OBJECTIVE

To determine whether healthy males who consumed increased amounts of dietary stearic acid compared with increased dietary palmitic acid exhibited any changes in their platelet aggregability, platelet fatty acid profiles, platelet morphology, or haemostatic factors.

METHODS

A randomized cross-over dietary intervention.

METHODS

Thirteen free-living healthy males consumed two experimental diets for 4 weeks with a 7 week washout between the two dietary periods. The diets consisted of approximately 30% of energy as fat (66% of which was the treatment fat) providing approximately 6.6% of energy as stearic acid (diet S) or approximately 7.8% of energy as palmitic acid (diet P). On days 0 and 28 of each dietary period, blood samples were collected and anthropometric and physiological measurements were recorded.

RESULTS

Stearic acid was increased significantly in platelet phospholipids on diet S (by 22%), while on diet P palmitic acid levels in platelet phospholipids also increased significantly (8%). Mean platelet volume, coagulation factor FVII activity and plasma lipid concentrations were significantly decreased on diet S, while platelet aggregation was significantly increased on diet P.

CONCLUSIONS

Results from this study indicate that stearic acid (19g/day) in the diet has beneficial effects on thrombogenic and atherogenic risk factors in males. The food industry might wish to consider the enrichment of foods with stearic acid in place of palmitic acid and trans fatty acids.

FSAP is a plasma serine protease for which a potential role in the regulation of coagulation and fibrinolysis is postulated, based on its property to activate factor VII (FVII) as well as pro-urokinase (uPA). In clinical studies, the G534E single nucleotide polymorphism (Marburg I) of FSAP has been linked to late complications of atherothrombosis and is associated with a low proteolytic activity, particularly, towards pro-uPA. This has stimulated much interest in a search for additional functions of FSAP in the cardiovascular system. FSAP is a potent inhibitor of vascular smooth muscle cell proliferation and migration in vitro and local application of FSAP (but not Marburg I variant) in animal models reduces neointima formation. This is due to a reduced proteolytic activity of the variant isoform towards platelet derived growth factor-BB, a key mediator of neointima development. Moreover, appreciable quantities of FSAP are localized to unstable atherosclerotic plaques and may contribute to plaque instability. These data indicate that the cellular regulatory effects of FSAP may be more important than its influence on haemostasis. In this review the contribution of FSAP to vascular fibroproliferative inflammatory diseases in the context of pericellular proteolysis of the extracellular matrix, growth factor activity and haemostasis will be highlighted.

Tissue factor (TF) is a transmembrane glycoprotein that localizes the coagulation serine protease factor VII/VIIa (FVII/VIIa) to the cell surface. The primary function of TF is to activate the clotting cascade. The TF:FVIIa complex also activates cells by cleavage of a G-protein coupled receptor called protease-activated receptor 2 (PAR2). TF is expressed by tumor cells and contributes to a variety of pathologic processes, such as thrombosis, metastasis, tumor growth, and tumor angiogenesis. For instance, tumor cells release TF-positive procoagulant microparticles into the circulation and these may trigger venous thromboembolism in patients with cancer. TF on circulating tumor cells also leads to the coating of the cells with fibrin that traps them within the microvasculature and facilitates hematogenous metastasis. In addition, TF:FVIIa-dependent activation of PAR2 on tumor cells increases tumor growth via an undefined mechanism. One possibility is that PAR2-dependent signaling increases the expression of proangiogenic proteins. Other studies have reported that endothelial cells in the tumor vasculature express TF and this may enhance angiogenesis. These results suggest that inhibition of TF should reduce several pathologic pathways that increase tumor growth and metastasis. This would represent a novel approach to anticancer therapy. Initial studies using inhibitors of the TF:FVIIa complex in mouse tumor models have produced encouraging results. Nevertheless, additional studies are needed to determine if this strategy can be successfully translated to the treatment of cancer patients.

