Tissue factor (TF) is believed to play an important role in coagulation, inflammation, angiogenesis and wound healing as well as in tumor growth and metastasis. To facilitate in vivo studies in experimental murine models, we have produced recombinant murine factor VII (FVII) and the ectodomain of murine TF, TF(1-223). Murine FVII was activated to FVIIa with human factor Xa and upon reaction with FFR-chloromethyl ketone converted into an active site-blocked TF antagonist, FFR-FVIIa. The activity of murine FVIIa was characterized in factor X activation assays as well as in clot assays with murine and human thromboplastin in murine and human plasma. In these assays murine FVIIa exhibited a specific activity equivalent to or higher than human FVIIa. Further analysis showed that murine FVIIa binds with high affinity to both murine and human TF, whereas the association of human FVIIa to murine TF is about three orders of magnitude weaker than the association to human TF. This difference was further emphasized by the effect of murine-and human FFR-FVIIa on bleeding in an in vivo mouse model. Intra-peritoneal administration of 1 mg/kg murine FFR-FVIIa significantly prolonged the tail-bleeding time, whereas no effect on bleeding was observed with a 25-times higher dose of the human FFR-FVIIa. Together, these data confirms the notion of poor species compatibility between human FVII and murine TF and emphasizes the requirement for autologous FVIIa in studies on the role of the TF in experimental in vivo pharmacology.

We studied the relationships between albuminuria, tissue factor-induced coagulation, and endothelial cell dysfunction in 67 patients with non-insulin-dependent diabetes mellitus (NIDDM) who were divided into three groups on the basis of their urinary albumin excretion rate (AER). To assess the early phase of tissue factor-induced coagulation, activated factor VII (FVIIa) levels in plasma were measured by a direct fluorogenic assay. As markers of endothelial cell dysfunction, levels of von Willebrand factor (vWF), tissue-type plasminogen activator-plasminogen activator inhibitor-1 (TPA-PAI-1) complex, PAI-1, and tissue factor pathway inhibitor (TFPI) were measured. FVIIa levels were increased in normoalbuminuric NIDDM patients (AER < 15 micrograms/min) when compared with normal control subjects. This FVIIa increase was accompanied by an increase in thrombin-antithrombin III complex (TAT) levels, indicating increased activation of coagulation even in normoalbuminuric patients. In NIDDM patients with microalbuminuria (AER = 15-200 micrograms/min), the FVIIa level, the FVIIa-FVII antigen (Ag) ratio (an indicator of activation of FVII zymogen to FVIIa), and the TAT level were further increased. This group also had higher levels of endothelial cell-derived factors (vWF, TPA-PAI-1 complex, and PAI-1) than the control group. The levels of endothelial cell-derived factors (including TFPI) were highest in the NIDDM patients with overt albuminuria (AER>> 200 micrograms/min). In all 67 diabetic patients, AER showed a strong positive correlation with FVIIa (r = .574, P < .0001) and a weakly but still significant correlation with FVIIa-FVII:Ag (r = .365, P = .01), vWF (r = .315, P < .01), and TAT (r = .323, P < .01).(ABSTRACT TRUNCATED AT 250 WORDS)

BACKGROUND

Obstructive sleep apnoea (OSA) is associated with increased cardiovascular risk, however the mechanisms are not well established.

OBJECTIVE

This study aimed to determine whether treatment of OSA with nasal continuous positive airway pressure (CPAP) would favourably alter coagulability across the sleep-wake cycle.

METHODS

In a randomised crossover trial, 28 patients received therapeutic or placebo CPAP, each for 2 months with a 1 month washout between treatments. After each treatment period, a 24 h coagulation study was conducted in the laboratory. Plasminogen activator inhibitor-1 (PAI-1), D-dimer, fibrinogen, von Willebrand Factor (vWF), factor VIII (FVIII), factor VII (FVII) and factor V (FV) were determined at seven time points over the day and night.

RESULTS

At baseline, patients had severe OSA (Apnoea Hypopnoea Index 37.9 ± 23.9 events/h). Treatment of OSA with CPAP compared with placebo resulted in lower 24 h levels of vWF (-3.9%, p=0.013), FVIII (-6.2%, p=0.007) and FV (-4.2%, p<0.001). The greatest difference occurred during the nocturnal and early morning periods. In contrast, fibrinogen, D-dimer, FVII and PAI-1 did not differ between treatments, however all markers displayed diurnal variability independent of treatment.

CONCLUSIONS

In this randomised, placebo-controlled crossover trial, treatment of OSA with CPAP reduced the early morning level of vWF, and nocturnal levels of FVIII and FV. These findings suggest that CPAP may reduce cardiovascular risk in OSA, in part through reducing risk of thrombosis.

