We have detected versican, a member of the large chondroitin sulfate proteoglycans, and its degraded C-terminal G3 fragments in human plasma and observed that the versican G3 domain promoted blood coagulation. Silencing G3 expression with small interfering RNA reduced the effect of G3 on coagulation. Plasma coagulation assays suggest that G3 enhances coagulation irrespective of its actions on platelets and white blood cells. To examine how versican affected blood coagulation, we used normal human plasma and different types of coagulation factor-deficient plasmas. The experiments indicated that versican enhanced coagulation through the extrinsic pathway, and that Factor VII was the target molecule. FVII activity assays showed that G3 activated FVII in the presence of plasma but not with purified FVII directly. Yeast two-hybrid, immunoprecipitation, and gel co-migration assays showed that G3 interacted with the tissue factor pathway inhibitor-1 (TFPI-1). TFPI-1 activity assays suggested that G3 inhibited TFPI-1 activity, allowing FVIIa and FXa to facilitate the coagulation process. G3-induced blood coagulation was further confirmed with a mouse model in a real-time manner. Taken together, these results indicate that versican may represent a new target for the development of therapies against atherosclerosis.

OBJECTIVE

To explore the pharmacogenomics of warfarin using the extreme-discordant-phenotype (EDP) methodology.

METHODS

The target phenotype was the stable warfarin dose prescribed to 353 patients. Pharmacogenetic polymorphisms assessed were coagulation factor VII (FVII) -401G>T and FVII -402G>A, VKORC1 3673G>A, and CYP2C9*2, *3, *5, and *11 alleles. The EDP analyses contrasted the frequencies of these polymorphisms at different cutoff points (5th through 30th percentiles of the warfarin dose distribution) at opposite ends of the warfarin dose distribution.

RESULTS

Significant differences existed in FVII -402G>A genotype frequency at the 5th percentile with an over-representation of the wildtype GG genotype at low warfarin doses and in VKORC1 3673G>A and CYP2C9 polymorphisms at all cutoff points where the variant alleles were overrepresented at low warfarin doses.

CONCLUSIONS

The EDP methodology provides increased statistical power for detection of small contributions of genetic polymorphisms to multiple drug-response phenotypes, such as warfarin dose requirement for adequate anticoagulation.

Blood coagulation is initiated in response to vessel damage in order to preserve the integrity of the mammalian vascular system. The coagulation cascade can also be initiated by mediators of the inflammatory response, and fibrin deposition has been noted in a variety of pathological states. The cascade of coagulation zymogen activations which leads to clot formation is initiated by exposure of flowing blood to tissue factor (TF), the cellular receptor and cofactor for factor VII (FVII). FVII binds to the receptor in a 1:1 stoichiometric complex and is rapidly activated. FVIIa undergoes an active site transition upon binding TF in the presence of calcium which enhances the fundamental properties of the enzyme. This results in rapid autocatalytic activation of FVII to VIIa thereby amplifying the response by generating more TF-VIIa complexes. The TF-VIIa activates both FIX and FX. Further FXa generation by the IXa-VIIIa-Ca(2+)-phospholipid complex is required to sustain the coagulation mechanism, since the TF-VIIa complex is rapidly inactivated. Structure and function studies have identified a number of regions on both TF and FVII involved in this interaction. It is clear, however, that the molecular structures of TF, FVII and the TF-VII complex will have to be solved before we fully understand this complex interaction. The activity of the TF-VIIa complex is controlled by two inhibitors:tissue factor pathway inhibitor (TFPI) and antithrombin III (AT-III). TFPI circulates in plasma, is associated with vascular cell surface and is released from platelets following stimulation by thrombin. TFPI requires the formation of an active TF-VIIa complex and FXa generation before inhibition can occur. Similarly, AT-III which is unable to inhibit circulating FVIIa requires the formation of the TF-VIIa complex. TFPI prevents further participation of TF in the coagulation process by forming a stable quaternary complex, TF-VIIa-Xa-TFPI. In contrast, the AT-III-VIIa complex is thought to dissociate from TF allowing it to interact with additional FVII-VIIa. TFPI has been considered the primary regulator of TF-VIIa activity during haemostasis. Whether AT-III in the presence of glycosaminoglycans on cell surfaces expressing TF can function as an auxiliary second physiological regulator is not known.

