasma with varying concentrations of purified fibrinogen (10–200 μg/mL) and assayed for D-dimer antigen levels pre- and post- plasmin treatment. D-dimer levels were determined by a quantitative latex agglutination assay (MiniQuant® D-dimer, Ireland). The assay utilizes a monoclonal antibody specific for an epitope that is present on plasmin modified crosslinked D-dimer domain on either fibrinogen or fibrin. We found that generation of cryptic D-dimer antigen was a) dependent on FXIIIa, b) uncovered only after plasmin digestion and c) increased with escalating fibrinogen concentration. The level of cryptic D-dimer antigen increased from 0.45 μg/ml to 5.4 μg/ml in the presence of fibrinogen (10–200 μg/ml), FXIIIa and 5 mM calcium chloride. We had earlier confirmed that the cryptic D-dimer antigen could be detected in both EDTA and citrated plasmas and the exposure of this antigen was not modified by calcium present during plasmin digestion. We also determined that the cryptic D-dimer antigen was generated 150 sec before a visible clot appeared in recalcifed plasma in plastic tubes. We then investigated whether cryptic D-dimer antigen circulated in healthy individuals and if these levels changed in patients. Cryptic D-dimer antigen was detected (mean level 1 μg/ml