BACKGROUND

Activation of the coagulation cascade has been demonstrated in pulmonary fibrosis. In addition to its procoagulant function, various coagulation proteases exhibit cellular effects that may also contribute to fibrotic processes in the lung.

OBJECTIVE

To investigate the importance of protease-activated receptor (PAR)-2 and its activators, coagulation factor VIIa (FVIIa)/tissue factor (TF), in the development of idiopathic pulmonary fibrosis (IPF).

METHODS

Expression and localization of PAR-2 and its activators were examined in IPF lung tissue. The ability of PAR-2 to mediate various cellular processes was studied in vitro.

RESULTS

Expression of PAR-2 was strongly elevated in IPF lungs and was attributable to alveolar type II cells and fibroblasts/myofibroblasts. Transforming growth factor-β(1), a key profibrotic cytokine, considerably enhanced PAR-2 expression in human lung fibroblasts. FVIIa stimulated proliferation of human lung fibroblasts and extracellular matrix production in a PAR-2-dependent manner, but did not initiate differentiation of fibroblasts into myofibroblasts. PAR-2/FVIIa-driven mitogenic activities were mediated via the p44/42 mitogen-activated protein kinase pathway and were independent of factor Xa and thrombin production. Proproliferative properties of FVIIa were markedly potentiated in the presence of TF and abrogated by TF antisense oligonucleotides. Hyperplastic alveolar type II cells overlying fibroblastic foci were found to be the source of FVII in IPF lungs. Moreover, TF colocalized with PAR-2 on fibroblasts/myofibroblasts in IPF lungs.

CONCLUSIONS

The PAR-2/TF/FVIIa axis may contribute to the development of pulmonary fibrosis; thus, interference with this pathway confers novel therapeutic potential for the treatment of IPF.

A new inhibitory peptide binding exosite on the protease domain of coagulation Factor VIIa (FVIIa) has been identified. A novel series of peptide inhibitors of FVIIa, termed the "A-series" peptides, identified from peptide phage libraries and exemplified by peptide A-183 [Dennis, M. S., Roberge, M., Quan, C., and Lazarus, R. A. (2001) Biochemistry 40, 9513-9521], specifically bind at a site that is distinct from both the active site and the exosite of another recently described peptide inhibitor of FVIIa, E-76 [Dennis, M. S., Eigenbrot, C., Skelton, N. J., Ultsch, M. H., Santell, L., Dwyer, M. A., O'Connell, M. P., and Lazarus, R. A. (2000) Nature 404, 465-4701. Peptide A-183 prolonged TF-dependent clotting in human, but not rabbit plasma. Thus, a panel of human FVIIa mutants, containing 70 of the 76 rabbit sequence differences in the protease domain, localized the binding site to residues in the 60s loop and the C-terminus. The location of the exosite was refined by a series of FVIIa alanine mutants, which showed that proximal residues Trp 61 and Leu 251 were critical for binding. Kinetic and equilibrium binding constants for zymogen FVII, FVIIa and TF x FVIIa were determined using immobilized N-terminal biotinylated A-183 by surface plasmon resonance. No peptide binding to nine other human serine proteases was observed. Key residues on the peptide were determined from binding to FVIIa and inhibition of FX activation using a series of alanine mutants of A-183 fused to the Z domain of protein A. Analysis of the mutagenesis data is presented in the context of a crystal structure of A-183 in complex with a version of zymogen FVII [Eigenbrot, C., Kirchhofer, D., Dennis, M. S., Santell, L., Lazarus, R. A., Stamos, J., and Ultsch, M. H. (2001) Structure 9, 627-636]. The shape and proximity of this exosite to the active site may lend itself towards the design of new anticoagulants that inhibit FVIIa.

The genetic diagnosis of a single family with combined vitamin K-dependent clotting factor deficiency (VKCFD2, OMIM #607473) finally led to the identification and molecular characterization of vitamin K epoxide reductase (VKORC1). VKORC1 is the key enzyme of the vitamin K cycle and the molecular target of coumarins, which represent the most commonly prescribed drugs for therapy and prevention of thromboembolic conditions. However, coumarins are known to have a narrow therapeutic window and a considerable risk of bleeding complications caused by a broad variation of intra- and inter-individual drug requirement. Now, 3 years after its identification, VKORC1 has greatly improved our understanding of the vitamin K cycle and has led to the translation of basic research into clinical practise in at least three directions: (i) Mutations within VKORC1 have been shown to cause a coumarin-resistant phenotype and a single SNP (rs9923231) within the VKORC1 promoter region has been identified as the major pharmacodynamic determinant of coumarin dose. Together with the previously described CYP2C9 variants and other dose-influencing factors, such as age, gender and weight, individualized dosing algorithms have become available. (ii) Preliminary studies indicate that concomitant application of low-dose vitamin K (80-100 microg day(-1)) and warfarin significantly improves INR stability and time of INR within the therapeutic range. (iii) Co-expression studies of FIX and FX with VKORC1 have shown that VKOR activity is the rate-limiting step in the synthesis of biologically active vitamin K-dependent factors. Thus, co-expression of VKORC1 leads to a more efficient production of recombinant vitamin K-dependent coagulation factors such as FIX and FVII. This could improve production of recombinant factor concentrates in the future.

