Antigenic Cross-Reactivity Between SARS-CoV-2 S1-RBD and Its Receptor ACE2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emerging virus responsible for the ongoing COVID-19 pandemic. SARS-CoV-2 binds to the human cell receptor angiotensin-converting enzyme 2 (ACE2) through its receptor-binding domain in the S1 subunit of the spike protein (S1-RBD). The serum levels of autoantibodies against ACE2 are significantly higher in patients with COVID-19 than in controls and are associated with disease severity. However, the mechanisms through which these anti-ACE2 antibodies are induced during SARS-CoV-2 infection are unclear. In this study, we confirmed the increase in antibodies against ACE2 in patients with COVID-19 and found a positive correlation between the amounts of antibodies against ACE2 and S1-RBD. Moreover, antibody binding to ACE2 was significantly decreased in the sera of some COVID-19 patients after preadsorption of the sera with S1-RBD, which indicated that antibodies against S1-RBD can cross-react with ACE2. To confirm this possibility, two monoclonal antibodies (mAbs 127 and 150) which could bind to both S1-RBD and ACE2 were isolated from S1-RBD-immunized mice. Measurement of the binding affinities by Biacore showed these two mAbs bind to ACE2 much weaker than binding to S1-RBD. Epitope mapping using synthetic overlapping peptides and hydrogen deuterium exchange mass spectrometry (HDX-MS) revealed that the amino acid residues P463, F464, E465, R466, D467 and E471 of S1-RBD are critical for the recognition by mAbs 127 and 150. In addition, Western blotting analysis showed that these mAbs could recognize ACE2 only in native but not denatured form, indicating the ACE2 epitopes recognized by these mAbs were conformation-dependent. The protein-protein interaction between ACE2 and the higher affinity mAb 127 was analyzed by HDX-MS and visualized by negative-stain transmission electron microscopy imaging combined with antigen-antibody docking. Together, our results suggest that ACE2-cross-reactive anti-S1-RBD antibodies can be induced during SARS-CoV-2 infection due to potential antigenic cross-reactivity between S1-RBD and its receptor ACE2.

Proteome profiling reveals novel biomarkers to identify complicated parapneumonic effusions.

Patients with pneumonia and parapneumonic effusion (PPE) have elevated mortality and a poor prognosis. The aim of this study was to discover novel biomarkers to help distinguish between uncomplicated PPE (UPPE) and complicated PPE (CPPE). Using an iTRAQ-based quantitative proteomics, we identified 766 proteins in pleural effusions from PPE patients. In total, 45 of these proteins were quantified as upregulated proteins in CPPE. Four novel upregulated candidates (BPI, NGAL, AZU1, and calprotectin) were selected and further verified using enzyme-linked immunosorbent assays (ELISAs) on 220 patients with pleural effusions due to different causes. The pleural fluid levels of BPI, NGAL, AZU1, and calprotectin were significantly elevated in patients with CPPE. Among these four biomarkers, BPI had the best diagnostic value for CPPE, with an AUC value of 0.966, a sensitivity of 97%, and a specificity of 91.4%. A logistic regression analysis demonstrated a strong association between BPI levels > 10 ng/ml and CPPE (odds ratio = 341.3). Furthermore, the combination of pleural fluid BPI levels with LDH levels improved the sensitivity and specificity to 100% and 91.4%, respectively. Thus, our findings provided a comprehensive effusion proteome data set for PPE biomarker discovery and revealed novel biomarkers for the diagnosis of CPPE.

A novel small-form NEDD4 regulates cell invasiveness and apoptosis to promote tumor metastasis.

Despite numerous investigations on metastasis, the determinants of metastatic processes remain unclear. We aimed to identify the metastasis-associated genes in hepatocellular carcinoma (HCC). Potent metastatic SK-hep-1 (SK) cells, designated 'SKM', were generated using Transwell assay followed by selection in a mouse model. Genes expressed differentially in SKM and SK cells were identified via microarray analyses. A small form of Neural precursor cell-expressed developmentally downregulated 4 (sNEDD4) was identified to be overexpressed in SKM cells, which was confirmed as a novel transcript using liquid chromatography-mass spectrometry. In clinical specimens, sNEDD4 was significantly overexpressed in tumors and serves as a poor prognostic factor for male patients with HCC (P = 0.035). Upon subcutaneous introduction of sNEDD4-overexpressing SK cells into flanks of nude mice, tumors grew faster than those of the control group. Furthermore, sNEDD4-mediated promotion of tumor metastasis was demonstrated in the orthotopic mouse model. Overexpression of sNEDD4 increased the invasive ability of SK cells through upregulation of matrix metalloproteinase 9 and inhibited serum deprivation-induced apoptosis via upregulation of myeloid cell leukemia 1. In conclusion, sNEDD4 is a novel metastasis-associated gene, which prevents apoptosis under nutrient restriction conditions. The present findings clearly support the prognostic potential of sNEDD4 for HCC.

