Estrogenic regulation of tissue factor and tissue factor pathway inhibitor in platelets

Oral estrogen treatment increases thrombotic risk. Tissue factor (TF), tissue factor pathway inhibitor (TFPI), and platelet interaction with leukocytes are important determinants of thrombogenesis. Therefore, the present study was designed to define and compare platelet TF and TFPI mRNA and adhesion protein expression in platelets derived from animals treated with different types of oral estrogens. Ovariectomized pigs were treated with 17beta-estradiol (2 mg/day), conjugated equine estrogen (CEE; 0.625 mg/day), or raloxifene (60 mg/day) for 4 wk. Compared with intact animals, ovariectomy and treatment differentially affected populations of leukocytes: neutrophils decreased whereas lymphocytes increased significantly 4 wk after ovariectomy and with 17beta-estradiol and CEE treatments; eosinophils increased only with 17beta-estradiol treatment. Content of TF protein increased in platelets from 17beta-estradiol- and raloxifene-treated pigs, whereas TF mRNA was detected only in platelets from 17beta-estradiol- and CEE treated pigs. TFPI mRNA increased in platelets after ovariectomy and estrogen treatment. Only a trace of TFPI protein was detected, but a higher-molecular-mass protein was observed in all treatment groups. Expression of CD40 and CD40 ligand increased with ovariectomy and decreased with 17beta-estradiol and CEE treatments more than with raloxifene. The ratio of activated to basal P-selectin expression decreased with ovariectomy and increased with raloxifene treatments. These results suggest that estrogenic formulations may affect individual thrombotic risk by different mechanisms that regulate TF and platelet-leukocytic interactions. These studies provide the rationale for evaluation of interactions among platelets and TF and TFPI expression on thrombin generation during estrogen treatment in humans.     

A phase II trial of sorafenib in metastatic melanoma with tissue correlates

Background

Sorafenib monotherapy in patients with metastatic melanoma was explored in this multi-institutional phase II study. In correlative studies the impact of sorafenib on cyclin D1 and Ki67 was assessed.

Methodology/Principal Findings

Thirty-six patients treatment-naïve advanced melanoma patients received sorafenib 400 mg p.o. twice daily continuously. Tumor BRAF^V600E mutational status was determined by routine DNA sequencing and mutation-specific PCR (MSPCR). Immunohistochemistry (IHC) staining for cyclin D1 and Ki67 was performed on available pre- and post treatment tumor samples. The main toxicities included diarrhea, alopecia, rash, mucositis, nausea, hand-foot syndrome, and intestinal perforation. One patient had a RECIST partial response (PR) lasting 175 days. Three patients experienced stable disease (SD) with a mean duration of 37 weeks. Routine BRAF^V600E sequencing yielded 27 wild-type (wt) and 6 mutant tumors, whereas MSPCR identified 12 wt and 18 mutant tumors. No correlation was seen between BRAF^V600E mutational status and clinical activity. No significant changes in expression of cyclin D1 or Ki67 with sorafenib treatment were demonstrable in the 15 patients with pre-and post-treatment tumor samples.

Conclusions/Significance

Sorafenib monotherapy has limited activity in advanced melanoma patients. BRAF^V600E mutational status of the tumor was not associated with clinical activity and no significant effect of sorafenib on cyclin D1 or Ki67 was seen, suggesting that sorafenib is not an effective BRAF inhibitor or that additional signaling pathways are equally important in the patients who benefit from sorafenib.

Trial registration

Clinical Trials.gov {"type":"clinical-trial","attrs":{"text":"NCT00119249","term_id":"NCT00119249"}}NCT00119249     

Differential expression of tissue factor and tissue factor pathway inhibitor in metastatic melanoma lesions

Tissue factor (TF) is involved in tumor progression and metastatic potency in some malignant tumors and its function is regulated by tissue factor pathway inhibitor (TFPI) therefore the interaction of both molecules is crucial for their functional role. We evaluated the clinical relevance of TF and TFPI expression in benign and malignant melanocytic lesions. Expression of both was examined by immunoperoxidase staining using serial tissue sections in 16 nevi, 34 primary and 15 metastatic melanoma lesions. TF and TFPI were ubiquitously expressed in benign and malignant melanocytic lesions. This finding was confirmed by Western blot analysis using cultured human melanocytes, nevi cells (NCN) and melanoma cell lines. Although TF expression was not associated with malignant transformation and disease progression, TFPI expression in primary and metastatic melanoma lesions was significantly lower and weaker than that in nevi lesions in terms of intensity and percentage of stained cells. In addition, TFPI expression in metastatic lesions was significantly lower and weaker than that of TF. These results suggest that the relative expression of TF to TFPI may play a crucial role in the malignant transformation and metastatic potency in melanocytic cells.     