Exposure of tissue factor (TF)-bearing cells to blood is the initial event in coagulation and intravascular thrombus formation. However, the mechanisms which determine thrombus growth remain poorly understood. To explore whether the procoagulant activity of vessel wall-bound cells regulates thrombus expansion, we studied in vitro spatial clot growth initiated by cultured human cells of different types in contact pathway-inhibited, non-flowing human plasma. Human aortic endothelial cells, smooth muscle cells, macrophages and lung fibroblasts differed in their ability to support thrombin generation in microplate assay with peaks of generated thrombin of 60 +/- 53 nmol L(-1), 135 +/- 57 nmol L(-1), 218 +/- 55 nmol L(-1) and 407 +/- 59 nmol L(-1) (mean +/- SD), respectively. Real-time videomicroscopy revealed the initiation and spatial growth phases of clot formation. Different procoagulant activity of cell monolayers was manifested as up to 4-fold difference in the lag times of clot formation. In contrast, the clot growth rate, which characterized propagation of clotting from the cell surface to plasma, was largely independent of cell type (< or = 30% difference). Experiments with factor VII (FVII)-, FVIII-, FX- or FXI-deficient plasmas and annexin V revealed that (i) cell surface-associated extrinsic Xase was critical for initiation of clotting; (ii) intrinsic Xase regulated only the growth phase; and (iii) the contribution of plasma phospholipid surfaces in the growth phase was predominant. We conclude that the role of TF-bearing initiator cells is limited to the initial stage of clot formation. The functioning of intrinsic Xase in plasma provides the primary mechanism of sustained and far-ranging propagation of coagulation leading to the physical expansion of a fibrin clot.

Low-density lipoprotein receptor-related protein-1 (LRP1) is a large endocytic and signaling receptor that is widely expressed. In the liver, LRP1 plays an important role in regulating the plasma levels of blood coagulation factor VIII (fVIII) by mediating its uptake and subsequent degradation. fVIII is a key plasma protein that is deficient in hemophilia A and circulates in complex with von Willebrand factor. Because von Willebrand factor blocks binding of fVIII to LRP1, questions remain on the molecular mechanisms by which LRP1 removes fVIII from the circulation. LRP1 also regulates cell surface levels of tissue factor, a component of the extrinsic blood coagulation pathway. This occurs when tissue factor pathway inhibitor bridges the fVII/tissue factor complex to LRP1, resulting in rapid LRP1-mediated internalization and downregulation of coagulant activity. In the vasculature LRP1 also plays protective role from the development of aneurysms. Mice in which the lrp1 gene is selectively deleted in vascular smooth muscle cells develop a phenotype similar to the progression of aneurysm formation in human patient, revealing that these mice are ideal for investigating molecular mechanisms associated with aneurysm formation. Studies suggest that LRP1 protects against elastin fiber fragmentation by reducing excess protease activity in the vessel wall. These proteases include high-temperature requirement factor A1, matrix metalloproteinase 2, matrix metalloproteinase-9, and membrane associated type 1-matrix metalloproteinase. In addition, LRP1 regulates matrix deposition, in part, by modulating levels of connective tissue growth factor. Defining pathways modulated by LRP1 that lead to aneurysm formation and defining its role in thrombosis may allow for more effective intervention in patients.

Our research aims to provide quantitatively transparent, biologically realistic descriptions of the processes involved in hemostasis which will permit predictions of the behavior of the coagulation system in normal and pathologic states. We use four models of coagulation: (1) numerical approximations of the tissue factor (Tf) pathway of thrombin generation based upon mechanism and dynamics; (2) Tf activation of the "blood coagulation proteome" from isolated cells and proteins; (3) Tf activated contact pathway inhibited whole blood in vitro; and (4) blood shed from standardized microvascular wounds in vivo. The results from these models are integrated in interactive assessments aimed at achieving convergence of biochemical rigor and biological authenticity. Microvascular injury is the most biologically secure but least accessible to mechanistic study. Numerical models while quantitatively transparent are biologically limited. By the integrated analyses of all four models, we establish observations which require inclusion or discovery of new parameters to achieve mechanistically interpretable biological reality. Discoveries made in this fashion have included thrombin's role in the initiation phase, TFPI/ATIII/APC synergy interactions, rfVIIa in fVII deficiency, the roles of fVIII and fIX in the Tf reaction, and the cleavage of fIX by fXa membrane. Ideally, our results will provide descriptions which predict the behavior of the biological blood coagulation system under normal and pathologic conditions.

