Expression and methylation status of tissue factor pathway inhibitor-2 gene in non-small-cell lung cancer

Tissue factor pathway inhibitor-2 (TFPI-2) is a Kunitz-type serine proteinase inhibitor that inhibits plasmin-dependent activation of several metalloproteinases. Downregulation of TFPI-2 could thus enhance the invasive potential of neoplastic cells in several cancers, including lung cancer. In this study, TFPI-2 mRNA was measured using a real-time PCR method in tumours of 59 patients with non-small-cell lung cancer (NSCLC). Tumour TFPI-2 mRNA levels appeared well correlated with protein expression evaluated by immunohistochemistry and were 4–120 times lower compared to those of nonaffected lung tissue in 22 cases (37%). Hypermethylation of the TFPI-2 gene promoter was demonstrated by restriction enzyme-polymerase chain reaction in 12 of 40 cases of NSCLC (30%), including nine of 17 for whom tumour TFPI-2 gene expression was lower than in noncancerous tissue. In contrast, this epigenetic modification was shown in only three of 23 tumours in which no decrease in TFPI-2 synthesis was found (P=0.016). Decreased TFPI-2 gene expression and hypermethylation were more frequently associated with stages III or IV NSCLC (eight out of 10, P=0.02) and the TFPI-2 gene promoter was more frequently hypermethylated in patients with lymph node metastases (eight out of 16, P=0.02). These results suggest that silencing of the TFPI-2 gene by hypermethylation might contribute to tumour progression in NSCLC

Syncytiotrophoblast Microvesicles Released from Pre-Eclampsia Placentae Exhibit Increased Tissue Factor Activity

Background: Pre-eclampsia is a complication of pregnancy associated with activation of coagulation. It is caused by the placenta, which sheds increased amounts of syncytiotrophoblast microvesicles (STBM) into the maternal circulation. We hypothesized that STBM could contribute to the haemostatic activation observed in pre-eclampsia. Methodology/Principal Findings: STBM were collected by perfusion of the maternal side of placentae from healthy pregnant women and women with pre-eclampsia at caesarean section. Calibrated automated thrombography was used to assess thrombin generation triggered by STBM-borne tissue factor in platelet poor plasma (PPP). No thrombin was detected in PPP alone but the addition of STBM initiated thrombin generation in 14/16 cases. Pre-eclampsia STBM significantly shortened the lag time (LagT, P = 0.01) and time to peak thrombin generation (TTP, P = 0.005) when compared to normal STBM. Blockade of tissue factor eliminated thrombin generation, while inhibition of tissue factor pathway inhibitor significantly shortened LagT (p = 0.01) and TTP (P,0.0001), with a concomitant increase in endogenous thrombin potential. Conclusions/Significance: STBM triggered thrombin generation in normal plasma in a tissue factor dependent manner, indicating that TF activity is expressed by STBM. This is more pronounced in STBM shed from pre-eclampsia placentae. As more STBM are shed in pre-eclampsia these observations give insight into the disordered haemostasis observed in thi

Computer model of the interaction of human TFPI-2 Kunitz-type serine protease inhibitor with human plasmin