In-depth proteomic analysis of six types of exudative pleural effusions for nonsmall cell lung cancer biomarker discovery.

Pleural effusion (PE), a tumor-proximal body fluid, may be a promising source for biomarker discovery in human cancers. Because a variety of pathological conditions can lead to PE, characterization of the relative PE proteomic profiles from different types of PEs would accelerate discovery of potential PE biomarkers specifically used to diagnose pulmonary disorders. Using quantitative proteomic approaches, we identified 772 nonredundant proteins from six types of exudative PEs, including three malignant PEs (MPE, from lung, breast, and gastric cancers), one lung cancer paramalignant PE, and two benign diseases (tuberculosis and pneumonia). Spectral counting was utilized to semiquantify PE protein levels. Principal component analysis, hierarchical clustering, and Gene Ontology of cellular process analyses revealed differential levels and functional profiling of proteins in each type of PE. We identified 30 candidate proteins with twofold higher levels (q<0.05) in lung cancer MPEs than in the two benign PEs. Three potential markers, MET, DPP4, and PTPRF, were further verified by ELISA using 345 PE samples. The protein levels of these potential biomarkers were significantly higher in lung cancer MPE than in benign diseases or lung cancer paramalignant PE. The area under the receiver-operator characteristic curve for three combined biomarkers in discriminating lung cancer MPE from benign diseases was 0.903. We also observed that the PE protein levels were more clearly discriminated in effusions in which the cytological examination was positive and that they would be useful in rescuing the false negative of cytological examination in diagnosis of nonsmall cell lung cancer-MPE. Western blotting analysis further demonstrated that MET overexpression in lung cancer cells would contribute to the elevation of soluble MET in MPE. Our results collectively demonstrate the utility of label-free quantitative proteomic approaches in establishing differential PE proteomes and provide a new database of proteins that can be used to facilitate identification of pulmonary disorder-related biomarkers.

Targeted proteomics pipeline reveals potential biomarkers for the diagnosis of metastatic lung cancer in pleural effusion.

The ability to discriminate lung cancer malignant pleural effusion (LC-MPE) from benign pleural effusion has profound implications for the therapy and prognosis of lung cancer. Here, we established a pipeline to verify potential biomarkers for this purpose. In the discovery phase, label-free quantification was performed for the proteome profiling of exudative pleural effusion in order to select 34 candidate biomarkers with significantly elevated levels in LC-MPE. In the verification phase, signature peptides for 34 candidates were first confirmed by accurate inclusion mass screening (AIMS). To quantify the candidates in PEs, multiple reaction monitoring mass spectrometry (MRM-MS) with stable isotope-labeled standards (SIS) peptides was performed for the 34 candidate biomarkers using the QconCAT approach for the generation of the SIS peptides. The results of the MRM assay were used to prioritize candidates based on their discriminatory power in 82 exudative PE samples. The five potential biomarkers (ALCAM, CDH1, MUC1, SPINT1, and THBS4; AUC > 0.7) and one three-marker panel (SPINT1/SVEP1/THBS4; AUC = 0.95) were able to effectively differentiate LC-MPE from benign PE. Collectively, these results demonstrate that our pipeline is a feasible platform for verifying potential biomarkers for human diseases.

Comparative and targeted proteomic analyses of urinary microparticles from bladder cancer and hernia patients.