Hypoxia-inducible factors (HIF)-1α and HIF2α are major transcription factors required for adaptive responses to hypoxia. HIFs form a complex with aryl hydrocarbon receptor nuclear translocator (ARNT) to bind to the regulatory regions of target genes. The acetylation of histones by histone acetyltransferases (HATs) is one of the epigenetic marks associated with active chromatin. Indeed, HIFs recruit p300 HAT to hypoxia response elements (HREs) within gene regulatory regions. Here, we report an unusual HIF-mediated transcriptional activation in ovarian clear cell carcinoma (CCC). While characterizing coagulation factor VII (FVII) gene induction during hypoxic conditions, we observed that the interaction of HIF2α with Sp1, but not with ARNT, could induce transcription of FVII in a HRE-independent manner. Unexpectedly, this gene activation is associated with histone deacetylation. We found that a class II HDAC, HDAC4, is recruited with HIF2α to the FVII promoter as a co-activator, while p300 HAT negatively regulated this process. Furthermore, this mechanism can be synergistically enhanced via a deacetylation-dependent pathway when cells are simultaneously exposed to hypoxic and serum-free conditions. These results suggest the presence of a stress-responsive transcription mediated by the HIF2α/Sp1/HDAC4 network and explain how CCC shed their procoagulant activity under hypoxia.

BACKGROUND

The objective of this study was to develop a ligand-targeted photodynamic therapy (tPDT) by conjugating factor VII (fVII) protein with photosensitiser verteporfin in order to overcome the poor selectivity and enhance the effect of non-targeted PDT (ntPDT) for cancer. fVII is a natural ligand for receptor tissue factor (TF) with high affinity and specificity. The reason for targeting receptor TF for the development of tPDT is that TF is a common but specific target on angiogenic tumour vascular endothelial cells (VEC) and many types of tumour cells, including solid tumours and leukaemia.

METHODS

Murine factor VII protein (mfVII) containing a mutation (Lys341Ala) was covalently conjugated via a cross linker EDC with Veterporfin (VP) that was extracted from liposomal Visudyne, and then free VP was separated by Sephadex G50 spin columns. fVII-tPDT using mfVII-VP conjugate, compared to ntPDT, was tested in vitro for the killing of breast cancer cells and VEGF-stimulated VEC and in vivo for inhibiting the tumour growth of breast tumours in a mouse xenograft model.

RESULTS

We showed that: (i) fVII protein could be conjugated with VP without affecting its binding activity; (ii) fVII-tPDT could selectively kill TF-expressing breast cancer cells and VEGF-stimulated angiogenic HUVECs but had no side effects on non-TF expressing unstimulated HUVEC, CHO-K1 and 293 cells; (iii) fVII targeting enhanced the effect of VP PDT by three to four fold; (iii) fVII-tPDT induced significantly stronger levels of apoptosis and necrosis than ntPDT; and (iv) fVII-tPDT had a significantly stronger effect on inhibiting breast tumour growth in mice than ntPDT.

CONCLUSIONS

We conclude that the fVII-targeted VP PDT that we report here is a novel and effective therapeutic with improved selectivity for the treatment of breast cancer. Since TF is expressed on many types of cancer cells including leukaemic cells and selectively on angiogenic tumour VECs, fVII-tPDT could have broad therapeutic applications for other solid cancers and leukaemia.

Tissue factor (TF) is a 263 amino acid membrane-bound procoagulant protein that serves as a cofactor for the serine protease factor VII (fVII). Recombinant human TF (rTF) produced in both human kidney 293 cells and Escherichia coli has been immunoaffinity purified by using a TF-specific monoclonal antibody. Recombinant TF produced in 293 cells is glycosylated and migrates on reducing SDS-PAGE with an apparent molecular weight (Mr) of 45K. Some interchain disulfide-bonded rTF dimers are observed under nonreducing conditions. The E. coli produced rTF has a molecular weight of 33K and 35K, with the 33K band missing nine amino acids at the carboxy terminus. Although the E. coli produced rTF does not contain any carbohydrate, it is fully functional in both a chromogenic assay and a one-stage prothrombin time assay. A variant has been constructed wherein the cytoplasmic cysteine (residue 245) has been mutagenized to a serine residue. The amount of disulfide-linked aggregates is dramatically reduced following immunoaffinity purification of this four-cysteine variant (C2455), which is active in the chromogenic and prothrombin time assays.

