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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., Competing Interests: The authors have declared that no competing interests exist, (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

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.

Physicians need to recognize and manage Gulf War illness and similar postdeployment, chronic, multisymptom diseases among veterans of recent military operations., Competing Interests: Author disclosures The authors report no actual or potential conflicts of interest with regard to this article.

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.

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.

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 (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.

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.

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.

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.

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.

Using a novel whole blood assay, we recently demonstrated that tissue factor procoagulant activity (TF PCA) is present in normal individuals. Preliminary experiments suggested that this activity is localized in the mononuclear cell fraction. Postulating that whole blood TF PCA would therefore be undetectable when monocytes and neutrophils are absent from peripheral blood, we assayed TF PCA during the peri-transplant period in 15 consecutive patients undergoing allogeneic (n = 12) or autologous (n = 3) bone marrow transplantation (BMT) or peripheral blood stem cell transplantation (PBSCT). Baseline (pre-transplant) mean TF PCA was higher in patients compared to normal controls (P <0.005). Unexpectedly, although TF PCA during the period of profound aplasia was significantly reduced compared to baseline (p <0.05), fully 55% of the initial activity remained detectable. During the engraftment phase, TF PCA returned to pre-transplant levels, with a linear correlation between monocyte counts and TF PCA (r = 0.63). In contrast to normal whole blood, incubation of aplastic samples with E. Coli lipopolysaccharide ex vivo failed to induce TF PCA. Throughout the period of study--but especially during the aplastic phase--the absolute number of circulating endothelial cells (CECs) that were TF antigen-positive was increased compared to normals (P <0.001). However, removal of these cells from whole blood samples failed to significantly diminish total TF PCA indicating that CECs alone could not account for the detectable TF PCA during aplasia. We conclude that neither circulating mature myelo-monocytic cells nor endothelial cells can account for all the functionally intact TF in peripheral blood. Further studies are needed to identify the other source(s) of TF PCA.

Human granulocytic ehrlichiosis (HGE) is a recently recognized rickettsial tick-borne febrile illness that may occasionally be complicated by coagulopathy. The agent of HGE (aHGE) is an obligate intracellular pathogen, which replicates in endosomes within neutrophils and their precursors. We hypothesized that aHGE might cause DIC via induction of monocyte tissue factor procoagulant activity (TF PCA). Peripheral blood mononuclear cells (PBMNC) and HL-60 cells were used to model the effect of aHGE infection on monocytes/macrophages. Mononuclear cells inoculated with aHGE in vitro demonstrated approximately a 12-15-fold increase in TF PCA, with peak activity occurring at 8-12 h. HL-60 cells inoculated with aHGE also manifested a 4-6 fold induction of TF PCA, with maximal activity occurring at about 8 h. By comparison, E. Coli lipopolysaccharide (LPS) also induced an increase in TF PCA of an equivalent magnitude, and with a similar time course. Induction of TF did not require inoculation of HL-60 cells with live organism, since heat-inactivated aHGE still stimulated TF PCA expression in the target cells. Furthermore, filtered supernatants from heat-inactivated organisms induced TF PCA suggesting that the effect is due to a soluble mediator produced by the organism. Although aHGE is a gram negative organism, the soluble mediator did not appear to be classic endotoxin in that the supernatants tested negative for endotoxin by the Limulus Amoebocyte assay, and polymixin had no inhibitory effect on aHGE supernatants. We conclude that aHGE induces cells of the myelo-monocytic lineage to synthesize TF, which may contribute to the clinical coagulopathy that can be observed in this condition. An atypical soluble mediator or cellular component of the organism appears to be critically important in TF induction by aHGE.