Bladder cancer is a common urologic cancer whose incidence continues to rise annually. Urinary microparticles are an attractive material for noninvasive bladder cancer biomarker discovery. In this study, we applied isotopic dimethylation labeling coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to discover bladder cancer biomarkers in urinary microparticles isolated from hernia (control) and bladder cancer patients. This approach identified 2964 proteins based on more than two distinct peptides, of which 2058 had not previously been reported as constituents of human urine exosomes/microparticles. A total of 107 differentially expressed proteins were identified as candidate biomarkers. Differences in the concentrations of 29 proteins (41 signature peptides) were precisely quantified by LC-MRM/MS in 48 urine samples of bladder cancer, hernia, and urinary tract infection/hematuria. Concentrations of 24 proteins changed significantly (p<0.05) between bladder cancer (n=28) and hernia (n=12), with area-under-the-curve values ranging from 0.702 to 0.896. Finally, we quantified tumor-associated calcium-signal transducer 2 (TACSTD2) in raw urine specimens (n=221) using a commercial ELISA and confirmed its potential value for diagnosis of bladder cancer. Our study reveals a strong association of TACSTD2 with bladder cancer and highlights the potential of human urinary microparticles in the noninvasive diagnosis of bladder cancer.

Overexpression of ADP-ribosylation factor 1 in human gastric carcinoma and its clinicopathological significance.

Gastric cancer is the sixth leading cause of cancer-related death in Taiwan, and the identification of related factors is essential to increase patient survival. ADP-ribosylation factor 1 (ARF1) was initially identified using 2-D electrophoresis combined with MALDI-time-of-flight mass spectrometry. ADP-ribosylation factor 1 belongs to the Ras superfamily or GTP-binding protein family and has been shown to enhance cell proliferation. In the current study, we evaluated the potential of ARF1 as a biomarker for gastric cancer detection. ADP-ribosylation factor 1 mRNA was upregulated in tumor tissues (compared with adjacent non-tumor tissues, n = 55) in approximately 67.2% of gastric cancer patients. Expression of ARF1 protein was additionally observed using Western blot and immunohistochemistry (IHC) analyses. The clinicopathological correlations of ARF1 were further evaluated. Elevated ARF1 expression was strongly correlated with lymph node metastasis (P = 0.008), serosal invasion (P = 0.046), lymphatic invasion (P = 0.035), and pathological staging (P = 0.010). Moreover, the 5-year survival rate for the lower ARF1 expression group (n = 50; IHC score < 90) was higher than that of the higher expression group (n = 60; IHC score ≥ 90) (P = 0.0228, log-rank test). To establish the specific function of ARF1 in human gastric cancer, isogenic ARF1-overexpressing cell lines were prepared. Our results showed that ARF1-overexpressing clones display enhanced cell proliferation, migration, and invasion. Furthermore, ARF1-overexpression might contribute to poor prognosis of patients. These findings collectively support the utility of ARF1 as a novel prognostic marker for gastric cancer and its role in cell invasion.

Comprehensive proteome analysis of malignant pleural effusion for lung cancer biomarker discovery by using multidimensional protein identification technology.

Malignant pleural effusion (MPE) obtained from lung adenocarcinoma may contain potentially useful biomarkers for detection of lung cancer. In this study, we used a removal system for high-abundance proteins followed by one-dimensional SDS-PAGE combined with nano-LC-MS/MS to generate a comprehensive MPE proteome data set with 482 nonredundant proteins. Next, we integrated the MPE proteome and secretome data sets from three adenocarcinoma cell lines, with a view to identifying potential PE biomarkers originating from malignant cells. Four potential candidates, alpha-2-HS-glycoprotein (AHSG), angiogenin, cystatin-C, and insulin-like growth factor-binding protein 2, (IGFBP2), were isolated for preclinical validation using ELISA. Both AHSG and IGFBP2 levels were increased in lung patients with MPE (n = 68), compared to those with nonmalignant pleural effusion (n = 119). Notably, the IGFBP2 level was higher in MPE, compared with that in benign diseases (bacteria pneumonia and tuberculosis pleuritis), and significantly associated with malignancy, regardless of the cancer type. Our data additionally support an extracellular function of IGFBP2 in migration in lung cancer cells. These findings collectively suggest that the adenocarcinoma MPE proteome provides a useful data set for malignancy biomarker research.

Comprehensive proteomic analysis of mineral nanoparticles derived from human body fluids and analyzed by liquid chromatography-tandem mass spectrometry.