The transmembrane glycoprotein tissue factor (TF) is the initiator of the coagulation cascade in vivo. When TF is exposed to blood, it forms a high-affinity complex with the coagulation factors factor VII/activated factor VIIa (FVII/VIIa), activating factor IX and factor X, and ultimately leading to the formation of an insoluble fibrin clot. TF plays an essential role in hemostasis by restraining hemorrhage after vessel wall injury. An overview of biological and physiological aspects of TF, covering aspects consequential for thrombosis and hemostasis such as TF cell biology and biochemistry, blood-borne (circulating) TF, TF associated with microparticles, TF encryption-decryption, and regulation of TF activity and expression is presented. However, the emerging role of TF in the pathogenesis of diseases such as sepsis, atherosclerosis, certain cancers and diseases characterized by pathological fibrin deposition such as disseminated intravascular coagulation and thrombosis, has directed attention to the development of novel inhibitors of tissue factor for use as antithrombotic drugs. The main advantage of inhibitors of the TF*FVIIa pathway is that such inhibitors have the potential of inhibiting the coagulation cascade at its earliest stage. Thus, such therapeutics exert minimal disturbance of systemic hemostasis since they act locally at the site of vascular injury.

Since the discovery of RNA interference (RNAi), researchers have identified a variety of small interfering RNA (siRNA) structures that demonstrate the ability to silence gene expression through the classical RISC-mediated mechanism. One such structure, termed "Dicer-substrate siRNA" (dsiRNA), was proposed to have enhanced potency via RISC-mediated gene silencing, although a comprehensive comparison of canonical siRNAs and dsiRNAs remains to be described. The present study evaluates the in vitro and in vivo activities of siRNAs and dsiRNAs targeting Phosphatase and Tensin Homolog (PTEN) and Factor VII (FVII). More than 250 compounds representing both siRNA and dsiRNA structures were evaluated for silencing efficacy. Lead compounds were assessed for duration of silencing and other key parameters such as cytokine induction. We identified highly active compounds from both canonical siRNAs and 25/27 dsiRNAs. Lead compounds were comparable in potency both in vitro and in vivo as well as duration of silencing in vivo. Duplexes from both structural classes tolerated 2'-OMe chemical modifications well with respect to target silencing, although some modified dsiRNAs demonstrated reduced activity. On the other hand, dsiRNAs were more immunostimulatory as compared with the shorter siRNAs, both in vitro and in vivo. Because the dsiRNA structure does not confer any appreciable benefits in vitro or in vivo while demonstrating specific liabilities, further studies are required to support their applications in RNAi therapeutics.

Tissue factor (TF) is a transmembrane receptor for FVII that triggers blood coagulation. It is not normally exposed to circulating blood, but may be produced by endothelium and monocytes under pathological conditions. Platelets take up TF-positive microparticles from leukocytes and TF appears on platelets adhering to leukocytes following collagen stimulation of blood. However, the presence of TF in circulating platelets has not been directly demonstrated. In this study, flow cytometric analysis of washed platelets from five healthy adult volunteers demonstrated TF-antigen on both resting platelets and platelets activated by thrombin (0.1 U/ml), collagen (5 microg/ml) or ADP (5 microM). TF released by platelets was demonstrated in the supernatants of non-activated and activated washed platelets by dot-immunoblotting and Western blotting. The amount of TF released from non-activated and activated platelets was quantitated using an enzyme-linked immunosorbent assay (ELISA). Washed non-activated and platelets activated by thrombin, collagen or ADP released 27-35 pg TF per mg protein. TF associated with the platelet surface was biologically inactive, although released TF was functionally active as determined by a two-stage factor X activation assay. We conclude that platelets contain an inactive form of TF that may develop functional activity following its release. However, the role of platelet TF in health and disease remains to be determined.

Overexpression of tissue factor (TF) is characteristically observed in advanced pancreatic cancer and has been associated with invasion and metastasis. Functional responses of TF activation are here investigated using as a model system the human pancreatic cancer cell lines SW979 (which overexpresses TF) and MIAPaCa2 (which does not express detectable levels). After stimulation of these cell lines with factor VIIa (FVIIa), the only known TF ligand, expression of urokinase receptor (uPAR) gene was up-regulated in SW979 cells in a dose-dependent manner but not in MIAPaCa2 cells. Interestingly, urokinase (uPA) and its specific inhibitor PAI-1 were not up-regulated. Exposure to functionally inactivated FVIIa did not show any effect on uPAR expression on SW979 cells despite binding to TF with higher efficiency. The neutralizing anti-TF antibody 5G9 blocked the FVIIa-induced up-regulation of uPAR completely, whereas hirudin failed to block this up-regulation. Treatment of SW979 cells with Factor Xa did not up-regulate the expression of uPAR gene, whereas treatment with FVII induced the same level of enhanced uPAR gene expression as that with FVIIa. In the matrigel invasion assay, enhanced invasion of SW979 cell line induced by FVIIa was completely inhibited by anti-TF antibody and alpha2-antiplasmin. Moreover, the endogenous levels of uPAR gene expression were significantly correlated with the level of TF gene expression in 19 human cancer cell lines (P < 0.05). These data suggest that up-regulation of uPAR expression by tumor cells leading to tumor invasion is induced through the TF-FVIIa pathway rather than TF-initiated thrombin generation. This is the first report that TF may be one of the key receptors that can up-regulate expression of the plasminogen activator receptor in human cancer cells to enhance tumor invasion and metastasis.