Tissue factor (TF), the cellular receptor and cofactor for clotting factor VII/VIIa (FVII/VIIa), is known mainly as the initiator of the coagulation protease cascade. Recently, it was shown that inactivation of the murine TF gene (TF-/-) results in embryonic lethality which is most likely due to some failure of vascular integrity. On the other hand, gene disruption in mice of coagulation proteins like FVII, prothrombin, and fibrinogen results in phenotypes of embryonic development that contrast with that of TF-/-, suggesting a role for TF beyond fibrin formation in embryogenesis. In addition, there is a growing body of evidence that cellular TF may be involved in nonhemostatic functions. To determine the microtopography of membrane TF with regard to the cytoskeleton organization, we examined the expression patterns of TF and cytoskeletal proteins in various cell lines by means of double immunofluorescence and electron microscopy (EM). In spreading cells, a granular membrane TF expression of the cell cortex and a pronounced granular TF staining of microspikes, lamellipodes, and ruffled membrane areas were observed. Especially, actin and alpha-actinin were in close proximity to TF in these regions. Colocalization of TF and nonmuscle filamin (ABP-280) at the leading edge of spreading cells indicated an association of TF with the actin filament system, too. Using scanning EM we found gold-labeled TF at long processes and actin-filament-containing microspikes of neighboring cells in both branching and contact sites. By the means of immunogold EM we observed that TF is localized at the cell surface in a spotty pattern, at the base and at the top of budding processes. The observed staining pattern points to a connection of TF with elements of the cytoskeleton in these highly dynamic membrane regions, a fact which is underlined by the recently described molecular interaction of TF's cytoplasmic domain with ABP-280. In cells undergoing cytokinesis, we detected also strong TF expression in dynamic membrane areas and protrusions of the midbodies, indicating an accumulation of TF in actin-rich membrane areas with high contractile activity. In addition, we were able to demonstrate that immobilized ligands for TF, both catalytically active and inactive FVIIa or anti-TF mAbs, accelerated adhesion and spreading of TF-expressing cancer cells. Thus, our findings support the contention that ligation of cellular TF may be involved in morphogenic processes such as adhesion and spreading by an association to cytoskeletal structures. On the other hand, incubation of these cells with proteolytically active FVIIa but not with covalently inactivated FVIIa (DEGR-FVIIa) or anti-TF mAbs in solution resulted in increased motility of these cells, indicating that not only ligation of TF but also the proteolytic activity of TF-FVIIa complex is involved in cell migration.

Initiation of haemostasis involves the formation of a complex between tissue factor (TF) and activated factor VII (FVIIa) following injury. TF is found in the deeper layers of the vessel wall, in atherosclerotic plaques and in some types of tumour cell and is only exposed to circulating blood after tissue damage. Likewise, FVII is only enzymatically active when complexed with TF (TF/FVIIa). It has recently been shown that the administration of recombinant activated FVII (rFVIIa) in high doses (approximately 100 microg/kg) can induce haemostasis in the absence of FVIII and FIX. In addition, from in-vitro studies it appears that rFVIIa can bind with low affinity to the activated platelet surface and, independently of TF, induce the thrombin burst needed for haemostasis. The ability of rFVIIa to compensate for FVIII/FIX deficiency has been proven clinically in haemophilia patients with life- and limb-threatening bleeds. In addition, patients with congenital FVII deficiency have been successfully treated for bleeds with rFVIIa. Recombinant FVIIa has been used in patients with platelet disorders; five patients with Glanzmann's thrombasthenia and one with Bernard-Soulier's thrombasthenia have had bleeding episodes managed effectively. Recombinant FVIIa has also been shown to normalize prothrombin time in patients with liver disease and in warfarin-treated individuals.

Fibrinogen, coagulation factor VII (FVII), and factor VIII (FVIII) and its carrier von Willebrand factor (vWF) play key roles in hemostasis. Previously identified common variants explain only a small fraction of the trait heritabilities, and additional variations may be explained by associations with rarer variants with larger effects. The aim of this study was to identify low-frequency (minor allele frequency [MAF] ≥0.01 and <0.05) and rare (MAF <0.01) variants that influence plasma concentrations of these 4 hemostatic factors by meta-analyzing exome chip data from up to 76,000 participants of 4 ancestries. We identified 12 novel associations of low-frequency (n = 2) and rare (n = 10) variants across the fibrinogen, FVII, FVIII, and vWF traits that were independent of previously identified associations. Novel loci were found within previously reported genes and had effect sizes much larger than and independent of previously identified common variants. In addition, associations at KCNT1, HID1, and KATNB1 identified new candidate genes related to hemostasis for follow-up replication and functional genomic analysis. Newly identified low-frequency and rare-variant associations accounted for modest amounts of trait variance and therefore are unlikely to increase predicted trait heritability but provide new information for understanding individual variation in hemostasis pathways.