Mineralo-protein nanoparticles (NPs) formed spontaneously in the body have been associated with ectopic calcifications seen in atherosclerosis, chronic degenerative diseases, and kidney stone formation. Synthetic NPs are also known to become coated with proteins when they come in contact with body fluids. Identifying the proteins found in NPs should help unravel how NPs are formed in the body and how NPs in general, be they synthetic or naturally formed, interact within the body. Here, we developed a proteomic approach based on liquid chromatography (LC) and tandem mass spectrometry (MS/MS) to determine the protein composition of carbonate-apatite NPs derived from human body fluids (serum, urine, cerebrospinal fluid, ascites, pleural effusion, and synovial fluid). LC-MS/MS provided not only an efficient and comprehensive determination of the protein constituents, but also a semiquantitative ranking of the identified proteins. Notably, the identified NP proteins mirrored the protein composition of the contacting body fluids, with albumin, fetuin-A, complement C3, α-1-antitrypsin, prothrombin, and apolipoproteins A1 and B-100 being consistently associated with the particles. Since several coagulation factors, calcification inhibitors, complement proteins, immune regulators, protease inhibitors, and lipid/molecule carriers can all become NP constituents, our results suggest that mineralo-protein complexes may interface with distinct biochemical pathways in the body depending on their protein composition. We propose that LC-MS/MS be used to characterize proteins found in both synthetic and natural NPs.

Quantitative plasma proteome analysis reveals aberrant level of blood coagulation-related proteins in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC), one of the most common cancers in Southeast Asia, is not easily diagnosed until advanced stages. To discover potential biomarkers for improving NPC diagnosis, we herein identified the aberrant plasma proteins in NPC patients. We first removed the top-seven abundant proteins from plasma samples of healthy controls and NPC patients, and then labeled the samples with different fluorescent cyanine dyes. The labeled samples were then mixed equally and fractionated with ion-exchange chromatography followed by SDS-PAGE. Proteins showing altered levels in NPC patients were identified by in-gel tryptic digestion and LC-MS/MS. When the biological roles of the 45 identified proteins were assessed via MetaCore™ analysis, the blood coagulation pathway emerged as the most significantly altered pathway in NPC plasma. Plasma kallikrein (KLKB1) and thrombin-antithrombin III complex (TAT) were chosen for evaluation as the candidate NPC biomarkers because of their involvement in blood coagulation. ELISAs confirmed the elevation of their plasma levels in NPC patients versus healthy controls. Western blot and activity assays further showed that the KLKB1 active form was significantly increased in NPC plasma. Collectively, our results identified the significant alteration of blood coagulation pathway in NPC patients, and KLKB1 and TAT may represent the potential NPC biomarkers.

Importin subunit alpha-2 is identified as a potential biomarker for non-small cell lung cancer by integration of the cancer cell secretome and tissue transcriptome.

The cancer cell secretome may contain potentially useful biomarkers. In this study, we integrated the profiles of secreted proteins in lung cancer cell lines with mRNA expression levels from pulmonary adenocarcinoma tissue, with a view to identify effective biomarkers for non-small cell lung cancer (NSCLC). Among the novel candidates isolated, importin subunit alpha-2 (also known as karyopherin subunit alpha [KPNA]-2), was selected for further validation. Immunohistochemical staining revealed overexpression of KPNA2 in the nuclei of tumor cells, compared with adjacent normal cells. A sandwich ELISA assay developed to detect KPNA2 levels in serum samples showed significantly higher serum KPNA2 in NSCLC patients than in healthy controls. A combination of serum KPNA2 and carcinoembryonic antigen displayed higher diagnostic capacity than either marker alone. Importantly, protein levels of KPNA2 in pleural effusion from NSCLC patients were also significantly higher than those from non-lung cancer. Moreover, knockdown of KPNA2 inhibited the migration ability and viability of lung cancer cells. Our results collectively suggest that integration of the cancer cell secretome and transcriptome datasets provides an efficient means of identifying novel biomarkers for NSCLC, such as KPNA2.

Discovery of novel bladder cancer biomarkers by comparative urine proteomics using iTRAQ technology.

A urine sample preparation workflow for the iTRAQ (isobaric tag for relative and absolute quantitation) technique was established. The reproducibility of this platform was evaluated and applied to discover proteins with differential levels between pooled urine samples from nontumor controls and three bladder cancer patient subgroups with different grades/stages (a total of 14 controls and 23 cancer cases in two multiplex iTRAQ runs). Combining the results of two independent clinical sample sets, a total of 638 urine proteins were identified. Among them, 55 proteins consistently showed >2-fold differences in both sample sets. Western blot analyses of individual urine samples confirmed that the levels of apolipoprotein A-I (APOA1), apolipoprotein A-II, heparin cofactor 2 precursor and peroxiredoxin-2 were significantly elevated in bladder cancer urine specimens (n = 25-74). Finally, we quantified APOA1 in a number of urine samples using a commercial ELISA and confirmed again its potential value for diagnosis (n = 126, 94.6% sensitivity and 92.0% specificity at a cutoff value of 11.16 ng/mL) and early detection (n = 71, 83.8% sensitivity and 94.0% specificity). Collectively, our results provide the first iTRAQ-based quantitative profile of bladder cancer urine proteins and represent a valuable resource for the discovery of bladder cancer markers.