The vasculature of mouse breast tumor spheroids grown on mammary fat pad tissue in an intravital microscopy (IVM) viewing chamber was shown to derive from infiltrating angiogenic mammary vessels. The receptors tissue factor (TF), alpha V beta 3 integrin and Tie-2 were expressed on the vascular endothelium in the periphery but not in the center of the tumor spheroids nor in the mammary tissue nor in smooth muscle tissue, whereas Tie-1 and PCAM-1 were expressed extensively in the entire tumor and in the vascular endothelium of the entire tumor nodule and in normal mammary tissue. TF is a specific target for adenoviral vector-mediated cancer immunotherapy. Subcutaneous injection of the AdfVII/IgG(1)Fc vector leads to the release into the system circulation of a fVII/IgG(1)Fc immunoconjugate molecule that binds specifically and tightly to TF on vascular endothelial cells and tumor cells, activating a cytolytic immune response against the targeted cells. We show that a single administration of the AdfVII/IgG(1)Fc vector destroys the peripheral but not the central vasculature of a tumor spheroid, causing partial tumor regression; additional administrations prevent regeneration of the peripheral vasculature and regrowth of the tumor. These findings indicate that a critical parameter for optimizing tumor damage is the schedule for successive administrations of the AdfVII/IgG(1)Fc, which should coincide with the regeneration of the peripheral vasculature and continue until the tumor is destroyed.

The transmembrane protein tissue factor (TF) is the cell surface receptor for coagulation factor VII (FVII) and activated factor VII (FVIIa). Recently, TF has been identified as a regulator of angiogenesis, tumor growth, and metastasis. This study was designed to link the binding of FVII(a) to its receptor, TF, with the subsequent triggering of angiogenesis through vascular endothelial growth factor (VEGF) production by human lung fibroblasts. We report that incubation of fibroblasts, which express constitutive surface TF, with FVII(a) induces VEGF synthesis. FVII(a)-induced VEGF secretion, assessed by a specific enzyme-linked immunosorbent assay, was time- and concentration-dependent. VEGF secretion was maximal after 24 hours of incubation of the cells with 100 nmol/L FVII(a) and represented a threefold induction of the basal VEGF level. Reverse transcriptase-polymerase chain reaction analysis of VEGF detected three mRNA species of 180, 312, and 384 bp corresponding, respectively, to VEGF121, VEGF165, and VEGF189. A 2.5- to 3.5-fold increase was observed for the 180- and 312-bp transcripts at 12 and 24 hours, respectively. FVII(a)-dependent VEGF production was inhibited by a pool of antibodies against TF, pointing to the involvement of this receptor. On specific active-site inhibition with dansyl-glutamyl-glycinyl-arginyl chloromethyl ketone, FVIIa lost 70% of its capacity to elicit VEGF production. Consistent with this, the native form (zymogen) of FVII only had a 1.8-fold stimulating effect. Protein tyrosine kinase and protein kinase C are involved in signal transduction leading to VEGF production, as shown by the inhibitory effects of genistein and GF 109203X. The results of this study indicate that TF is essential for VIIa-induced VEGF production by human fibroblasts and that its role is mainly linked to the proteolytic activity of the TF-VIIa complex.

The incidence of venous thromboembolism (VTE) is well recognized to increase with aging. Concurrently, the plasma concentrations of many coagulation factors (e.g., fibrinogen, factor [F] V, FVII, FVIII, and FIX) increase with aging, as does von Willebrand factor (VWF), thrombin generation, and platelet activation. Data are conflicting regarding age-related changes in the natural anticoagulants, including protein C, protein S, and antithrombin. Changes are also observed with components of the fibrinolytic pathway. All in all, aging is associated with a variety of hemostasis changes that on balance reflects a heightened procoagulant status compared with earlier age. It has to be recognized that as this occurs in the otherwise normal general population, this can also be considered a normal phenomenon of progressive life. An element of this heightened procoagulant status may reflect ongoing inflammatory processes, given some markers, notably FVIII and fibrinogen, are acute phase reactants. A variety of acquired prothrombotic risk factors (e.g., cancer, autoimmune disorders, and diabetes) also gradually develop with aging, some of which may induce profound abnormalities of hemostasis, and confound the age-related changes in hemostasis, as well as their influence on thrombotic risk. In this article, we review the changes in hemostasis markers measurable within many hemostasis laboratories, and consider many of the important implications for clinical and laboratory practice. Apart from representing an increased thrombotic risk, additional considerations entail the potential need (1) to utilize age-adjusted normal ranges (e.g., for D-dimer), (2) to consider the consequence on previous diagnoses (e.g., "mild type 1" von Willebrand disease [VWD], where VWF test results may "normalize" with aging), and (3) to consider the effect of these changes of risk factors on the (perceived) therapeutic efficacy of antithrombotic medications such as aspirin.

