Ronald R. Bach

Tumor necrosis factor enhances expression of tissue factor mRNA in endothelial cells

We have examined the effect of recombinant tumor necrosis factor on the expression of tissue factor activity and tissue factor mRNA levels in vascular endothelial cells. Following exposure of human umbilical vein endothelial cells to this cytokine, the appearance of tissue factor procoagulant activity was detected following cell disruption, and was maximal at 6 hours. Northern blot analysis of cytokine-treated cells demonstrated a similar increase in the synthesis of tissue factor mRNA, followed by a gradual decline to the basal level by 18 hours. Cycloheximide by itself induced the accumulation of high levels of tissue factor mRNA in these cells. This result suggests that the proteins necessary for transcription of the tissue factor gene are present in the endothelial cell prior to cytokine stimulation, and synthesis of the tissue factor mRNA may be controlled, in part, by a labile repressor protein.

Tumor-derived tissue factor activates coagulation and enhances thrombosis in a mouse xenograft model of human pancreatic cancer.

Cancer patients often have an activated clotting system and are at increased risk for venous thrombosis. In the present study, we analyzed tissue factor (TF) expression in 4 different human pancreatic tumor cell lines for the purpose of producing derivative tumors in vivo. We found that 2 of the lines expressed TF and released TF-positive microparticles (MPs) into the culture medium. The majority of TF protein in the culture medium was associated with MPs. Only TF-positive cell lines activated coagulation in nude mice, and this activation was abolished by an anti-human TF Ab. Of the 2 TF-positive lines, only one produced detectable levels of human MP TF activity in the plasma when grown orthotopically in nude mice. Surprisingly, < 5% of human TF protein in plasma from tumor-bearing mice was associated with MPs. Mice with TF-positive tumors and elevated levels of circulating TF-positive MPs had increased thrombosis in a saphenous vein model. In contrast, we observed no difference in thrombus weight between tumor-bearing and control mice in an inferior vena cava stenosis model. The results of the present study using a xenograft mouse model suggest that tumor TF activates coagulation, whereas TF on circulating MPs may trigger venous thrombosis.

Tissue factor gene expression in acute myeloblastic leukemia

We have studied tissue factor gene expression in the leukocytes of 22 patients with acute myeloblastic leukemia (AML). Total RNA from peripheral blood or bone marrow cells depleted of monocytes was analyzed by Northern blot analysis using a 32P-labeled tissue factor cDNA probe. Cells from 10 cases expressed tissue factor mRNA and positive cases were distributed among the myeloblastic, myelomonocytic, and monocytic subtypes of AML. Tissue factor transcripts were not detected in cells derived from normal bone marrow. The expression of this gene product on the surface of leukemic cells could contribute to the excessive thrombin generation that has been observed in some individuals with this disorder.

Tissue factor activity of blood mononuclear cells is increased after total knee arthroplasty

Tissue factor (TF) is present in small quantities in normal blood and is reported to be elevated in arterial and venous thrombosis. Patients undergoing total knee arthoplasty (TKA) are at high risk of post-operative venous thromboembolism (VTE). To evaluate the possible contribution of elevated blood TF to VTE risk, we performed serial studies of peripheral blood mononuclear cell (PBMC) functional TF procoagulant activity (PCA) in 19 patients after TKA. PBMC and platelet TF PCA were measured by a functional, clot-based assay following decryption with a calcium ionophore. Plasma TF antigen levels were measured by ELISA. All subjects received chemoprophylaxis and none had VTE. After TKA total TF PCA of PBMC was elevated in 19 of 19 subjects. The peak increase above preoperative levels was 1.1-13.6 fold (>two-fold in 58% and >three-fold in 42%). Median TF PCA of PBMC was not elevated following tourniquet removal, but it was significantly elevated on postoperative days 1 and 2. Thereafter, it decreased to near preoperative values at day 6. Neither platelet TF PCA nor plasma TF antigen levels increased significantly. Since the PBMC count did not rise, the increase in TF PCA was attributable to cell synthesis. The increase in blood TF PCA preceded the median time of diagnosis of venous thromboembolism after TKA established previously. These observations indicate a) TKA stimulates synthesis of encrypted PBMC TF PCA which is likely to contribute to the pathophysiology of VTE; b) TF antigen is not a reliable indicator of TF PCA.

Synthesis of tissue factor messenger RNA and procoagulant activity in breast cancer cells in response to serum stimulation.