The aim was to review the pregnancy and obstetric outcome in women with factor VII (FVII) deficiency. The study group contained women with FVII deficiency, registered with Haemophilia centre and Haemostasis Unit at the Royal Free Hospital, London. The women were interviewed and case notes were reviewed. The main outcome measures were changes in FVII levels in pregnancy, maternal and perinatal outcome. The FVII levels ranged from 7-36 IU dL(-1) in the 13 women included in the study. There were a total of 14 pregnancies in seven women. Ten pregnancies in four women were prior to the diagnosis of FVII deficiency. Following the diagnosis of FVII deficiency, there were four pregnancies in three women. There was an increase in the FVII level during pregnancy in these women from a mean baseline level of 33 IU dL(-1) to a mean of 73 IU dL(-1). These women received recombinant FVIIa replacement during labour and delivery. There were two early pregnancy losses, both associated with excessive haemorrhage. There was only one postpartum haemorrhage in the study. There is a significant increase in FVII levels in pregnancy in women with heterozygous FVII deficiency. The risk of bleeding in early pregnancy might be higher than that at term, due to inadequate rise in the FVII level in early pregnancy.

Lung cancer is the second leading type of cancer, with venous thromboembolism being the second leading cause of death. Studies have shown increased levels of microparticles and cell-free DNA (CFDNA) in cancer patients, which can activate coagulation through extrinsic and intrinsic pathways, respectively. However, the impact of lung cancer chemotherapy on microparticle and/or CFDNA generation is not completely understood. The aim of the study was to study the effects of platinum-based chemotherapeutic agents on generation of procoagulant microparticles and CFDNA in vitro and in vivo. Microparticles were isolated from chemotherapy-treated monocytes, human umbilical vein endothelial cells, or cancer cells. Tissue factor (TF) and phosphatidylserine levels were characterized and thrombin/factor Xa generation assays were used to determine microparticle procoagulant activity. CFDNA levels were isolated from cell supernatants and plasma. A murine xenograft model of human lung carcinoma was used to study the procoagulant effects of TF microparticles and CFDNA in vivo. In vitro, platinum-based chemotherapy induced TF/phosphatidylserine microparticle shedding from A549 and A427 lung cancers cells, which enhanced thrombin generation in plasma in a FVII-dependent manner. CFDNA levels were increased in supernatants of chemotherapy-treated neutrophils and plasma of chemotherapy-treated mice. TF microparticles were elevated in plasma of chemotherapy-treated tumour-bearing mice. Plasma CFDNA levels are increased in chemotherapy-treated tumour-free mice and correlate with increased thrombin generation. In tumour-bearing mice, chemotherapy increases plasma levels of CFDNA and TF/phosphatidylserine microparticles. Platinum-based chemotherapy induces the shedding of TF/phosphatidylserine microparticles from tumour cells and the release of CFDNA from host neutrophils.