We developed a simple assay for the measurement of tissue factor procoagulant activity (TF PCA) in whole blood samples that avoids the need for mononuclear cell isolation. This method combines convenience of sample collection and processing with a high degree of sensitivity and specificity for TF. Using this method, we have determined that TF PCA is detectable in whole blood samples from normal individuals, which is itself a novel observation. Essentially all PCA could be shown to be localized in the mononuclear cell fraction of blood. Compared with controls, whole blood TF levels were significantly (P < .000001) elevated in patients with sickle cell disease (SCD), regardless of the subtype of hemoglobinopathy (SS or SC disease). No significant difference in TF PCA was observed between patients in pain crisis compared with those in steady-state disease. Because TF functions as cofactor in the proteolytic conversion of FVII to FVIIa in vitro, it was expected that an increase in circulating TF PCA would lead to an increased in vivo generation of FVIIa. On the contrary, FVIIa levels were actually decreased in the plasma of patients with SCD. Plasma TF pathway inhibitor (TFPI) antigen levels were normal in SCD patients, suggesting that accelerated clearance of FVIIa by the TFPI pathway was not responsible for the reduced FVIIa levels. We propose that elevated levels of circulating TF PCA may play an important role in triggering the activation of coagulation known to occur in patients with SCD. Because TF is the principal cellular ligand for FVIIa, it is possible that increased binding to TF accounts for the diminished plasma FVIIa levels.

The activation of tissue factor (TF) procoagulant activity (PCA) on the surface of cells may be the first step in the extrinsic pathway of blood coagulation. The latent TF PCA on intact cells is expressed following mechanical disruption or exposure to ionomycin, a Ca2+ ionophore. Within seconds of ionophore addition, an increase in TF PCA of more than 100-fold is observed. The ionophore effect is blocked by pretreating the cells with calmodulin inhibitors. Complexes of TF and activated factor VII (FVIIa) form on both untreated and ionophore-treated cells. However, pseudosubstrate inhibitors bind only to TF-FVIIa on ionophore-treated cells. When proteins on unperturbed cells are crosslinked with 3-3'-dithiobis(sulphosuccinimidylpropionate), crosslinked homodimeric TF is produced. This TF crosslinking is prevented by first treating the cells with ionomycin. Thus, there is an apparent change in TF quaternary structure that is coincident with the ionophore-induced change in TF PCA. These results suggest a simple mechanism for the activation of TF PCA on cells which is triggered by Ca2+ influx into the cytosol. A Ca2+-binding protein, possibly calmodulin, appears to be an essential link in the signal transduction pathway going from increased cytosolic Ca2+ to increased expression of TF PCA on the cell surface. This activation of TF PCA may result from exposure of an essential macromolecular-substrate-binding site on the TF-FVIIa complex that is the direct result of a change in TF quaternary structure, i.e. the conversion of cryptic TF dimers to procoagulant TF monomers.

Tissue factor (TF) procoagulant activity (PCA) on the surface of intact HL-60 cells is encrypted. This latent TF PCA was activated by exposing the cells to ionomycin, a calcium ionophore. Within seconds an increase in TF PCA of greater than 100-fold was observed. The ionomycin effect was blocked by pretreating the cells with calmidazolium, a calmodulin inhibitor. Changes in TF structure and function, coincident with the ionophore-induced increase in TF PCA, were identified. TF-factor VIIa complexes formed on both untreated and ionophore-treated cells, but pseudosubstrate inhibitors only bound to TF-factor VIIa on the ionophore-treated cells. TF PCA was inhibited by reacting cells with sulfosuccinimidyl-6-(biotinamido)hexanoate, and the rate of this reaction increased twofold after cells were exposed to ionomycin. When proteins on the surface of untreated cells, expressing minimal TF PCA, were cross-linked with 3-3'-dithiobis(sulfosuccinimidylpropionate), cross-linked TF dimers were produced. TF cross-linking was prevented by first treating the cells with ionomycin. These results suggest a mechanism for the ionomycin-induced increase in TF PCA. TF activation appears to be a calmodulin-dependent process, which exposes an essential macromolecular substrate binding site on TF, possibly as the result of a change in TF quaternary structure.