Catabolism of factor VIIa bound to tissue factor in fibroblasts in the presence and absence of tissue factor pathway inhibitor

Vascular injury leads to the exposure of blood to fibroblasts and smooth muscle cells within the vessel wall. These cells constitutively express tissue factor (TF), the cellular receptor for plasma clotting factor VIIa (FVIIa). Formation of TF.FVIIa complexes on cell surfaces triggers the blood coagulation cascade. In the present study, we have investigated the fate of TF.FVIIa complexes formed on the cell surface of fibroblasts in the presence and absence of plasma inhibitor, tissue factor pathway inhibitor (TFPI). FVIIa bound to TF on the cell surface was internalized and degraded without depleting the cell surface TF antigen and activity. TFPI significantly enhanced the TF-specific internalization and degradation of FVIIa. TFPI-enhanced internalization and degradation of FVIIa requires the C-terminal domain of TFPI and factor Xa. TFPI. Xa-mediated internalization of FVIIa was associated with the depletion of TF from the cell surface. A majority of the internalized FVIIa was degraded, but a small portion of the internalized FVIIa recycles back to the cell surface as an intact protein. In addition to TF, other cell surface components, such as low density lipoprotein receptor-related protein (LRP) and heparan sulfates, are essential for TFPI.Xa-induced internalization of FVIIa. Acidification of cytosol, which selectively inhibits the endocytotic pathway via coated pits, inhibited TFPI.Xa-mediated internalization but not the basal internalization of FVIIa. Overall, our data support the concept that FVIIa bound to cell surface TF was endocytosed by two different pathways. FVIIa complexed with TF in the absence of the inhibitor was internalized via a LRP-independent and probably noncoated pit pathway, whereas FVIIa complexed with TF along with the inhibitor was internalized via LRP-dependent coated pit pathway.     

Determination of the disulfide bond pairings in human tissue factor pathway inhibitor purified fromEscherichia coli

The disulfide bond assignments of human alanyl tissue factor pathway inhibitor purified fromEscherichia coli have been determined. This inhibitor of the extrinsic blood coagulation pathway possesses three Kunitz-type inhibitor domains, each containing three disulfide bonds. The disulfide bond pairings in domains 1 and 3 were determined by amino acid sequencing and mass spectrometry of peptides derived from a thermolysin digest. However, thermolysin digestion did not cleave any peptide bonds within domain 2. The disulfide bond pairings in domain 2 were determined by isolating it from the thermolysin treatment and subsequently cleaving it with pepsin and trypsin into peptides which yielded the three disulfide bond pairings in this domain. These results demonstrate that the disulfide pairings in each of the three domains of human tissue factor pathway inhibitor purified fromEscherichia coli are homologous to each other and also to those in bovine pancreatic trypsin inhibitor.     

Impact of the tissue factor pathway inhibitor gene on apoptosis in human vascular smooth muscle cells

Tissue factor pathway inhibitor (TFPI) plays a vitally important role in the blood coagulation pathway. Recent studies indicated that TFPI induces apoptosis in vascular smooth-muscle cells (VSMCs) in animals. The present study investigated whether the TFPI gene could also induce apoptosis in human vascular smooth-muscle cells (hVSMCs). Such cells were isolated from human umbilical arteries and subsequently transfected with pIRES-TFPI plasmid (2 μg/mL). MTT assaying and cell counting were applied to measure cell viability and proliferation, RT-PCR was utilized to analyze TFPI gene expression in the cells. Apoptosis was analyzed by fluorescence activated cell sorting (FACS). Several key proteins involved in apoptosis were examined through Western blotting. It was shown that TFPI gene transfer led to its increased cellular expression, with a subsequent reduction in hVSMC proliferation. Further investigation demonstrated that TFPI gene expression resulted in lesser amounts of procaspase-3, procaspase-8 and procascase-9, and an increased release of mitochondrial cytochrome c (cyt-c) into cytoplasm, thereby implying the involvement of both extrinsic and intrinsic pathways in TFPI gene-induced apoptosis in hVSMCs.     