Candidate serological biomarkers for cancer identified from the secretomes of 23 cancer cell lines and the human protein atlas.

Although cancer cell secretome profiling is a promising strategy used to identify potential body fluid-accessible cancer biomarkers, questions remain regarding the depth to which the cancer cell secretome can be mined and the efficiency with which researchers can select useful candidates from the growing list of identified proteins. Therefore, we analyzed the secretomes of 23 human cancer cell lines derived from 11 cancer types using one-dimensional SDS-PAGE and nano-LC-MS/MS performed on an LTQ-Orbitrap mass spectrometer to generate a more comprehensive cancer cell secretome. A total of 31,180 proteins was detected, accounting for 4,584 non-redundant proteins, with an average of 1,300 proteins identified per cell line. Using protein secretion-predictive algorithms, 55.8% of the proteins appeared to be released or shed from cells. The identified proteins were selected as potential marker candidates according to three strategies: (i) proteins apparently secreted by one cancer type but not by others (cancer type-specific marker candidates), (ii) proteins released by most cancer cell lines (pan-cancer marker candidates), and (iii) proteins putatively linked to cancer-relevant pathways. We then examined protein expression profiles in the Human Protein Atlas to identify biomarker candidates that were simultaneously detected in the secretomes and highly expressed in cancer tissues. This analysis yielded 6-137 marker candidates selective for each tumor type and 94 potential pan-cancer markers. Among these, we selectively validated monocyte differentiation antigen CD14 (for liver cancer), stromal cell-derived factor 1 (for lung cancer), and cathepsin L1 and interferon-induced 17-kDa protein (for nasopharyngeal carcinoma) as potential serological cancer markers. In summary, the proteins identified from the secretomes of 23 cancer cell lines and the Human Protein Atlas represent a focused reservoir of potential cancer biomarkers.

Discovery of retinoblastoma-associated binding protein 46 as a novel prognostic marker for distant metastasis in nonsmall cell lung cancer by combined analysis of cancer cell secretome and pleural effusion proteome.

Nonsmall cell lung cancer (NSCLC) is the most common type of lung cancer, which is one of the most prominent causes of cancer-related mortality worldwide. Discovery of serum tumor markers could facilitate early NSCLC detection and metastatic prognosis. Here, we simultaneously analyzed the NSCLC cell secretome and proteomic profiles of pleural effusion from lung adenocarcinoma patients for NSCLC biomarker discovery. Retinoblastoma-associated binding protein 46 (RbAp46), one of the proteins detected both in NSCLC cell secretome and pleural effusion proteome, was chosen for further evaluation. Both of RbAp46 mRNA and protein levels were upregulated significantly in NSCLC cancer tissues. Serum levels of RbAp46 were markedly higher in NSCLC patients than in healthy controls, and a combination of RbAp46 and CEA could outperform CEA alone in discriminating NSCLC patients from healthy persons. Importantly, elevated serum RbAp46 level was highly correlated with NSCLC distant metastasis. Moreover, knockdown of RbAp46 inhibited the migration ability of lung cancer cells. Our data collectively suggest that RbAp46 serves as a novel biomarker and prognosticator for NSCLC, and is involved in lung cancer cell migration.

Direct regulation of androgen receptor-associated protein 70 by thyroid hormone and its receptors.