Tissue factor (TF), the physiologic initiator of coagulation, is present on the surface of cells but is not fully active unless the cell is lysed. This phenomenon, termed TF encryption, may be regulated by changes in membrane structure. Because apoptosis is associated with cell membrane alterations and conditions associated with apoptosis have also been associated with TF de-encryption, we hypothesized that apoptosis would result in enhanced TF procoagulant activity. Cultured human fibroblasts and endotoxin-stimulated endothelial cells were treated to induce apoptosis as evidenced by morphologic and DNA changes. Under the same conditions, changes in the level of TF activity were measured. Conditions that resulted in endothelial apoptosis were associated with de-encryption of TF activity. Similar results were obtained in fibroblasts except that only the morphologic changes, not the alterations in DNA size characteristic of apoptosis in other cells, were found. The data suggest an association between apoptosis and expression of cell surface tissue factor activity. Because of the recognized linkage of the coagulation system with wound healing and neoplasia, we speculate that this association may help to regulate the flux of cells in tissues being remodelled by apoptosis.

Bacterial lipopolysaccharide (LPS) initiates the cascade of inflammatory events that, in infected patients, often result in a lethal systemic inflammatory response known as the sepsis syndrome. We studied LPS-stimulated expression of tissue factor (TF) in human peripheral blood mononuclear cells (PBMCs) and cultured endothelial cells or tumor necrosis factor-alpha (TNF-alpha) in PBMCs. CD14, a PBMC membrane protein, is involved in LPS signaling and is also present as a soluble molecule in serum. CD14 is absent from endothelial cells and, in varying degrees, from monocytes of patients with paroxysmal nocturnal hemoglobinuria (PNH). LPS stimulation of TF in normal monocytes was enhanced > 30-fold by serum at low concentrations of LPS (< or = 10 ng/ml). The serum dependence of endothelial cells was even more pronounced; a full response to LPS was not observed in endothelium under serum-free conditions, even with LPS concentrations as high as 100 ng/ml. To better define the role of CD14, CD14-deficient PBMCs from two patients with PNH were compared with normal PBMCs. Although less than 3% of PNH monocytes expressed CD14, LPS-induced synthesis of TF and TNF-alpha by PBMCs from PNH patients was inhibited by anti-CD14 antibodies. Because patient serum samples were found to contain soluble CD14, we sought to determine whether PNH monocytes might respond to LPS through an activation pathway dependent on soluble CD14. Recombinant soluble CD14 substituted for serum to enable LPS stimulation of endothelium, PNH PBMCs, and surprisingly, CD14-replete normal PBMCs. In addition, a truncated sCD14 containing the N-terminal 152 amino acids similarly enabled LPS stimulation of normal PBMCs. These data underscore the importance of soluble CD14 and suggest that CD14 present in serum enables LPS responses in PNH monocytes and endothelial cells and may even influence the effects of LPS in normal human phagocytes.

Procoagulant activity of pairs of cell lines, which were derived from the same original cell type but which possess different growth characteristics and metastatic properties, was examined. The following characteristics were considered suggestive of a greater likelihood of metastatic potential: high histological grade; establishment of the line from a metastatic rather than a nonmetastatic cancer; increased tumorigenicity in nude mice; and/or estrogen receptor-negative mammary cancer. Procoagulant activity was evaluated by a two stage clotting assay. Procoagulant activity was highly variable, with up to a 1,300-fold difference, among the cancer cell lines examined. The rate of clot formation was factor VII dependent and was totally inhibited by an anti tissue factor monoclonal antibody, indicating that tissue factor was the only significant procoagulant present in these cancer cells. Tissue factor antigen expression, evaluated by ELISA, correlated with procoagulant activity. In all pairs of cancer cell lines, those with characteristics of increased proliferative potential had increased tissue factor levels compared to cell lines that originated from the same cell type, but which possess less aggressive characteristics. Tissue factor activity in these cancer cells was increased by cell lysis or by exposure of intact cells to a calcium ionophore, similar to results previously obtained in fibroblasts. Tissue factor mRNA was evaluated by northern blot analysis using a specific probe complementary to tissue factor mRNA. The previously described predominant tissue factor mRNA species of 2.2 kb was identified in the majority of cancer cell lines examined, but tissue factor mRNA species of 3.2 to 3.4 kb were also identified.(ABSTRACT TRUNCATED AT 250 WORDS)