The C-terminus of tissue factor pathway inhibitor is essential to its anticoagulant activity

Tissue factor pathway inhibitor (TFPI) from different cell lines shows up to 15-fold differences in the ratio of anticoagulant to chromogenic activity. The anticoagulant activity was dependent on the purification procedure used and it was possible to isolate two fractions of recombinant TFPI. Only one of these fractions showed anticoagulant activity comparable with TFPI from normal human plasma, and Western blotting showed that the low-activity fraction did not react with an antibody raised against a peptide of TFPI located near the C-terminal. Analysis by mass spectroscopy of peptides from V8 protease digests showed that C-terminal amino acids could only be identified from the high-activity form, while heterologous fragmentation had taken place in the form with low anticoagulant activity. Previously published studies on TFPI have been performed using material of low anticoagulant activity compared with plasma TFPI, and we suggest that these studies have been performed with material degraded in the C-terminus.     

A phase I/II study of recombinant tumor necrosis factor and recombinant interferon gamma in patients with AIDS-related complex

A clinical trial was conducted to determine the tolerance and toxicity of recombinant tumor necrosis factor (rTNF) and recombinant interferon gamma (rIFN-gamma) when administered concurrently by continuous intravenous infusion to 11 patients with the AIDS-related complex (ARC). In addition, HIV culture, p24 antigen levels, and CD4 positive lymphocytes were monitored to obtain preliminary evidence of antiviral and immunologic effects. Two 5-day treatment cycles were separated by a 9-day washout period. Two patients were entered at each dosage level and each patient received the two 5-day treatment cycles at two sequential dose levels ranging from 1 to 25 micrograms/m2. Two patients did not complete their second treatment cycle--one due to the development of a rash, the second due to central venous catheter discomfort. The occurrence of phlebitis with peripheral vein administration of these agents necessitated administration via central venous catheter. With the exception of a single patient who developed severe headache at the 25 micrograms/m2 dose, severe clinical toxicities were not observed. Fever, chills, headache, and myalgias were the most significant clinical toxicities observed and all were dose dependent. The percentage fall in total granulocytes was dose dependent and ranged from 17% at the 1 microgram/mm2 dose to 48% at both the 15 and 25 micrograms/mm2 dose levels. The mean nadir granulocyte count was 1694/mm3. No significant renal or hepatic toxicity was observed. Of 22 treatment cycles the CD4 cell number was increased in 11, unchanged in 7, and decreased in 4. The mean CD4 cell number did not change significantly (176 +/- 143/mm3 pretherapy versus 279 +/- 305/mm3 posttherapy).(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of the disulfide bond pairings in human tissue factor pathway inhibitor purified fromEscherichia coli

The disulfide bond assignments of human alanyl tissue factor pathway inhibitor purified fromEscherichia coli have been determined. This inhibitor of the extrinsic blood coagulation pathway possesses three Kunitz-type inhibitor domains, each containing three disulfide bonds. The disulfide bond pairings in domains 1 and 3 were determined by amino acid sequencing and mass spectrometry of peptides derived from a thermolysin digest. However, thermolysin digestion did not cleave any peptide bonds within domain 2. The disulfide bond pairings in domain 2 were determined by isolating it from the thermolysin treatment and subsequently cleaving it with pepsin and trypsin into peptides which yielded the three disulfide bond pairings in this domain. These results demonstrate that the disulfide pairings in each of the three domains of human tissue factor pathway inhibitor purified fromEscherichia coli are homologous to each other and also to those in bovine pancreatic trypsin inhibitor.     

Structural mechanism for heparin-binding of the third Kunitz domain of human tissue factor pathway inhibitor.

Tissue factor pathway inhibitor (TFPI) inhibits the activity of coagulation factor VIIa and Xa through its K1 and K2 domain, respectively, and the inhibitory activity is enhanced by heparin. The function of the K3 domain of TFPI has not been established, but the domain probably harbors a heparin binding site (HBS-2). We determined the three-dimensional solution structure of the TFPI K3 domain (Glu182-Gly242) by heteronuclear multidimensional NMR. The results showed that the molecule is composed of one antiparallel beta-sheet and one alpha-helix, and in overall structure is very similar to the K2 domain, with the rms deviation of 1.55 A for the 58 defined C(alpha) positions. However, the surface electrostatic properties of both domains are different each other. The lack of inhibitory activity of the K3 domain is explained by the absence of electrostatic interaction with factor Xa over a large surface area. A titration experiment with size-fractionated heparin showed that a heparin binding site was located in the vicinity of the alpha-helix. In this region, a positively charged cluster is formed by Lys213, Lys232, and Lys240, and the negatively charged sulfate groups of heparin bind there. The enhancement of inhibitory activity by heparin probably was not due to a conformational change to TFPI itself. It is likely that heparin simply increases the local concentration of TFPI on the cell surface and stabilizes the initial complex that forms.     