Inherited deficiencies of plasma proteins involved in blood coagulation generally lead to lifelong bleeding disorders, whose severity is inversely proportional to the degree of factor deficiency. Haemophilia A and B, inherited as X-linked recessive traits, are the most common hereditary hemorrhagic disorders caused by a deficiency or dysfunction of blood coagulation factor VIII (FVIII) and factor IX (FIX). Together with von Willebrand's disease, a defect of primary haemostasis, these X-linked disorders include 95% to 97% of all the inherited deficiencies of coagulation factors. The remaining defects, generally transmitted as autosomal recessive traits, are rare with prevalence of the presumably homozygous forms in the general population of 1:500,000 for FVII deficiency and 1 in 2 million for prothrombin (FII) and factor XIII (FXIII) deficiency. Molecular characterization, carrier detection and prenatal diagnosis remain the key steps for the prevention of the birth of children affected by coagulation disorders in developing countries, where patients with these deficiencies rarely live beyond childhood and where management is still largely inadequate. These characterizations are possible by direct or indirect genetic analysis of genes involved in these diseases, and the choice of the strategy depends on the effective available budget and facilities to achieve a large benefit. In countries with more advanced molecular facilities and higher budget resources, the most appropriate choice in general is a direct strategy for mutation detection. However, in countries with limited facilities and low budget resources, carrier detection and prenatal diagnosis are usually performed by linkage analysis with genetic markers. This article reviews the genetic diagnosis of haemophilia, genetics and inhibitor development, genetics of von Willebrand's disease and of rare bleeding disorders.

The congenital FVII deficiency (FVIID) is a rare haemorrhagic disorder with an autosomal recessive pattern of inheritance. Data on phenotype and the genotype from 717 subjects in Central Europe (six countries), Latin America (Costa Rica, Venezuela) and United States, enrolled in the Greifswald Registry of FVII Deficiency were analysed. We detected 131 different mutations in 73 homozygous, 145 compound heterozygous and 499 heterozygous subjects. Regional differences were observed in the mutation pattern and the clinical profile of the evaluated patients. Seventy-one per cent of homozygous and 50% of compound heterozygous subjects were symptomatic. The clinical manifestations of the homozygous subjects were characterized by intracranial haemorrhage (2%), gastrointestinal bleeding (17%), haemarthrosis (13%), epistaxis (58%), gum bleeding (38%), easy bruising (37%), haematoma (15%), haematuria (10%) and menorrhagia (19 of 26 females, 73%). The clinical variability and genotype-phenotype correlation was evaluated in the homozygous subjects. The pattern of bleeding symptoms among compound heterozygous patients was severe and similar to that of the homozygous patients. The large-scale analysis of 499 heterozygous subjects shows that 93 (19%) presented with spontaneous bleeding symptoms such as haemarthrosis (4%), epistaxis (54%), gum bleeding (14%), easy bruising (38%), haematoma (23%), haematuria (5%) and menorrhagia (19 of 45 females; 42%). The severe haemorrhages - intracranial and gastrointestinal - were not reported in heterozygous subjects. The clinical variability and the regional differences in the mutation pattern are discussed regarding care and treatment.

Protein glycosylation is post-translational modification (PTM) which is important for pharmacokinetics and immunogenicity of recombinant glycoprotein therapeutics. As a result of variations in monosaccharide composition, glycosidic linkages and glycan branching, glycosylation introduces considerable complexity and heterogeneity to therapeutics. The host cell line used to produce the glycoprotein has a strong influence on the glycosylation because different host systems may express varying repertoire of glycosylation enzymes and transporters that contributes to specificity and heterogeneity in glycosylation profiles. In this review, we discuss the types of host cell lines currently used for recombinant therapeutic production, their glycosylation potential and the resultant impact on glycoprotein properties. In addition, we compare the reported glycosylation profiles of four recombinant glycoproteins: immunoglobulin G (IgG), coagulation factor VII (FVII), erythropoietin (EPO) and alpha-1 antitrypsin (A1AT) produced in different mammalian cells to establish the influence of mammalian host cell lines on glycosylation.

