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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.

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 (TTG) is a calcium dependent enzyme that covalently crosslinks a wide variety of ECM proteins producing a protease resistant matrix. TTG is secreted by inflammatory and endothelial cells, involved in activating transforming growth factor beta-1 (TGF beta-1) and expressed during wound healing response. In this study, we investigated how TTG 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 TTG into F-ZC resulted in a dose-dependent, 2-fold increase in granulation tissue thickness by day 6 of wound healing (p

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

SummaryHuman tissue transglutaminase (TGM2) is a calcium-dependent crosslinking enzyme involved in the posttranslational modification of intra- and extracellular proteins and implicated in several neurodegenerative diseases. To find specific inhibitors to TGM2, two structurally diverse chemical libraries (LOPAC and Prestwick) were screened. We found that ZM39923, a Janus kinase inhibitor, and its metabolite ZM449829 were the most potent inhibitors with IC50 of 10 and 5 nM, respectively. In addition, two other inhibitors, including tyrphostin 47 and vitamin K3, were found to have an IC50 in the micromolar range. These agents used in part a thiol-dependent mechanism to inhibit TGM2, consistent with the activation of TGM2 by reduction of an intramolecular disulfide bond. These inhibitors were tested in a polyglutamine-expressing Drosophila model of neurodegeneration and found to improve survival. The TGM2 inhibitors we discovered may serve as valuable lead compounds for the development of orally active TGM2 inhibitors to treat human diseases.

The development of hepatocellular carcinoma (HCC) is probably related to infection with hepatitis B virus (HBV). Hepatocytes in livers of patients with HCC have been reported to show putative preneoplastic changes such as hyperplasia, dysplasia, or adenomatous regeneration. To determine quantitatively whether these morphologic changes are associated with HBV-infected cells, the authors performed morphometry of hepatitis B surface antigen (HBsAg)-positive hepatocytes in the nontumorous portion of 10 livers with HCC and in 10 livers without HCC. The diameter of nuclei and cytoplasm of HBsAg-positive hepatocytes was measured after demonstration of HBsAg by the peroxidase-antiperoxidase method. As controls, HBsAg-negative hepatocytes in the same liver sections were measured as well as hepatocytes of 20 age-matched HBsAg-negative patients with normal liver or alcoholic cirrhosis. HBsAg-positive hepatocytes exhibited significantly larger nuclei and a higher nucleocytoplasmic ratio than control hepatocytes. In addition, HBsAg-positive cells were often arranged in foci that consisted of two cell populations: hypertrophic (enlarged nuclei and nucleocytoplasmic ratio) and hyperplastic (two-cell-thick plates of small cells with a high nucleocytoplasmic ratio). While precancerous cells have been difficult to identify, these morphologic changes are frequently associated with the development of malignant neoplasia.