Crystal structure of human factor VIIa/tissue factor in complex with peptide mimetic inhibitor

The 3D structure of human factor VIIa/soluble tissue factor in complex with a peptide mimetic inhibitor, propylsulfonamide-D-Thr-Met-p-aminobenzamidine, is determined by X-ray crystallography. As compared with the interactions between thrombin and thrombin inhibitors, the interactions at S2 and S3 sites characteristic of factor VIIa and factor VIIa inhibitors are revealed. The S2 site has a small pocket, which is filled by the hydrophobic methionine side chain in P2. The small S3 site fits the small size residue, D-threonine in P3. The structural data and SAR data of the peptide mimetic inhibitor show that these interactions in the S2 and S3 sites play an important role for the improvement of selectivity versus thrombin. The results will provide valuable information for the structure-based drug design of specific inhibitors for FVIIa/TF.     

Tissue factor, tissue factor pathway inhibitor and cytoadhesive molecules in patients with an acute coronary syndrome

The tissue factor plays a crucial role in initiating blood coagulation after plaque rupture in patients with acute coronary syndrome. It is abundant in atherosclerotic plaques. Moreover, P-selectin, some cytokines, endotoxin and immune complexes can stimulate monocytes and induce the tissue factor expression on their surface. The aim of the study was to compare plasma levels of the tissue factor, tissue factor pathway inhibitor, P-selectin, E-selectin and ICAM-1 in patients with acute myocardial infarction, unstable angina pectoris, stable coronary artery disease and normal control subjects. In addition, plasma levels of the tissue factor, tissue factor pathway inhibitor, P-selectin, E-selectin and ICAM-1 were measured in the blood withdrawn from the coronary sinus in a subgroup of patients with unstable angina pectoris and stable coronary artery disease in which the difference between concentrations in the coronary sinus and systemic blood was calculated. A significant increase in tissue factor pathway inhibitor plasma levels was detected in patients with acute myocardial infarction (373.3+/-135.1 ng/ml, p<0.01) and unstable angina pectoris (119.6+/-86.9 ng/ml, p<0.05) in contrast to the patients with stable coronary artery disease (46.3+/-37.5 ng/ml) and normal subjects (45.1+/-14.3 ng/ml). The plasma levels of tissue factor pathway inhibitor were significantly increased both in the coronary sinus and systemic blood in the patients with unstable angina pectoris. There was only a non-significant trend to higher plasma levels of the tissue factor in patients with acute myocardial infarction and unstable angina pectoris as compared to the patients with stable coronary artery disease and normal subjects, the values being 129.1+/-30.2 pg/ml, 130.5+/-57.8 pg/ml, 120.2+/-45.1 pg/ml and 124.9+/-31.8 pg/ml, respectively. Plasma levels of soluble P-selectin was only slightly, but non-significantly higher in patients with unstable angina pectoris and stable coronary artery disease (184.2+/-85.4 ng/ml and 201.6+/-67.9 ng/ml, respectively) than in patients with the acute myocardial infarction (157.4+/-88.4 ng/ml) or normal subjects (151.4+/-47.1 ng/ml). The difference in plasma levels of soluble ICAM-1 between the blood withdrawn from the coronary sinus and systemic circulation correlated significantly with the corresponding difference in plasma levels of soluble P-selectin and E-selectin. In conclusion, the tissue factor and the tissue factor pathway inhibitor play a crucial role in the initiation of arterial thrombosis. The tissue factor pathway inhibitor levels are increased both in the systemic blood and in the coronary sinus of patients with the acute coronary syndrome.     

A phase I trial of recombinant gamma interferon in patients with cancer

Summary A total of 11 patients were treated on an escalating, single dose trial of recombinant gamma interferon (rIFN-), 6 patients by the i.m. and 5 patients by the i.v. route of administration. Dose ranges within each individual were from 0.05 mg/m² of IFN (1 mg10×10⁶ units of IFN) escalating to 10 mg/m². All dosages were delivered twice weekly and the i.v. dose was infused over 5 min. The most common toxicities encountered included fever, chils, fatigue, anorexia, and granulocytopenia. The influenzalike symptoms were very similar to those encountered with IFN- but were generally less severe. The granulocytopenia was dose-related and transient with recovery generally seen within 48–72 h following administration of rIFN-. Absolute granulocyte counts only rarely dropped below 1000 mm³. Hepatotoxicity was not observed. IFN levels were determined by both a bioassay and an enzyme-linked immunosorbent assay. By the i.v. route, the peak level of IFN activity could usually be seen at completion of the infusion with a serum half-life of 30 min. By the i.m. route, the peak level of serum activity was generally detected between 4–8 h with a serum half-life of 4.5 h after the initial elimination phase. Peak IFN levels appeared to correlate with maximum toxicity. One patient with melanoma had a 25% reduction in a cutaneous lesion, but there were no other minimal, partial, or complete responses.This project has been funded at least in part with Federal funds from the Department of Health and Human Services, under contract number NO1-CO-23910 with Program Resources, Inc. The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.     