The structural determinants of lipopolysaccharide required for the induction of tissue factor in human umbilical vein endothelial cells were studied. Intact lipid A was essential for the induction of tissue factor whereas the incomplete lipid A precursors lipid IVA and lipid X, as well as monophosphoryl lipid A and acyloxyacyl hydrolase-treated lipopolysaccharide, were unable to induce tissue factor and tissue factor specific mRNA. However, the lipid A precursor, lipid IVA, was able to inhibit LPS-mediated induction of tissue factor; structural determinants distal to lipid A were found to be required for maximal induction of tissue factor activity and tissue factor mRNA. The presence of serum in the assay was found to amplify but was not obligate for tissue factor induction by LPS.

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.

Background: Herpes simplex virus (HSV)-infected endothelium is a model for vascular injury and possibly the development of atherosclerosis. In vitro infection of human umbilical vein endothelial cells (HUVEC) by HSV-1 results in a number of changes including the expression of a procoagulant activity (PCA) compatible with that due to tissue factor (TF) synthesis. In this study, we have further characterized this PCA using more stringent assays for TF, and examined whether virus rendered incapable of replication retains the ability to stimulate TF synthesis in HUVEC., Experimental Design: Confluent monolayers of HUVEC were exposed to intact or ultraviolet/heat-inactivated HSV-1. At appropriate time intervals, TF PCA was assessed by clotting assays, and TF antigen by an enzyme-linked immunosorbent assay specific for TF. The appearance of mRNA specific for TF was performed by Northern blotting., Results: TF activity was demonstrated by both 1-stage and 2-stage clotting assays; the dependence of the latter on factor VIIa, and the inhibition by specific blocking antibodies to human TF support the notion that the PCA is indeed due to TF. Furthermore, cellular TF antigen levels were found to parallel TF activity, and there was a transient de novo expression of TF mRNA. Tissue factor PCA in HSV-infected HUVEC remained "encrypted"; that is, full clotting activity was not expressed in the absence of cellular disruption in a situation analogous to that seen in all normal cells thus far examined that express TF PCA. However, this response did not appear to be dependent upon replicative infection of HSV-1 within the endothelial cell since a similar (although lesser) induction of TF PCA was present in cells that had been exposed to virus previously rendered incapable of replication., Conclusions: HSV-1 induces PCA in HUVEC which is clearly TF-dependent; this response does not require viral replication. These data indicate increased complexity in HSV interactions with vascular endothelium and imply induction of some procoagulant functions by nonproductive infection.

Tissue factor (TF) is an integral membrane glycoprotein which functions as an initiator of coagulation. Furthermore, it is probably the principal biological initiator of this essential hemostatic process. This article reviews the studies which form the basis for these assertions. The work on TF is traced from the 19th century discovery of the thromboplastic activity of tissues to the recent purification of the protein from bovine and human tissues and the isolation cDNA clones coding from human TF. The features of TF structure and function which tailor it to the role of initiator of the coagulation cascade are considered. For example, cell-surface TF and factor VII, the plasma serine proteases zymogen, form a proteolytic complex without prior proteolysis of either component. In addition, a kinetic model for the molecular mechanism of TF-initiated clotting is reviewed. The factors which control the expression of TF procoagulant activity by cultured cells are examined in light of the hypothesized role of TF in normal hemostasis. Also, the potential pathological consequences of aberrant TF expression, i.e., thrombosis and hemorrhage, are explored.