Thyroid hormone (T3) regulates multiple physiological processes during development, growth, differentiation, and metabolism. Most T3 actions are mediated via thyroid hormone receptors (TRs) that are members of the nuclear hormone receptor superfamily of ligand-dependent transcription factors. The effects of T3 treatment on target gene regulation was previously examined in TRalpha1-overexpressing hepatoma cell lines (HepG2-TRalpha1). Androgen receptor (AR)-associated protein 70 (ARA70) was one gene found to be up-regulated by T3. The ARA70 is a ligand-dependent coactivator for the AR and was significantly increased by 4- to 5-fold after T3 treatment by Northern blot analyses in the HepG2-TRalpha1 stable cell line. T3 induced a 1- to 2-fold increase in the HepG2-TRbeta1 stable cell line. Both stable cell lines attained the highest fold expression after 24 h treatment with 10 nM T3. The ARA70 protein was increased up to 1.9-fold after T3 treatment in HepG2-TRalpha1 cells. Similar findings were obtained in thyroidectomized rats after T3 application. Cycloheximide treatment did not suppress induction of ARA70 transcription by T3, suggesting that this regulation is direct. A series of deletion mutants of ARA70 promoter fragments in pGL2 plasmid were generated to localize the thyroid hormone response element (TRE). The DNA fragments (-234/-190 or +56/+119) gave 1.55- or 2-fold enhanced promoter activity by T3. Thus, two TRE sites exist in the upstream-regulatory region of ARA70. The TR-TRE interaction was further confirmed with EMSAs. Additionally, ARA70 could interfere with TR/TRE complex formation. Therefore, the data indicated that ARA70 suppresses T3 signaling in a TRE-dependent manner. These experimental results suggest that T3 directly up-regulates ARA70 gene expression. Subsequently, ARA70 negatively regulates T3 signaling.

Overexpression of CLIC1 in human gastric carcinoma and its clinicopathological significance.

Gastric cancer is the second most common cancer worldwide and the fifth leading cause of cancer-related death in Taiwan. Identification of biomarkers is essential to improve patient survival. Fifty aberrantly expressed proteins were identified using 2-DE combined with MALDI TOF MS and were grouped based on their function. The overexpression of proteins was confirmed using real-time quantitative RT-PCR, Western blot, and immunohistochemical analysis. The clinicopathological correlations and prognostic significance of these aberrantly expressed proteins were evaluated to determine the novel gastric cancer biomarkers. In this study, expression of chloride intracellular channel 1 (CLIC1) is significantly up-regulated in 67.9% of gastric patients and was selected for further study. The CLIC1 expression in tumor tissues was increased by 1.95-fold (range, 0.01-6.19-fold) compared with that expressed by adjacent noncancerous mucosa. Elevated CLIC1 expression was strongly correlated with lymph node metastasis, lymphatic invasion, perineural invasion, and pathological staging. Additionally, the 5-year survival rate for the low CLIC1 expression group (n = 28; <1.72-fold) was higher than that for the high CLIC1 expression group (n = 28; >or=1.72-fold) (log rank, p = 0.0300). Experimental results indicate that overexpression of CLIC1 is a potential prognostic marker for gastric cancer.

Thyroid hormone receptor-dependent transcriptional regulation of fibrinogen and coagulation proteins.

Thyroid hormone (T(3)) regulates growth, development, and differentiation. These activities are mediated by the nuclear thyroid hormone receptors (TRs), which belong to the steroid/TR superfamily of ligand-dependent transcription factors. The effect of T(3) treatment on target gene regulation was investigated in a TRalpha-overexpressing hepatoma cell line (HepG2-TRalpha), by performing cDNA microarrays. We demonstrate that 148 of the 7597 genes represented were up-regulated by T(3), including fibrinogen and several other components of the coagulation factor system. To confirm the microarray results, fibrinogen and a small number of the blood clotting components were further investigated using quantitative RT-PCR. The T(3)-induction ratios observed with quantitative RT-PCR for factors such as thrombin (8-fold), coagulation factor X (4.9-fold), and hepatoglobin (30-fold) were similar to those observed by the cDNA microarray analysis. Further investigation, using HepG2-TRalpha (cell lines, revealed a 2- to 3-fold induction of fibrinogen transcription after 24 h of T(3) treatment. In addition, T(3) treatment increased the level of fibrinogen protein expression 2.5- to 6-fold at 48 h. The protein synthesis inhibitor, cycloheximide, did not inhibit the induction of fibrinogen by T(3), indicating that this regulation was direct. Furthermore, transcription run-on experiments indicate that the induction of fibrinogen by T(3) is regulated largely at the level of transcription. Similar observations were made on the regulation of fibrinogen by T(3) using rats that received surgical thyroidectomy (TX) as an in vivo model. These results suggest that T(3) plays an important role in the process of blood coagulation and inflammation and may contribute to the understanding of the association between thyroid diseases and the misregulation of the inflammatory and clotting profile evident in the circulatory system of these patients.