The association between type 2 diabetes and cardiovascular disease is long recognized. Although perturbations of haemostatic markers have been shown to be associated with macrovascular disease in patients with type 2 diabetes, it is unclear whether these are primarily due to endothelial dysfunction or a result of inflammation. The present study was undertaken to elucidate whether elevated levels of factor VIII (FVIII) and von Willebrand factor (vWF) in women with type 2 diabetes represent endothelial dysfunction, inflammation or an alternate mechanism. Sixty-four women with type 2 diabetes were evaluated using ultrasonography Doppler for carotid intima-media thickness (IMT) and were classified as group A--having no (<1 mm), group B - mild (≥1 mm and no plaque) and group C--moderate (≥1 mm and presence of plaque and stenosis) macrovascular disease. Several haemostatic markers including, FVIII, vWF and fibrinogen were assessed. In addition, thrombomodulin, a marker for endothelial damage, and high-sensitivity C-reactive protein (hsCRP), an inflammatory marker, were also measured. A significant association of elevated FVIII was found in group B and C patients (i.e. patients with IMT ≥1 mm and with plaque). Elevated fibrinogen and vWF levels were also found but confined to group C patients. No significant difference among subgroups was found for any other variable evaluated (hsCRP, thrombomodulin and FVII). In conclusion, plasma FVIII levels are elevated in women with type 2 diabetes and macrovascular disease. It also appears that this is not mediated by inflammation or endothelial injury and is likely to be due to an alternate mechanism.

In the absence of definite guidelines in the area, we have carried a systemic review to provide a thorough overview concerning the efficacy and safety of recombinant activated factor VII (rFVIIa, NovoSeven®, Novo Nordisk A/S, Bagsværd, Denmark) in patients with Glanzmann's thrombasthenia (GT) and FVII deficiency, undergoing surgical procedures. PubMed, Web of Science, Scopus and EMBASE databases was employed for the search. Three multicenter registries were identified: the Glanzmann's Thrombasthenia Registry (GTR), the Seven Treatment Evaluation Registry (STER), and a German post-marketing surveillance registry (the WIRK study). In addition, data from 10 case-series and/or single-center experiences have been summarized. We have found that the following; perioperatively, the hemostatic effectiveness of rFVIIa was high in GT patients and in those with FVII deficiency undergoing both minor and major surgical procedures. Moreover, in all studies, rFVIIa was well tolerated. Thus, the current evidence shows an optimal perioperative safety/efficacy profile of rFVIIa in the setting of these rare bleeding disorders, and provides the rationale for further studies aimed at evaluating the optimal perioperative anti-hemorrhagic prophylaxis with rFVIIa in GT and in FVII deficient patients.

Factor VII (FVII) coagulant activity has been proven to be associated with the risk of future fatal coronary heart disease (CHD) in middle-aged men. Recent studies have emphasized the role of triglyceride-rich lipoproteins and FVII genotype in determining plasma levels of FVII protein and activity. The present study was undertaken to examine whether FVII activity state and protein concentration in fasting plasma are altered in young men with proven myocardial infarction (MI) and examined the relations of FVII to subfractions of apo B-containing lipoproteins and the Arg->>Gln polymorphism in the FVII gene. Activated FVII (FVIIa) was determined by a clotting assay using soluble, recombinant, truncated tissue factor. A total of 94 men with a first MI before the age of 45 (mean age +/- SD, 39.6 +/- 4.5 years) were included in the study along with 99 population-based, age-matched control subjects. In addition to FVIIa and FVII antigen (FVII:Ag), a panel of FVII activity assays were included for comparison with previous work in this field. The plasma level of FVII:Ag was higher in patients than in control subjects when the entire groups were compared (537 +/- 128 versus 479 +/- 93 ng/mL, P < .001), the differences being accounted for by patients with hypertriglyceridemic lipoprotein phenotypes. In contrast, FVIIa was similar in patients and control subjects (4.6 +/- 1.4 versus 4.3 +/- 1.3 ng/mL, NS), which means that the proportion of FVIIa molecules was unaltered or even lower in the patients. As expected, the Arg->>Gln polymorphism significantly influenced both FVII mass and activity levels. In addition, presence of the Gln allele appeared to be associated with a lower proportion of fully active FVII molecules. The polymorphism also affected the relation between the plasma concentration of VLDL and FVII:Ag. The triglyceride content and particle number of all VLDL subfractions, irrespective of particle size, correlated fairly strongly with FVII mass determinations but not at all with FVIIa. HDL cholesterol concentration, on the other hand, presumably reflecting the efficiency of lipoprotein lipase-mediated lipolysis of VLDL, related significantly to the FVIIa level. The Arg->>Gln polymorphism, independent of lipoprotein effects, explained 5% to 10% of the variation in FVII mass and activity. In conclusion, the present findings speak against a role of FVII as a risk factor for CHD, because a significantly increased potential for activation of coagulation (ie, raised basal concentration of FVIIa) was not observed among young postinfarction patients.(ABSTRACT TRUNCATED AT 400 WORDS)