Tissue factor/coagulation factor VII (fVII) complex formation on the surface of cancer cells plays important roles in cancer biology, such as cell migration and invasion, angiogenesis, and antiapoptotic effects. We recently found that various cancer cells ectopically synthesize fVII, resulting in activation of cell motility and invasion. Here, we characterized mechanisms of hepatic and ectopic fVII (FVII) gene expression to identify molecular targets enabling selective inhibition of the ectopic expression. Unlike hepatic expression, hepatocyte nuclear factor-4 binding to the promoter is not required for ectopic FVII expression, although Sp1 binding is essential. Furthermore, we found novel nuclear targets of basal hepatocytic and ectopic FVII expression. Notably, histone acetyltransferases p300 and cyclic AMP-responsive element binding protein-binding protein (CBP) are exclusively recruited to the promoter region of the FVII gene specifically in breast cancer cells. We further show that curcumin, a dietary compound, can selectively inhibit ectopic fVII expression by targeting p300/CBP activity. These results suggest a strategy to inhibit ectopic fVII-induced tumor progression without impairment of the physiologic hemostatic process.

Small nuclear U1-RNAs (snRNAs), the spliceosome components selectively recognizing donor splice sites (5'ss), were engineered to restore correct mRNA processing in a cellular model of severe coagulation factor VII (FVII) deficiency, caused by the IVS7 9726 + 5g/a change. Three U1-snRNAs, complementary to the mutated 5'ss (U1 + 5a) or to neighboring sequences were expressed with FVII minigenes in a hepatoma cell line. The U1-snRNAs reduced from 80% to 40% the exon 7 skipping, thus increasing exon definition. The U1 + 5a construct also dramatically increased recognition of the correct 5'ss over the 37-bp downstream cryptic site preferentially activated by the mutation, thus inducing appreciable synthesis of normal transcripts (from barely detectable to 50%). This effect, which was dose-dependent, clearly demonstrated that impaired recognition by the U1-snRNA was the mechanism responsible for FVII deficiency. These findings suggest compensatory U1-snRNAs as therapeutic tools in coagulation factor deficiencies caused by mutations at 5'ss, a frequent cause of severe defects.

Combined oral contraceptives (OC) are known to increase the risk of venous thromboembolism. The aim of this randomized, cycle-controlled, cross-over study in 28 healthy volunteers was to assess potential differences between the effects of an OC containing 150 microg levonorgestrel (as representative of the so-called second generation OC) and an OC containing 150 microg desogestrel (as representative of the third generation OC) in combination with 30 microg ethinylestradiol on several coagulation factors and markers of thrombin formation. All participants used each OC for two cycles, and were switched to the other OC after a washout period of two menstrual cycles. The plasma concentrations of factors II, VII, X, and fibrinogen significantly increased during use of both the levonorgestrel- and desogestrel-containing OC's. The plasma concentrations of factor VIII increased, and of factor V decreased, changes which only reached statistical significance during the use of the desogestrel-containing OC. During exposure to the desogestrel-containing OC, as compared with the levonorgestrel-containing OC, both factor VII and factor II showed a greater increase (FVII: 32% and 12% respectively; p <0.0001; FII: 16% and 12% respectively; p = 0.048), whereas factor V showed a greater decrease (-11% and -3% respectively; p = 0.010). Only one of the markers for ongoing coagulation (prothrombin fragment 1+2) showed a significant increase during OC use, whereas concentrations of thrombin-antithrombin complexes and soluble fibrin remained unchanged. For these markers, there was no difference between the tested OC's. We conclude that there are differences between the effects of levonorgestrel and desogestrel-containing OC's on some coagulation factors. Whether these changes provide a biological explanation for the reported differences in venous thromboembolic risk is as yet unclear. The real challenge now becomes to define a pattern of changes in the various systems which, if affected simultaneously, may tip the hemostatic balance towards a prethrombotic state and may lead to overt clinical venous thromboembolism.

OBJECTIVE

To determine the influence of physicochemical properties of lipid nanoparticles (LNPs) carrying siRNA on their gene silencing in vivo. Mechanistic understanding of how the architecture of the nanoparticle can alter gene expression has also been studied.

METHODS

The effect of 3-N-[(ω-methoxypoly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxy-propylamine (PEG-C-DMA) on hepatic distribution and FVII gene silencing was determined. FVII mRNA in hepatocytes and liver tissues was determined by Q-PCR. Hepatic distribution was quantified by FACS analysis using Cy5 labeled siRNA.

RESULTS

Gene silencing was highly dependent on the amount of PEG-C-DMA present. FVII gene silencing inversely correlated to the amount of PEG-C-DMA in LNPs. High FVII gene silencing was obtained in vitro and in vivo when the molar ratio of PEG-C-DMA to lipid was 0.5 mol%. Surprisingly, PEGylation didn't alter the hepatic distribution of the LNPs at 5 h post administration. Instead the amount of PEG present in the LNPs has an effect on red blood cell disruption at low pH.