Factor VII (FVII) deficiency is the most frequent among rare congenital bleeding disorders, accounting for one symptomatic individual per 500,000 population, apparently without any racial/ethnic predilection. FVII deficiency prevalence in the general population is probably higher because of the presence of asymptomatic and poorly symptomatic individuals. In accordance with the role of FVII as part of the initiating complex of the extrinsic coagulation pathway, laboratory diagnosis is easy, because FVII deficiency is the only congenital bleeding disorder characterized by isolated prolonged prothrombin time. Molecular diagnosis is available, and a broad spectrum of mutations has been characterized in the FVII gene, which is located in chromosome 13. Clinical manifestations are heterogeneous, ranging from severe life-threatening haemorrhages, such as cerebral, gastrointestinal, and joint haemorrhages, to miscellaneous minor bleeding. The main clinical features in our database (International Registry on Congenital FVII Deficiency database, n = 515) are as follows: (i) the absence of a clear-cut and consistent correlation between bleeding symptoms and FVII clotting levels; (ii) an excess of symptomatic women compared with men; (iii) frequent surgery-related bleeding, which is often a diagnostic tool in previously asymptomatic individuals. Several therapeutic options are possible, including plasma-derived and recombinant products, but therapeutic schedules, optimal dosages, and administration times still have to be precisely defined, and clinical studies, including online registries such as the Seven Treatment Evaluation Registry, are actually ongoing to achieve in a better manner a safe, rational and standardized substitution treatment for this congenital disorder.

BACKGROUND

Hemodilution and consumption of coagulation factors during cardiopulmonary bypass has been suggested to contribute to bleeding complications after cardiac surgery. The aim was to describe the activity of individual coagulation factors after CABG in relation to hemodilution and postoperative bleeding.

METHODS

Plasma concentrations of fibrinogen and plasma activity of FII, FV, FVII, FVIII, FIX, FX, FXI and FXIII adjusted for hemodilution were analysed in 57 CABG patients before, and 2h and 24h after surgery. Postoperative bleeding was registered and correlations to coagulation factor activity were calculated.

RESULTS

Adjusted plasma concentration of fibrinogen (-14+/-6%), and plasma activity of FII (-9+/-6%), FV (-13+/-8%), FX (-13+/-7%) and FXIII (-9+/-14%) were reduced two hours after surgery compared to baseline (all p<0.001). FVII (+3+/-12%, p=0.34) and FXI (+1+/-19%, p=0.50) were unchanged, while FVIII (+23+/-44%, p=0.006) and FIX (+23+/-17%, p<0.001) increased. Twenty-four hours after surgery fibrinogen (+45+/-27%), FVIII (+93+/-66%) and FIX (+33+/-26%) were all increased (all p<0.001), while FVII (-37+/-14%, p<0.001), FXI (-4+/-18%, p=0.02) and FXIII (-6+/-15%, p=0.004) were decreased. Median postoperative blood loss was 380 ml/12h. There were significant inverse correlations between postoperative blood loss and fibrinogen concentration 2h after surgery (r=-0.33, p=0.019) and between postoperative blood loss and pre- and postoperative FXIII activity (r=-0.34, p=0.009 and r=-0.41, p=0.003, respectively), but not between blood loss and any of the other factors.

CONCLUSIONS

There is a marked dissociation in plasma activity of individual coagulation factors after CABG. Plasma concentration of fibrinogen and factor XIII activity correlates inversely to postoperative blood loss after CABG.