Essentials The procoagulant effects of microparticles (MPs) on coagulation in endotoxemia are not known. MPs from endotoxemia volunteers were evaluated for procoagulant activity in a plasma milieu. MPs from endotoxemia volunteers shortened clotting times and enhanced thrombin generation. MP procoagulant effects were mediated in a factor XI-dependent manner.

Background Human endotoxemia is characterized by acute inflammation and activation of coagulation, as well as increased numbers of circulating microparticles (MPs). Whether these MPs directly promote coagulation and through which pathway their actions are mediated, however, has not been fully explored. Objectives In this study, we aimed to further characterize endotoxin-induced MPs and their procoagulant properties using several approaches. Methods Enumeration and characterization of MPs were performed using a new-generation flow cytometer. Relative contributions of the extrinsic and intrinsic pathways in MP-mediated procoagulant activity were assessed using plasmas deficient in factor (F) VII or FXI or with blocking antibodies to tissue factor (TF) or FXIa. Results Total MPs and platelet MPs were significantly elevated in plasma at 6 h after infusion of endotoxin in healthy human subjects. MPs isolated from plasma following endotoxin infusion also demonstrated increased TF activity in a reconstituted buffer system. When added to recalcified platelet-poor plasma, these MPs also promoted coagulation, as judged by a decreased clotting time with shortening of the lag time and time to peak thrombin using calibrated automated thrombography (CAT). However, the use of FVII-deficient plasma or blocking antibody to TF did not inhibit these procoagulant effects. In contrast, plasma clotting time was prolonged in FXI-deficient plasma and a blocking antibody to FXIa inhibited all MP-mediated parameters in the CAT assay. Conclusions The initiation of coagulation by cellular TF in endotoxemia is in contrast to (and presumably complemented by) the intrinsic pathway-mediated procoagulant effects of circulating MPs.

A model solution structure for the complete tissue factor-free calcium ion-bound human zymogen FVII (residues 1-406) (FVII) has been constructed to study possible conformational changes associated with the activation process and tissue factor (TF) binding. The initial structure for the present model was constructed using the X-ray crystallographic structure of human coagulation FVIIa/TF complex bound with calcium ions (Banner et al., Nature 1996, 380, 41-46). This model was subsequently subjected to lengthy molecular dynamics simulations. The Amber force field in conjunction with the PME electrostatic summation method was employed. The estimated TF free solution structure was then compared with the currently available X-ray crystal structures of FVIIa (with or without TF, variable inhibitor bound) to estimate the restructuring of FVII due to TF binding and activation. The solution structure of the zymogen FVII in the absence of TF is predicted to be an extended domain structure similar to that of the TF-bound X-ray crystal structure. An additional extension of the serine protease (SP) domain of the zymogen above a reference lipid surface by approximately 7 A was in agreement with experiment. Significant Gla-EGF1 and EGF1-EGF2 interdomain motions in the zymogen were observed. Carbohydrate dimers attached to Ser-52 and Ser-60 did not cause restructuring in this domain. Minimal restructuring of the SP domain is found upon inference of the zymogen from the activated form. The catalytic triad residues maintain the H-bonded network while Lys-341 occupies the S1 specific site in the zymogen.