CONCLUSIONS

Low but sufficient PEG-C-DMA amount in LNPs plays an important role for efficient FVII gene silencing in vivo. PEGylation did not alter the hepatic distribution of LNPs, but altered gene silencing efficacy by potentially reducing endosomal disruption.

All-trans-retinoic acid (ATRA) downregulates the expression of two cellular procoagulants, tissue factor (TF) and cancer procoagulant (CP), in human promyelocytic leukemia cells. To evaluate whether or not changes of the procoagulant activities (PCAs) may share mechanisms with the ATRA-induced cyto-differentiation process, we have characterized the effect of ATRA on the TF and CP expression by NB4 cells, an ATRA maturation-inducible cell line, and two NB4-derived cell lines resistant to ATRA-induced maturation, the NB4. 306 and NB4.007/6 cells. Next, we evaluated the effect on the PCAs of the NB4 parental cells of three synthetic retinoid analogues, ie: AM580 (selective for the retinoic acid receptor [RAR] alpha), capable to induce the granulocytic differentiation of NB4 cells; and CD2019 (selective for RARbeta) and CD437 (selective for RARgamma), both lacking this capability. Cells were treated with either ATRA or the analogues (10(-6) to 10(-8) mol/L) for 96 hours. The effect on cell differentiation was evaluated by morphologic changes, cell proliferation, nitro blue tetrazolium reduction assay, and flow cytometry analysis of the CD33 and CD11b surface-antigen expression. PCA was first measured in 20 mmol/L Veronal Buffer cell extracts by the one-stage clotting assay of normal and FVII-deficient plasmas. Further TF and CP have been characterized and quantified in cell-sample preparations by chromogenic and immunological assays. In the first series of experiments, ATRA downregulates both TF and CP in NB4 parental cells, as expected. However, in the differentiation-resistant cell lines, it induced a significant loss of TF but had little or no effect on CP. In a second series of experiments, in the NB4 parental cells, the RARalpha agonist (AM580) induced cell maturation and reduced 91% CP expression, whereas CD437 and CD2019 had no cyto-differentiating effects and did not affect CP levels. On the other hand, in the same cells the TF expression was reduced by ATRA and AM580, but also by the RARbeta agonist CD2019, which did not induce cell maturation. These data indicate that in NB4 cells, ATRA modulation of CP occurs in parallel with signs of cell differentiation, while the regulation of TF appears to be at least in part independent from these processes, and involves both alpha and beta nuclear retinoid receptors.

OBJECTIVE

Obesity and the metabolic syndrome are established risk factors of venous thromboembolism. As most coagulation factors are produced exclusively by the liver and non-alcoholic fatty liver disease (NAFLD) is tightly related to metabolic disorders, we aimed at studying the association of liver fat with various coagulation factor activities.

METHODS

Plasma prothrombin (PT) and activated partial thromboplastin time, activities of vWF:RCo, FVII, FVIII, FIX, FXI, FXII, FXIII, fibrinogen and D-dimer concentrations were measured in 54 subjects with and 44 without NAFLD diagnosed by proton magnetic resonance spectroscopy. Subjects were recruited retrospectively for metabolic studies in our laboratory. The body composition and features of insulin resistance were measured in all subjects.

RESULTS

FVIII (107±30 vs. 84±22%, P<0.001), FIX (110±14 vs. 94±16%, P<0.001), FXI (109±16 vs. 96±19%, P=0.001) and FXII (113±21 vs. 99±32%, P=0.002) activities were consistently elevated in subjects with as compared with those without NAFLD. Liver fat percentage was positively related to FVIII (r=0.28, P=0.005), FIX (r=0.36, P=0.0003), FXI (r=0.29, P=0.004) and FXII (r=0.30, P=0.003) activities, again independent of age, gender and body mass index (BMI). PT%, vWF:RCo activity and fibrinogen were higher in subjects with as compared with those without NAFLD, but this difference disappeared after adjusting for age, gender and BMI.

CONCLUSIONS

FVIII, FIX, FXI and FXII activities are increased in human NAFLD and correlate with the features of insulin resistance. The relationships between NAFLD and these coagulation factors are independent of age, gender and BMI, suggesting that the fatty liver can contribute to the risk of thrombosis.

BACKGROUND

The INTERCEPT Blood System, a photochemical treatment (PCT) process, has been developed to inactivate pathogens in platelet concentrates. These studies evaluated the efficacy of PCT to inactivate pathogens in plasma and the effect of PCT on plasma function.