Contribution of regions distal to glycine-160 to the anticoagulant activity of tissue factor pathway inhibitor

The functions of the first two Kunitz domains of tissue factor pathway inhibitor (TFPI) are well defined as active site-directed inhibitors of factor VIIa and factor Xa. The anticoagulant properties of the third Kunitz domain and C-terminal region were probed using altered forms of TFPI. TFPI-160 contains the first two Kunitz domains. K1K2C contains the first two Kunitz domains and the basic C-terminus. Neither TFPI-160 nor K1K2C contains the third Kunitz domain. In amidolytic assays containing calcium, TFPI-160 is a less potent inhibitor of factor Xa than TFPI. However, addition of the C-terminus in K1K2C nearly restores inhibitory activity to that of TFPI, indicating that the third Kunitz domain is not required for direct inhibition of factor Xa. When compared in assays containing phospholipids and factor Va, K1K2C and TFPI-160 are poor inhibitors compared to TFPI, demonstrating that the third Kunitz domain is required for the full anticoagulant activity of TFPI. TFPI was further characterized in amidolytic assays performed with Gla-domainless factor Xa and in prothrombin activation assays using submicellar concentrations of short-chain phospholipids (C6PS). TFPI and K1K2C are worse inhibitors of Gla-domainless factor Xa, compared to wild-type factor Xa, while TFPI-160 inhibits both forms of factor Xa equally, suggesting a C-terminus/Gla domain interaction. TFPI is a potent inhibitor of thrombin generation by prothrombinase assembled with C6PS, while TFPI-160 and K1K2C are not. Conversely, TFPI does not inhibit prothrombin activation by prothrombinase assembled on a two-dimensional lipid bilayer. Together, the data indicate that the region between Gly-160 and the end of the third Kunitz domain contributes to TFPI function by orienting the second Kunitz domain so that it can bind the active site of phospholipid-associated factor Xa prior to prothrombinase assembly and/or by slowing formation of the prothrombinase complex.     

In vitro phase II trial of recombinant interferon alpha-2 in gastrointestinal cancer

The antitumor effects of recombinant interferon alpha-2 (rIF) on clonogenic tumor cells were investigated in 29 cases of gastrointestinal cancer. An in vitro response (greater than or equal to 50% inhibition of tumor colony-forming units) was observed in 17% of the tumors, including 2 of 8 pancreatic, 2 of 6 gastric, and 1 of 10 colon cancer specimens. The relative efficacy of rIF in tissue cultures of pancreatic and gastric tumors was further substantiated by the resistance against simultaneously tested single conventional cytostatic drugs. Preliminary results of comparative studies of cloned interferon alpha-2 and human purified leukocyte interferon (hlIF) in 2 human colon cancer cell lines and 11 fresh tumor specimens suggest similar trends in terms of colony inhibition in individual assays. However, the interpatient differences indicate an overall superiority of the natural preparation (P less than 0.02).     

Detection of methylated tissue factor pathway inhibitor 2 and human long DNA in fecal samples of patients with colorectal cancer in China

Background: To investigate the feasibility of detecting methylated tissue factor pathway inhibitor (TFPI2) and quantifying human long DNA with fluorescent quantitative Alu PCR in fecal DNA as a non-invasive screening tool for colorectal cancer (CRC). Materials and Methods: Methylation-specific PCR (MSP) was performed to analyze TFPI2 gene promoter methylation status in a blinded fashion in stool samples taken from 30 endoscopically diagnosed healthy controls, 20 patients with adenomas, and 60 patients with colorectal cancer. Real-time Alu PCR was used to quantify human long DNA. Results: The specificity of fecal TFPI2 MSP assay and long DNA assay was 100% and 83.3%, respectively. The sensitivity of fecal TFPI2 MSP assay and long DNA assay was 68.3% and 53.3%, respectively. The sensitivity of fecal DNA assay (either marker being positive) was 86.7%, which was high for CRC. Conclusions: Our results have demonstrated the feasibility of using TFPI2 methylation and quantify human long DNA with fluorescent quantitative Alu PCR in fecal samples as a new noninvasive test for CRC.