This report documents our identification of a novel factor VII (FVII) gene mutation in a Japanese boy with FVII deficiency. The proband's FVII activity was 34% and his FVII antigen level was 40% of normal controls. DNA sequence analysis of the proband's FVII gene identified a C to G point mutation at nucleotide position 10 933 in exon 8, which results in the substitution of Cys (TGC) for Ser339 (TCC). Hinf I digestion results indicate the proband and his mother were heterozygous for the mutation. Both wild-type and mutant FVIIs were transiently expressed in COS-1 cells. FVII levels measured in the culture medium of FVII Ser339Cys mutants were markedly reduced as compared to those of cells with FVII wild-type. The amount of intracellular FVII in FVII Ser339Cys mutants was 80% of that in wild-type. In the wild-type FVII, Ser339 is juxtaposed to Asp338, which is positioned at the bottom of the substrate-binding pocket in the protease domain and located adjacent to FVII Cys340, that forms a disulphide bond with Cys368. We suspect that the creation of a novel unpaired cysteine through this mutation leads to abnormal disulphide bonding during protein folding, thereby reducing the secretion of FVII.

Tissue factor (TF) is an integral membrane protein widely expressed in normal human cells. Blood coagulation factor VII (fVII) is a key enzyme in the extrinsic coagulation cascade that is predominantly secreted by hepatocytes and released into the bloodstream. The TF-fVII complex is aberrantly expressed on the surface of cancer cells, including ovarian cancer cells. This procoagulant complex can initiate intracellular signaling mechanisms, resulting in malignant phenotypes. Cancer tissues are chronically exposed to hypoxia. TF and fVII can be induced in response to hypoxia in ovarian cancer cells at the gene expression level, leading to the autonomous production of the TF-fVII complex. Here, we discuss the roles of the TF-fVII complex in the induction of malignant phenotypes in ovarian cancer cells. The hypoxic nature of ovarian cancer tissues and the roles of TF expression in endometriosis are discussed. Arguments will be extended to potential strategies to treat ovarian cancers based on our current knowledge of TF-fVII function.

Factor VII (FVII) is a four-domain glycoprotein that plays a critical role in the initiation of blood coagulation. Hereditary deficiencies of this plasma protein results in a bleeding diathesis that varies in severity amongst affected patients. We have analysed the FVII gene in 27 patients with FVII deficiency from 21 unrelated families predominantly of Middle-Eastern extraction. A total of 19 different mutations were identified, of which 12 were novel and 7 had been previously reported. Nine of the 12 novel mutations were missense mutations located in the Gla domain (Ser23Pro), the second epidermal growth factor domain (Cys135Arg) and the catalytic serine protease domain (Arg247Cys, Arg277Cys, Ser282Arg, Pro303Thr, Ser363Ile, Trp364Cys, Trp364Phe), of which five are homozygous. Three novel splice mutations were identified in intron 1a (IVS1a+5), intron 2 (IVS2+1) and intron 6 (IVS6+1). Of the seven previously reported mutations, five were missense mutations of which three are homozygous (Gln100Arg, Arg152Gln, Arg304Gln, Cys310Phe and Thr359Met), one was a 17 bp deletion (10585del117bp) and one was a splice site mutation within intron 7 (IVS7+7). This study has significantly extended the current database of FVII mutations, including the number of known homozygous mutations. Conformational analyses of crystal structures for FVIIa and the FVIIa-tissue factor complex provided likely explanations for the effect of the missense mutations on FVIIa secretion or function. In particular, since 23 missense mutations were located to the serine protease domain, mostly to the region between the catalytic triad and the contact surface with tissue factor, this showed that the orientation of the serine protease domain relative to bound tissue factor in the complex is crucial for functional activity.

OBJECTIVE

Oat bran shows cholesterol-lowering properties, but its effects on other cardiovascular risk markers are less frequently investigated. This study examined the effects of oat bran on blood lipids, hemostatic factors and energy utilization.

