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.

Gulf war illness inflammation reduction trial: A phase 2 randomized controlled trial of low-dose prednisone chronotherapy, effects on health-related quality of life.

BACKGROUND: Gulf War illness (GWI) is a deployment-related chronic multisymptom illness impacting the health-related quality of life (HRQOL) of many U.S. Military Veterans of the 1990-91 Gulf War. A proinflammatory blood biomarker fingerprint was discovered in our initial study of GWI. This led to the hypothesis that chronic inflammation is a component of GWI pathophysiology. OBJECTIVES: The GWI inflammation hypothesis was tested in this Phase 2 randomized controlled trial (RCT) by measuring the effects of an anti-inflammatory drug and placebo on the HRQOL of Veterans with GWI. The trial is registered at ClinicalTrials.gov, Identifier: NCT02506192. RCT DESIGN AND METHODS: Gulf War Veterans meeting the Kansas case definition for GWI were randomized to receive either 10 mg modified-release prednisone or matching placebo. The Veterans RAND 36-Item Health Survey was used to assess HRQOL. The primary outcome was a change from baseline in the physical component summary (PCS) score, a measure of physical functioning and symptoms. A PCS increase indicates improved physical HRQOL. RESULTS: For subjects with a baseline PCS <40, there was a 15.2% increase in the mean PCS score from 32.9±6.0 at baseline to 37.9±9.0 after 8 weeks on modified-release prednisone. Paired t-test analysis determined the change was statistically significant (p = 0.004). Eight weeks after cessation of the treatment, the mean PCS score declined to 32.7±5.8. CONCLUSIONS: The prednisone-associated improvement in physical HRQOL supports the GWI inflammation hypothesis. Determining the efficacy of prednisone as a treatment for GWI will require a Phase 3 RCT.

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.

Gulf War Illness-associated increases in blood levels of interleukin 6 and C-reactive protein: biomarker evidence of inflammation.

OBJECTIVE: Gulf War Illness is a chronic multisymptom disorder severely impacting the health and well-being of many Veterans of the 1990-1991 Gulf War. Symptoms that define the disease include pain, fatigue, mood and memory impairments, gastrointestinal problems, lung disorders, and skin rashes. In our previous biomarker study, we discovered Gulf War Illness-associated proinflammatory blood biomarkers. Therefore, we hypothesized that chronic inflammation causes the symptoms that define this disorder. Testing the chronic inflammation hypothesis is the objective of this study. RESULTS: The biomarker fingerprint of Gulf War Illness is the end-product of a cascade of proinflammatory cytokine signals. In particular, the observed increase in C-reactive protein predicts a corresponding increase in interleukin 6, the cytokine that stimulates hepatocytes to produce C-reactive protein. Therefore, in this study we measured potential upstream cytokine signals in plasma samples from Gulf War Veterans. As predicted, a positive correlation between interleukin 6 and C-reactive protein was observed.

Improving Health Care for Veterans With Gulf War Illness.

Physicians need to recognize and manage Gulf War illness and similar postdeployment, chronic, multisymptom diseases among veterans of recent military operations.

Tissue factor encryption.

Tissue factor (TF) encryption is the post-translational suppression of TF procoagulant activity (PCA) on the cell surface. There is emerging evidence of encrypted TF in normal blood associated with monocytes and platelets. Expression of this latent TF PCA during the propagation phase of blood coagulation may contribute to hemostasis. One pathway leading to the decryption of TF PCA begins with an increase in cytosolic calcium. A large calcium influx triggers both the exposure of phosphatidylserine and the expression of TF PCA on cell surfaces. The connections between these events are reviewed along with evidence that lipid raft association may also contribute to TF encryption. The last step in the decryption of TF PCA is the proteolytic activation of zymogen factor VII. This event may be a key to understanding the different roles of intravascular and extravascular TF in the process of blood coagulation.

Tissue factor; from Morawitz to microparticles.

Tissue factor is the principal activator of blood coagulation in vivo. The existence of extravascular tissue factor has been recognized for over a century, but a rational role as a cell-based enzymatic cofactor in blood coagulation was first proposed by Paul Morawitz in 1905. By the close of the last century, very low levels of circulating tissue factor had been identified in the intravascular compartment, but its role in health and in hemostatic and thrombotic disorders continues to be debated. Nonetheless, ongoing research suggests that tissue factor may be a rational therapeutic target in a number of human disease states.

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 veteran's 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 µmol/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 µmol/l U46619 was significantly greater in patients whose CRP was at least 2 µg/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.

Large enhancement of functional activity of active site-inhibited factor VIIa due to protein dimerization: insights into mechanism of assembly/disassembly from tissue factor.

Active site-inhibited blood clotting factor VIIa (fVIIai) binds to tissue factor (TF), a cell surface receptor that is exposed upon injury and initiates the blood clotting cascade. FVIIai blocks binding of the corresponding enzyme (fVIIa) or zymogen (fVII) forms of factor VII and inhibits coagulation. Although several studies have suggested that fVIIai may have superior anticoagulation effects in vivo, a challenge for use of fVIIai is cost of production. This study reports the properties of dimeric forms of fVIIai that are cross-linked through their active sites. Dimeric wild-type fVIIai was at least 75-fold more effective than monomeric fVIIai in blocking fVIIa association with TF. The dimer of a mutant fVIIai with higher membrane affinity was 1600-fold more effective. Anticoagulation by any form of fVIIai differed substantially from agents such as heparin and showed a delayed mode of action. Coagulation proceeded normally for the first minutes, and inhibition increased as equilibrium binding was established. It is suggested that association of fVIIa(i) with TF in a collision-dependent reaction gives equal access of inhibitor and enzyme to TF. Assembly was not influenced by the higher affinity and slower dissociation of the dimer. As a result, anticoagulation was delayed until the reaction reached equilibrium. Properties of different dissociation experiments suggested that dissociation of fVIIai from TF occurred by a two-step mechanism. The first step was separation of TF-fVIIa(i) while both proteins remained bound to the membrane, and the second step was dissociation of the fVIIa(i) from the membrane. These results suggest novel actions of fVIIai that distinguish it from most of the anticoagulants that block later steps of the coagulation cascade.

Induction of microparticle- and cell-associated intravascular tissue factor in human endotoxemia.

The precise role of intravascular tissue factor (TF) remains poorly defined, due to the limited availability of assays capable of measuring circulating TF procoagulant activity (PCA). As a model of inflammation-associated intravascular thrombin generation, we studied 18 volunteers receiving an infusion of endotoxin. A novel assay that measures microparticle (MP)-associated TF PCA from a number of cellular sources (but not platelets) demonstrated an 8-fold increase in activity at 3 to 4 hours after endotoxin administration (P <.001), with a return to baseline by 8 hours. TF antigen-positive MPs isolated from plasma were visualized by electron microscopy. Interindividual MP-associated TF response to lipopolysaccharide (LPS) was highly variable. In contrast, a previously described assay that measures total (cell and MP-borne) whole-blood TF PCA demonstrated a more modest increase, with a peak in activity (1.3-fold over baseline; P <.000 01) at 3 to 4 hours, and persistence for more than 24 hours. This surprisingly modest increase in whole-blood TF activity is likely explained by a profound although transient LPS-induced monocytopenia. MP-associated TF PCA was highly correlated with whole-blood TF PCA and total number of circulating MPs, and whole-blood TF PCA was highly correlated with TF mRNA levels.