METHODS

Jumbo (600 mL) plasma units were inoculated with high titers of test pathogens and treated with 150 micromol per L amotosalen and 3 J per cm(2) long-wavelength ultraviolet light. The viability of each pathogen before and after treatment was measured with biological assays. Plasma function was evaluated through measurement of coagulation factors and antithrombotic protein activities.

RESULTS

The levels of inactivation expressed as log-reduction were as follows: cell-free human immunodeficiency virus-1 (HIV-1), greater than 6.8; cell-associated HIV-1, greater than 6.4; human T-lymphotropic virus-I (HTLV-I), 4.5; HTLV-II, greater than 5.7; hepatitis B virus (HBV) and hepatitis C virus, greater than 4.5; duck HBV, 4.4 to 4.5; bovine viral diarrhea virus, 6.0; severe acute respiratory syndrome coronavirus, 5.5; West Nile virus, 6.8; bluetongue virus, 5.1; human adenovirus 5, 6.8; Klebsiella pneumoniae, greater than 7.4; Staphylococcus epidermidis and Yersinia enterocolitica, greater than 7.3; Treponema pallidum, greater than 5.9; Borrelia burgdorferi, greater than 10.6; Plasmodium falciparum, 6.9; Trypanosoma cruzi, greater than 5.0; and Babesia microti, greater than 5.3. Retention of coagulation factor activity after PCT was expressed as the proportion of pretreatment (baseline) activity. Retention was 72 to 73 percent of baseline fibrinogen and Factor (F)VIII activity and 78 to 98 percent for FII, FV, FVII, F IX, FX, FXI, FXIII, protein C, protein S, antithrombin, and alpha2-antiplasmin.

CONCLUSIONS

PCT of plasma inactivated high levels of a wide range of pathogens while maintaining adequate coagulation function. PCT has the potential to reduce the risk of transfusion-transmitted diseases in patients requiring plasma transfusion support.

BACKGROUND

The purpose of this study was to test a novel, dual tumour vascular endothelial cell (VEC)- and tumour cell-targeting factor VII-targeted Sn(IV) chlorin e6 photodynamic therapy (fVII-tPDT) by targeting a receptor tissue factor (TF) as an alternative treatment for chemoresistant breast cancer using a multidrug resistant (MDR) breast cancer line MCF-7/MDR.

METHODS

The TF expression by the MCF-7/MDR breast cancer cells and tumour VECs in MCF-7/MDR tumours from mice was determined separately by flow cytometry and immunohistochemistry using anti-human or anti-murine TF antibodies. The efficacy of fVII-tPDT was tested in vitro and in vivo and was compared with non-targeted PDT for treatment of chemoresistant breast cancer. The in vitro efficacy was determined by a non-clonogenic assay using crystal violet staining for monolayers, and apoptosis and necrosis were assayed to elucidate the underlying mechanisms. The in vivo efficacy of fVII-tPDT was determined in a nude mouse model of subcutaneous MCF-7/MDR tumour xenograft by measuring tumour volume.

RESULTS

To our knowledge, this is the first presentation showing that TF was expressed on tumour VECs in chemoresistant breast tumours from mice. The in vitro efficacy of fVII-tPDT was 12-fold stronger than that of ntPDT for MCF-7/MDR cancer cells, and the mechanism of action involved induction of apoptosis and necrosis. Moreover, fVII-tPDT was effective and safe for the treatment of chemoresistant breast tumours in the nude mouse model.

CONCLUSIONS

We conclude that fVII-tPDT is effective and safe for the treatment of chemoresistant breast cancer, presumably by simultaneously targeting both the tumour neovasculature and chemoresistant cancer cells. Thus, this dual-targeting fVII-tPDT could also have therapeutic potential for the treatment of other chemoresistant cancers.

BACKGROUND

Chronic obstructive pulmonary disease (COPD) patients have increased risk for cardiovascular mortality and venous thromboembolism. Tissue factor (TF) is the physiological initiating mechanism for blood coagulation and is pro-inflammatory.

METHODS

We have studied circulating blood-borne TF-procoagulant activity (TF-PCA), plasma coagulation factors (F) VIIa and FVIII, and thrombin-antithrombin (TAT) complexes in 11 stable, moderate-severe COPD patients, 10 free of exacerbation for >3 weeks.