METHODS

A double-blind, randomized crossover study in 24 adults (age 25.2±2.7 years; body mass index: 24.9±2.9 kg/m2), who completed two 2-week dietary intervention periods: low-fiber diet (control) or an oat bran (control +102 g oat bran/day) diet. Fasting blood samples were drawn before and after each period, and 3-day fecal samples were collected during the last week of each period.

RESULTS

Total cholesterol decreased by 14% during the oat bran period compared with 4% during the control period (P<0.001). Non-high-density lipoprotein (HDL) cholesterol decreased by 16% in the oat bran period compared with 3% in the control period (P<0.01), as did total triacylglycerol (21 vs 10%, P<0.05) and very-low-density lipoprotein triacylglycerol 33 vs 9%, P<0.01). Plasminogen activator inhibitor-1 (PAI-1) and factor VII (fVII) levels decreased more during consumption of oat bran compared with the control period (PAI-1: 30 vs 2.3%, P<0.01; fVII: 15 vs 7.6%, <0.001). Fecal volume and dry matter were greater when consuming the oat bran diet compared with the control (P<0.001), and energy excretion was increased by 37% (1014 vs 638 kJ/day, P<0.001); however, changes in body weight did not differ (oat bran:-0.3±0.5 kg; control: 0.0±0.7 kg).

CONCLUSIONS

Addition of oat bran (6 g soluble fiber/day) to a low-fiber diet lowered total and non-HDL cholesterol, as well as hemostatic factors, and may affect energy balance through reduced energy utilization.

Hepatocellular carcinoma (HCC) is one of the most common and aggressive malignancies worldwide. Treatment outcomes remain poor mainly due to lack of good diagnostic/prognostic markers and limited therapeutic strategies. We previously characterized aberrant activation of the TF/FVII/PAR2 pathway, which subsequently results in decreased autophagy, as a crucial event in malignant progression of HCC. Here, we identified miR-135a as a highly upregulated miRNA in HCC in response to TF/FVII/PAR2 activation. Analyzing 103 HCC patient specimens, we confirmed that miR-135a was frequently elevated in HCC tissues with higher FVII expression compared to adjacent non-cancerous counterparts. Increased miR-135a levels in HCC were also associated with tumor staging, recurrence, microvascular invasion, and decreased disease-free survival. We subsequently identified Atg14, a key component that regulates the formation of autophagosome as a direct target of miR-135a. Ectopic expression of miR-135a suppressed Atg14 levels and inhibited the autophagic processes. Our results indicate strong positive correlations between miR-135a levels and malignant behaviors in HCC patients and also suggest novel functions of miR-135a in regulation of autophagy, which could be useful as a potential target for prognostic and therapeutic uses.

Coagulation factor VII contains ten gamma-carboxyglutamic acid residues in the N-terminal region (Gla-domain) which are essential for the hemostatic function of FVII. The present study shows that granulocyte cathepsin G degrades the Gla-domain of FVIIa in vitro. Characterization of the truncated FVIIa by SDS-PAGE and N-terminal amino acid sequence analysis revealed that cleavage had occurred between Tyr-44 and Ser-45 and that further cleavage was only obtained on extensive cathepsin G exposure. Cleavage of vitamin K-dependent coagulation factors by cathepsin G may play a role in vivo, and it offers a convenient way of obtaining proteins deprived of their Gla-domain for functional and structural studies.

We describe a polymorphism in the 5' region of the coagulation factor VII (FVII) gene, originating from a decanucleotide (CCTATATCCT) insert present in the less frequent allele. This marker can be detected by restriction analysis of polymerase chain reaction products.

We have studied the molecular basis of factor (F) VII deficiency in 11 unrelated Indian patients. Mutations were identified in all 11 and included 5 missense, 2 nonsense and a frame shift mutation. Five of these were novel. These mutations were considered to be causative of disease because of their nature, evolutionary conservation and molecular modeling. This is the first report of mutations in patients with FVII deficiency from southern India.