Upregulation of the activated Factor VII (FVIIa)/Tissue Factor complex, downregulation of natural anticoagulation pathways, and inhibition of fibrinolysis, are major contributors to coagulopathies associated with acute inflammation. Provision of FVIIa, and consequent downstream coagulation-related proteases, also stimulates further inflammatory changes, which can result in disseminated intravascular coagulation. Thus, the potential protective effects in vivo of a genetic-based reduction in FVII levels have been investigated in a murine model of acute inflammation, namely lipopolysaccharide (LPS)-induced lethal endotoxaemia. Mice with a total FVII deficiency do not survive the neonatal period. Therefore mice expressing low levels of FVII (FVII(tTA/tTA)), producing sufficient amounts of FVII for survival (approximately 5% of wild-type (WT) FVII), were employed to investigate in vivo pathways involved in the crosstalk between coagulation, inflammation, and survival, consequent to administration of a lethal dose of LPS. The FVII(tTA/tTA) mice presented with reduced mortality, coagulation, and inflammatory responses in comparison with similarly treated WT mice after administration of LPS. The attenuated inflammatory responses in FVII(tTA/tTA) mice were associated with downregulation of Egr-1 signalling. Administration, in vivo, of specific inhibitors of FXa and thrombin demonstrated that the inflammatory responses were unaltered in WT mice, but further reduced in FVII(tTA/tTA) mice. Therefore, a FVII deficiency enhances survival from lethal endotoxaemia both through attenuation of inflammatory responses that result directly from reduced FVIIa levels, and, indirectly, from downregulation of coagulation proteases downstream of the FVII-dependent cascade.

Overexpression and exposition of tissue factor (TF) in atherosclerotic plaques and/or arterial thrombi are critical events in atherothrombosis. TF, the receptor for factor VII (FVII) and activated factor VII (FVIIa), is the principal initiator of blood coagulation and induces thrombin generation leading to fibrin formation and platelet activation. TF also plays a major role in cell migration and angiogenesis. TF activity is downregulated by Tissue Factor Pathway Inhibitor (TFPI), a Kunitz-type inhibitor, which forms a neutralizing complex with TF, FVIIa and activated factor X. In physiological conditions, TF is absent from vascular cells which come into contact with flowing blood and is present as an inactive pool in fibroblasts and smooth muscle cells (SMC). In contrast, TF is widely expressed in atherosclerotic plaques and is found in macrophages, SMCs, and foam-cells and also in extracellular matrix and acellular lipid-rich core. TF expression is up-regulated by inflammatory cytokines and oxidized lipids. Plaque thrombogenicity is directly correlated to their TF content. After fibrous cap disruption, TF is exposed on plaque surface and triggers thrombus formation leading to arterial lumen occlusion and/or downstream embolization. In coronary and carotid plaques, TF content was found to be higher in plaques from symptomatic than asymptomatic patients. Soluble forms of TF and microparticles of monocyte and platelet origin, and bearing TF, constitute "blood-born TF". The contribution of this TF pool to arterial thrombosis is still under discussion. TF pathway is a target for new therapeutic agents that can decrease TF activity, such as active site-inactivated factor VIIa, recombinant TFPI and antibodies against TF or peptides interfering with TF-FVIIa complex activity.

OBJECTIVE

To measure the microtensile bond strength to ground enamel of different types of materials used as pit and fissure sealants in combination with different substrate conditioners.

METHODS

From 40 sound extracted molars, eight groups of five teeth were randomly formed. The experimental groups were: (1) (C) 37% phosphoric acid/ClinPro Sealant (3M ESPE); (2) (G) 37% phosphoric acid/Guardian Seal (Kerr); (3) (E/TF) 37% phosphoric acid/Excite/Tetric Flow (Ivoclar-Vivadent) (4) (OS/UF) 37% phosphoric acid/One Step (Bisco)/UniFil Flow (GC); (5) (OS/AE) 37% phosphoric acid/One Step/AEliteflo (Bisco); (6) (UB/UF) UniFil Bond/UniFil Flow (GC); (7) (CC/FVII) GC Cavity Conditioner/Fuji VII (GC); (8) (CC/FII) GC Cavity Conditioner/Fuji II LC Improved (GC). On the buccal of each tooth, a 5mm high build-up was created by incrementally adding layers of the sealing material on the conditioned enamel. By serially cutting the built-up tooth, multiple beam-shaped specimens about 1mm x 1mm in cross section were obtained, and loaded in tensile (0.5 mm/min) until failure occurred.

RESULTS

The bond strengths measured in MPa were: (C) 20.41+/-11.79; (G) 16.02+/-7.99; (E/TF) 24.06+/-9.67; OS/UF 15.63+/-9.00; (OS/AE) 9.31+/-6.05; (UB/UF) 4.96+/-3.46; (CC/FVII) 1.70+/-2.19; (CC/FII) 2.19+/-1.44.

CONCLUSIONS

The conventional and the resin-modified glass ionomers measured bond strengths significantly lower than those of any resin-based materials. Failure frequently occurred cohesively within the cement. Flowable composites in combination with phosphoric acid and a total-etch adhesive performed similarly to resin-based materials specifically conceived for sealings, such as ClinPro Sealant and Guardian Seal. The bond achieved by resin composite when treating enamel with the self-etching primer used in this study (UniFil Bond) was significantly lower than that developed when the substrate was etched with 37% phosphoric acid.