RESULTS

TF-PCA was increased in COPD patients (52.3+/-5.6 U/ml, (SE)) compared to control subjects (20.7+/-1.5, n=45, p<0.0001). TAT levels were increased (COPD patients: 2.99+/-0.65 ug/l; control subjects: 1.31+/-0.13, n=53, p<0.0001), indicating enhanced thrombin generation. Plasma FVIIa (the activated form of FVII) was higher in COPD (83+/-11 mU/ml; controls, 64+/-5 mU/ml, n=20) but did not reach statistical significance. Plasma FVIIc and FVIII were not increased. TF-PCA levels were inversely related to plasma FVIIa (r=-0.80, p=0.003) and FVIIc (r=-0.76, p=0.007).

CONCLUSIONS

Blood-borne TF-PCA is elevated and constitutes a prothrombotic and proinflammatory state in stable but moderate-severe COPD, and may contribute to the increased risk for vascular events.

Increasing evidence links blood coagulation proteins with the regulation of acute and chronic inflammatory disease. Of particular interest are vitamin K-dependent proteases, which are generated as a hemostatic response to vascular injury, but can also initiate signal transduction via interactions with vascular receptors. The endothelial cell protein C receptor (EPCR) is a multi-ligand vitamin K-dependent protein receptor for zymogen and activated forms of plasma protein C and factor VII. Although the physiological role of the EPCR-FVII(a) interaction is not well-understood, protein C binding to EPCR facilitates rapid generation of APC in response to excessive thrombin generation, and is a central requirement for the multiple signal-transduction cascades initiated by APC on both vascular endothelial and innate immune cells. Exciting recent studies have highlighted the emerging role of EPCR in modulating the cytoprotective properties of APC in a number of diverse inflammatory disorders. In this review, we describe the structure-function relationships, signal transduction pathways, and cellular interactions that enable EPCR to modulate the anticoagulant and anti-inflammatory properties of its vitamin K-dependent protein ligands, and examine the relevance of EPCR to both thrombotic and inflammation-associated disease.

Mild hemophilia A (HA), defined by clinical features and factor VIII coagulant activity (FVIII:C) between 0.05 and 0.40 IU mL(-1), is characteristically distinct from severe HA. Indeed, although the molecular characterization of mild HA has permitted the identification of specific underlying mutations, its clinical phenotype is strikingly different from that of patients with a severe FVIII defect, where spontaneous hemorrhages or recurrent joint bleeding are usual manifestations. With aging, mild HA patients may develop complications (i.e. cancers and cardiovascular disorders), the management of which may prove challenging due to the concomitant bleeding tendency. Furthermore, the development of inhibitors provides an additional major complication in these patients, because it increases the severity of the bleeding phenotype and complicates their management. Standard management of mild HA includes the use of desmopressin and antifibrinolytic agents for minor bleeding episodes or surgical procedures, whilst major bleeding or surgery requires replacement therapy with FVIII concentrates. As regards treatment of patients with inhibitors, bypassing agents (i.e. activated prothrombin complex concentrates and recombinant activated FVII) have proven effective in the treatment of bleeding episodes, but as there are insufficient data to determine the optimal approach to immune tolerance induction in this group of patients, their optimal management remains controversial. Rituximab is a newer, promising therapeutic option for inhibitor eradication in such patients. Many aspects concerning mild HA remain to be clarified, including the molecular basis, the natural history and the optimal diagnostic and therapeutic strategies. Only large prospective studies will shed light on this condition.

Thrombosis in congenital factor (F) VII deficiency was investigated through extensive phenotypic and molecular-genetic studies. Patients with a history of thrombosis among 514 entries in the FVII Deficiency Study Group database were evaluated. Thrombotic events were arterial in one case, disseminated intravascular coagulation in another and venous in seven. Gene mutations were characterized in eight patients: three were homozygous, three compound heterozygous and two heterozygous. FXa and IIa generation assays were consistent with the genetic lesions. One patient was heterozygous for the FV Leiden and one for the FIIG20210A mutation. In seven patients, surgical interventions and/or replacement therapies had a close temporal relationship with thrombosis, while in the remaining, events were apparently spontaneous. Thromboses were not associated with any specific age, phenotype, mutation zygosity or thrombophilic abnormalities. In particular, severe FVII deficiency did not seem to offer protection from strong thrombosis risk factors such as surgery and replacement therapy.