A total of 122 subjects, referred after presurgery screening or checkup for prolonged prothrombin time, were characterized for the presence of coagulation factor VII deficiency. Fourteen subjects carried a partial and asymptomatic deficiency, and in half of them dysfunctional molecules were detected in plasma. In nine subjects we found five missense mutations differing from those previously found in factor VII deficient patients. The others were homozygous for a common polymorphism (R353Q) that affects factor VII levels. A new codon dimorphism (A330) was also found in exon 8. Four mutations (R223W, M298I, R304Q, and R353Q) located at FVII-specific residues point out protein regions that are important for coagulation factor evolution, and two mutations (G342E and E265K) affect generic or partially generic residues. The newly reported mutations were combined with those we previously found, totalling 17 independent mutations responsible for FVII deficiency in 27 Italian pedigrees. We observed several similarities with the mutation pattern determined in factor IX, which include a high percentage of transitions at CpG doublets, the presence of hot spot sites affected by multiple substitutions, and of several topologically equivalent mutations.

Inherited factor (F)VII deficiency is rare in most populations but relatively common in Israel. The aim of this study was to characterize the molecular and functional defect in unrelated Israeli patients with FVII deficiency. Mutations were identified by direct sequencing of PCR-amplified genomic DNA fragments. Selected mutations were expressed in baby hamster kidney (BHK) cells and tested for binding to tissue factor (TF), activation by FXa and activation of FX. In 61 patients with FVII deficiency, the causative mutation in the FVII gene was discerned. The predominant mutation found in this and a previously reported cohort of 27 unrelated patients in Israel was Ala244Val substitution; of 121 independent mutant alleles defined in all 88 patients ascertained in Israel, 102 (84%) bore this alteration. Eleven additional mutations were identified of which one, Cys22Arg, is novel. Expression of the mutations in BHK cells revealed that four (Ala244Val, 11128delC, Leu300Pro and Cys22Arg) were cross-reacting material (CRM)- negative, and three (Ala294Val, Cys310Phe and Phe24del) were CRM-positive. As predicted by modeling, we observed no binding to TF of FVII Phe24del, diminished binding of FVII Cys310Phe and normal binding of FVII Ala294Val. The main defect of FVII Ala294Val was its inability to activate FX in the presence of TF. Coexpression of Ala294Val and Arg353Gln, a polymorphism known to affect FVII secretion, did not reveal an additive effect on FVII secretion, while coexpression of Ala244Val and Arg353Gln did yield an additive effect.

Expression of the human coagulation factor VII (FVII) gene by hepatoma cells was modulated in concert with levels of glucose and insulin in the culture medium. In low glucose medium without insulin, amounts of both FVII mRNA and secreted FVII protein were coordinately increased; in the presence of glucose with insulin, both were decreased. Analysis of the FVII promoter showed that these effects could be reproduced in a reporter-gene system, and a small promoter element immediately upstream of the translation start site of the gene, which mediated these effects, was identified. Mutation of this element largely abrogated the glucose/insulin-responsive change in expression of the reporter gene. Several members of the CCAAT/enhancer-binding protein family were found to be capable of binding the identified sequence element but not the mutated element. The expression of a FVII minigene directed by a segment of the native FVII promoter responded to co-expressed activating and inhibiting forms of CCAAT/enhancer-binding protein beta.

BACKGROUND

A reduced sensitivity for activated protein C (APC) is associated with an increased risk of venous thrombosis even in the absence of the factor (F)V Leiden mutation. This risk has been demonstrated with two APC sensitivity tests, which quantify the effects of APC on the activated partial thromboplastin time (APTT) and the endogenous thrombin potential (ETP), respectively.

OBJECTIVE

We examined determinants of both APC sensitivity tests in the control group of the Leiden Thrombophilia Study (LETS).