International audienceComputer model of the interaction of human TFPI-2 Kunitz-type serine protease inhibitor with human plasmin Dear Sir, Tissue factor pathway inhibitor-2 (TFPI-2) is a 32-kDa serine proteinase inhibitor that strongly inhibits trypsin, plasmin, factor XIa and plasma kallikrein [1], and is mainly associated with extracellular matrices [2]. By inhibiting plasmin, TFPI-2 thus efficiently decreases activation of several metalloproteinases [2] and reduces tumor invasion and metastasis [3,4]. In addition, TFPI-2 can also regulate plasmin in atherosclerotic plaques by modulating extracel-lular proteolytic mechanisms [5]. TFPI-2, which is a member of the Kunitz-type serine proteinase inhibitor family, consists of three tandemly repeated Kunitz-type domains, the first of which (K1) exhibits similarities in amino acid sequence to TFPI-1 and bovine pancreatic trypsin inhibitor (BPTI) (45% and 40%, respectively). The inhibition of serine proteinases by Kunitz-type inhibitors involves their binding to K1 and P1 residue that interacts with the proteinase pocket previously identified to be an arginine or a lysine residue [6-9]. Using site-directed mutagenesis experiments, Kamei et al. [10] reported that a TFPI-2 mutant with glutamine substituted for arginine lost its inhibitory activity towards trypsin and plasmin. Several structures showing the interaction of one serine proteinase with a Kunitz-type inhibitor, i.e. thrombin with BPTI [6], factor Xa/VIIa with TFPI-1 [7,8] and alpha-chymotrypsin with BPTI [9], have been determined crystallographically. However , TFPI-2 structure and plasmin complexed with a Kunitz-type inhibitor have not to date been described. This report is the first to address the structural basis for molecular recognition of plasmin by the K1 domain of TFPI-2 (TFPI-2/K1). The 3D models of TFPI-2/K1 was built using the automatic comparative modeling server swissPDB (http:// www.expasy.org). The structure of TFPI-1/K2 (44% identity with TFPI-2/K1 in 55 residues) was used as template to determine TFPI-2/K1. The resulting model was further energy-minimized using the steepest descent algorithm (SYBYL 6.9, Tripos, http://www.tripos.com), the atoms of the backbone being constrained during this step, and un-constrained using the conjugate gradient algorithm until the maximum derivative was < 0.1 kcal/mol/A ˚. After checking the stereochemical quality (WHATIF, http://www.cmbi. kun.nl/), all parameters were good for ensemble structure. The TFPI-2/K1 model and plasmin (coordinates extracted from the PDB file 1 bml) were superimposed on the Ca atoms of the TFPI-1/trypsin complex (coordinates extracted from the PDB file 1tfx). Finally, the few atoms that overlapped at the complex interface were relieved by energy minimization. The electrostatic potentials of TFPI-2/K1 and plasmin were calculated with swissPDB viewer 3.7b software and using formal charges at pH 7.4 (arginine, lysine, N-terminus, + 1; glutamate, aspartate and the C-terminus, À 1; and histidine, neutral), ionic strength in the aqueous environment of 0.15 M and dielectric constant of 80. The three-dimensional structure of TFPI-2/K1 exhibited the typical Kunitz-type proteinase inhibitor folding, i.e. a double stranded anti-parallel h-sheet from Arg20 to Phe33, and an a-helix from Trp48 to Ala54. The core of the domain, comprising the secondary structure elements and the three disulfide bonds, was highly conserved. TFPI-2/K1 exhibited the same overall fold as BPTI [6] and TFPI-1/K2 [7], with rms deviations (root-mean-square) for all 55 Ca atom positions of 0.70 and 1.09 A ˚ , respectively. These values are in agreement with the 40% identity with BPTI and 45% identity with TFPI-1/K2. The binding loop of TFPI-2/K1 (Leu19-Tyr33) was in direct contact with the active site of plasmin involving the characteristic main-main conforma-tional and intermolecular hydrogen bond interactions of canonical binding proteinase inhibitors [7-9]. Indeed, as shown in Table 1, TFPI-2/K1 residues positioned close to the active site of plasmin (PV2, PV1, P1 and P3 residues) were involved in hydrogen bonds. As compared with the TFPI-1/trypsin complex [7], two additional hydrogen bonds were found at the interface between TFPI-2/K1 and plasmin, probably improving the complementarity between the two molecules. The side-chain of TFPI-2 P1 residues (Arg15) extended into the plasmin-specific pocket, with its guanidi-nium group forming a salt bridge with the carboxylate group 0049-3848/$-see front matter

Improved PCR Method for Amplification of GC-Rich DNA Sequences

Most housekeeping genes, tumor-suppressor genes, and approx 40 % of tissue-specific genes contain CpG islands in their promoter region that are limited regions of high-density CpG residues

Characterization and functional analysis of TFPI-2 gene promoter in a human choriocarcinoma cell line

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Tissue factor pathway inhibitor and cancer

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