It has been shown earlier that mice with a total targeted deletion of the factorVII gene (FVII(-/-)) die perinatally, thereby precluding study of adult animals with this total deficiency. Consequently, mice producing very low levels of FVII were developed by targeted replacement of the wild-type (WT) murine FYII gene with its corresponding cDNA, under control of the tetracycline transactivator (tTA) promoter. When backcrossed into the C57BI/6 strain, unchallenged mice containing two replaced FVII(tTA) alleles (FVII(tTA/tTA) produce approximately 0.7% of WT FVII levels, but yet live to adulthood despite displaying severely downregulated overall thrombin production and spontaneously developing cardiac fibrosis at a young adult age. This genetically-altered mouse line provides an excellent animal model to study consequences of a severe FVII deficiency in unchallenged mice and in mice subjected to a variety of experimental challenges.

Inhibitors to factor VIII (FVIII) or IX (FIX) in patients with haemophilia A or B create a challenging problem for the treatment of these patients. Recombinant FVIIa (rFVIIa; NovoSeven, Novo Nordisk A/S, Bagsvaerd, Denmark) is a realistic treatment option, owing to its specific mode of action and lack of immunogenicity. This was a multicentre, open-label, compassionate-use trial in patients with severe haemophilia A (FVIII:C < 1%) or B (FIX:C < 1%) with inhibitors, acquired antibodies to FVIII or FIX, or FVII deficiency (FVII:C < 5%), for whom alternative therapies had failed or were contraindicated. Patients received rFVIIa treatment for life- or limb-threatening bleeding episodes or for coverage during essential surgery. The mean rFVIIa dose was approximately 90 microg kg-1 for haemophilia A/B and acquired inhibitor patients, and 25 microg kg-1 for FVII-deficient patients. Efficacy data for 67 treatment episodes (45 bleeding episodes, 22 surgical procedures) are presented; seven patients were treated for a concurrent serious bleeding episode and surgical procedure. At the end of treatment, rFVIIa was effective or partially effective in 85% of serious bleeding episodes. During surgery, bleeding was assessed as none or less than or equivalent to normal in 91% of surgical procedures; postoperatively, 91% of procedures were associated with no or minimal oozing. During 60 separate treatment episodes, 26 adverse events (22 nonserious, four serious) were reported in 15 patients, during 17 bleeding episodes or surgical procedures. Only 10 were considered as having a possible, probable, or unknown relationship with rFVIIa; of these, fever (n=2) and thrombophlebitis (n=3) were the most common. There was no evidence of disseminated intravascular coagulation. In conclusion, rFVIIa is an effective, well-tolerated treatment for serious bleeding episodes and bleeding associated with surgical procedures in patients with severe haemophilia A/B with inhibitors, acquired inhibitors, or FVII deficiency.

Patients with diabetes have an increased prevalence of premature atherosclerotic vascular disease, and alterations in plasma coagulation proteins have been incriminated as a possible cause. The roles of hyperglycemia and hyperinsulinemia in the pathogenesis of these changes are unknown. To examine the effects of prolonged hyperglycemia and of selective hyperinsulinemia on the tissue factor pathway of blood coagulation, nine healthy young men were infused with glucose to maintain levels at 11.1 mmol/l (approximately 200 mg/dl) for 18-72 h (hyperglycemia-hyperinsulinemia group). Five normal men were infused with regular insulin to maintain levels comparable to that in the previous group (900 pmol/l, approximately 150 microU/ml) and with glucose to maintain levels at 5.6 mmol/l (approximately 100 mg/dl) (euglycemia-hyperinsulinemia group). Measured were plasma activated factor VII activity (FVIIa), FVII coagulant (FVIIC) activity, FVIII coagulant (FVIIIC) activity, tissue factor pathway inhibitor (TFPI) antigen, and thrombin markers; and serum glucose, insulin, and electrolytes. Plasma FVIIa, FVIIC, FVIIIC, and TFPI rose during hyperglycemic-hyperinsulinemia but not during euglycemic-hyperinsulinemia. Markers of thrombin generation rose transiently and inconsistently during hyperglycemia-hyperinsulinemia. We concluded that in normal subjects, hyperglycemia-hyperinsulinemia induced activation of the tissue factor pathway, reflected by increases in plasma FVIIa, FVIIC, and TFPI. This activation was independent of hyperinsulinemia, hypertriglyceridemia, and hyperosmolality. The elevations in plasma coagulation factors during hyperglycemia-hyperinsulinemia, characteristic of type 2 diabetes, may constitute a potential for enhanced thrombin generation and thrombosis when triggered by exposure of tissue factor, such as during arterial plaque rupture.