METHODS

Multiple linear regression analysis was performed with normalized APC-SR(APTT) or APC-SR(ETP) as dependent variable and putative determinants [levels of FII, FV, FVII, FVIII, FIX, FX, FXI, FXII, FXIII A subunit, FXIII B subunit, protein S total, protein S free, protein C, tissue factor pathway inhibitor (TFPI) total, TFPI free, antithrombin and fibrinogen] as independent variables.

CONCLUSIONS

The major determinant of the APTT-based test was FVIII level, followed by FII level. The ETP-based test was influenced most by free protein S and free TFPI levels. In both tests FXa formation plays a major role, as the effect of FVIII and TFPI on the tests seems to be executed via FXa. The ETP-based test was also strongly influenced by oral contraceptive use, even when we adjusted for all the clotting factors listed above. This means that the effect of oral contraceptives on the ETP-based test is not fully explained by the changes of coagulation factor levels investigated in this study, and that the molecular basis of acquired APC resistance during use of oral contraceptives remains to be established.

BACKGROUND

Declined lung function is a risk factor for particulate matter associated respiratory diseases like asthma and chronic obstructive pulmonary disease (COPD). Carbon nanoparticles (CNP) are a prominent component of outdoor air pollution that causes pulmonary toxicity mainly through inflammation. Recently we demonstrated that mice (C3H/HeJ) with higher than normal pulmonary function resolved the elicited pulmonary inflammation following CNP exposure through activation of defense and homeostasis maintenance pathways. To test whether CNP-induced inflammation is affected by declined lung function, we exposed JF1/Msf (JF1) mice with lower than normal pulmonary function to CNP and studied the pulmonary inflammation and its resolution.

METHODS

5 μg, 20 μg and 50 μg CNP (Printex 90) were intratracheally instilled in JF1 mice to determine the dose response and the time course of inflammation over 7 days (20 μg dosage). Inflammation was assessed using histology, bronchoalveolar lavage (BAL) analysis and by a panel of 62 protein markers.

RESULTS

24 h after instillation, 20 μg and 50 μg CNP caused a 25 fold and 19 fold increased polymorphonuclear leucocytes (PMN) respectively while the 5 μg represented the 'no observable adverse effect level' as reflected by PMN influx (9.7 × 10E3 vs 8.9 × 10E3), and BAL/lung concentrations of pro-inflammatory cytokines. Time course assessment of the inflammatory response revealed that compared to day1 the elevated BAL PMN counts (246.4 × 10E3) were significantly decreased at day 3 (72.9 × 10E3) and day 7 (48.5 × 10E3) but did not reach baseline levels indicating slow PMN resolution kinetics. Strikingly on day 7 the number of macrophages doubled (455.0 × 10E3 vs 204.7 × 10E3) and lymphocytes were 7-fold induced (80.6 × 10E3 vs 11.2 × 10E3) compared to day1. At day 7 elevated levels of IL1B, TNF, IL4, MDC/CCL22, FVII, and vWF were detected in JF1 lungs which can be associated to macrophage and lymphocyte activation.

CONCLUSIONS

This explorative study indicates that JF1 mice with impaired pulmonary function also exhibits delayed resolution of particle mediated lung inflammation as evident from elevated PMN and accumulation of macrophages and lymphocytes on day 7. It is plausible that elevated levels of IL1B, IL4, TNF, CCL22/MDC, FVII and vWF counteract defense and homeostatic pathways thereby driving this phenomenon.

Canine coagulation factor VII (FVII) deficiency can be hereditary or acquired and may cause life threatening bleeding episodes if untreated. FVII procoagulant activity can be measured by FVII activity (FVII:C), but assays for measurement of canine specific FVII antigen (FVII:Ag) have not been available to date. In this study, a canine specific ELISA for measurement of FVII:Ag in plasma was developed and validated. The FVII:Ag ELISA correctly diagnosed homozygous and heterozygous hereditary FVII deficiency. Together with activity based assays, such as FVII:C, the FVII:Ag ELISA should be valuable in the diagnosis of hereditary canine FVII deficiency.