The procoagulant activity observed in many types of tissue and cultured cells is due to tissue factor, a 30 kd transmembrane protein. The mRNA for tissue factor is a 2.2-kb species, which in some non-cancer cells can be up-regulated or induced by cytokines or by serum stimulation. In this study, induction of procoagulant activity in cancer cells was evaluated using the breast cancer cell line, MCF-7, and an adriamycin resistant subline, AdrRMCF-7, which has increased tumorigenicity in nude mice compared to the parental cell line. Procoagulant activity was factor VIIa dependent and was inhibited by an anti-tissue factor antibody. MCF-7 cells had minimal tissue factor activity, while AdrRMCF-7 cells had an 10-fold increase compared to the parental line. This increase was not observed in MCF-7 cells transfected with the multi-drug resistant gene, which is associated with adriamycin resistance. Serum stimulation of quiescent MCF-7 cells increased tissue factor activity 5-fold over baseline level, but did not increase activity in cells grown in serum-replete medium. Tissue factor activity of AdrRMCF-7 quiescent cells and AdrMCF-7 cells grown in serum-replete medium was enhanced 2-fold by serum stimulation. The predominant tissue factor mRNA species in MCF-7 cells was a 3.2 to 3.4-kb band, which increased in response to serum stimulation of cells grown in serum-replete medium. The mature 2.2-kb tissue factor mRNA band was detected in quiescent MCF-7 cells within six hours of serum stimulation and remained present 24 hours after stimulation. Synthesis of the 2.2-kb tissue factor mRNA species in MCF-7 and AdrRMCF-7 cells correlated with appearance of procoagulant activity. Thus, while procoagulant activity correlates with the level of the 2.2-kb tissue factor mRNA species in these cancer cells, there are inherent differences in tissue factor activity, antigen, and mRNA levels, as well as in regulation of its synthesis between these cells.

Blood Biomarkers of Chronic Inflammation in Gulf War Illness.

Background More than twenty years following the end of the 1990–1991 Gulf War it is estimated that approximately 300,000 veterans of this conflict suffer from an unexplained chronic, multi-system disorder known as Gulf War Illness (GWI). The etiology of GWI may be exposure to chemical toxins, but it remains only partially defined, and its case definition is based only on symptoms. Objective criteria for the diagnosis of GWI are urgently needed for diagnosis and therapeutic research. Objective This study was designed to determine if blood biomarkers could provide objective criteria to assist diagnosis of GWI. Design A surveillance study of 85 Gulf War Veteran volunteers identified from the Department of Veterans Affairs Minnesota Gulf War registry was performed. All subjects were deployed to the Gulf War. Fifty seven subjects had GWI defined by CDC criteria, and 28 did not have symptomatic criteria for a diagnosis of GWI. Statistical analyses were performed on peripheral blood counts and assays of 61 plasma proteins using the Mann-Whitney rank sum test to compare biomarker distributions and stepwise logistic regression to formulate a diagnostic model. Results Lymphocyte, monocyte, neutrophil, and platelet counts were higher in GWI subjects. Six serum proteins associated with inflammation were significantly different in GWI subjects. A diagnostic model of three biomarkers—lymphocytes, monocytes, and C reactive protein—had a predicted probability of 90% (CI 76–90%) for diagnosing GWI when the probability of having GWI was above 70%. Significance The results of the current study indicate that inflammation is a component of the pathobiology of GWI. Analysis of the data resulted in a model utilizing three readily measurable biomarkers that appears to significantly augment the symptom-based case definition of GWI. These new observations are highly relevant to the diagnosis of GWI, and to therapeutic trials.

Elevated platelet count, C-reactive protein and thromboxane analog-induced platelet aggregation in patients with Gulf War veterans' illnesses: evidence of a chronic inflammatory state?

A previous study of Gulf War veterans illnesses (GWVI) observed evidence of platelet activation in a majority of patients with GWVI. To further characterize platelet function, we studied 43 patients (40 men) with GWVI (GWVI+) and 21 veterans who served concurrently in the Gulf War but who lacked criteria for GWVI (GWVI−). All participants were free of infection and known inflammatory diseases. Studies performed included platelet count, immature platelet fraction (IPF), plasma thrombopoietin (TPO), C-reactive protein (CRP), platelet aggregation and ATP secretion in response to six agonists, and spontaneous aggregation. Platelet counts and CRP were significantly elevated in GWVI+ compared to GWVI− patients without elevation in IPF or TPO. Platelet aggregation did not differ between GWVI+ and GWVI− patients except for spontaneous aggregation that was significantly greater in GWVI+ patients. Platelet ATP secretion was similar in the two groups, except the response to 50 &mgr;mol/l thrombin receptor agonist peptide 6 (TRAP 6) was significantly greater in GWVI+ patients. When platelet aggregation was analyzed in relation to CRP, the response to 0.5 &mgr;mol/l U46619 was significantly greater in patients whose CRP was at least 2 &mgr;g/ml. Therefore, GWVI+ patients had elevated platelet counts, spontaneous aggregation, TRAP 6-induced secretion, and CRP, but no impairment of platelet function. The increased platelet counts and U46619-induced aggregation appear to be consequences of an underlying inflammatory state in GWVI.