ABSTRACT
UNLABELLED: ESSENTIALS: The role of tissue factor (TF) in recombinant factor VIIa (rFVIIa) therapy in hemophilia is unclear. An acquired mouse hemophilia model with very low or normal levels of human TF was used in the study. rFVIIa is equally effective in correcting the bleeding in mice expressing low or normal levels of TF. Pharmacological doses of rFVIIa restore hemostasis in hemophilia independent of TF. BACKGROUND: Recombinant factor VIIa (rFVIIa) has been used widely for treating hemophilia patients with inhibitory autoantibodies against factor VIII or IX. Its mechanism of action is not entirely known. A majority of in vitro studies suggested that pharmacological concentrations of rFVIIa restore hemostasis in hemophilia in a phospholipid-dependent manner, independent of tissue factor (TF). However, a few studies suggested that a TF-dependent mechanism has a primary role in correction of bleeding by rFVIIa in hemophilia patients. Here, we investigated the potential contribution of TF in rFVIIa-induced hemostasis in hemophilia employing a model system of FVIII antibody-induced hemophilia in TF transgenic mice. METHODS: Mice expressing low levels of human TF (LTF mice), mice expressing relatively high levels of human TF (HTF mice) and wild-type mice (WT mice) had neutralizing anti-FVIII antibodies administered in order to induce hemophilia in these mice. The mice were then treated with varying concentrations of rFVIIa. rFVIIa-induced hemostasis was evaluated with the saphenous vein bleeding model. RESULTS: Administration of FVIII inhibitory antibodies induced the hemophilic bleeding phenotype in all three genotypes. rFVIIa administration rescued the bleeding phenotype in all three genotypes. No significant differences were observed in rFVIIa-induced correction of bleeding between LTF and HTF mice that had FVIII antibodies administered. CONCLUSIONS: Our results provide strong evidence supporting the suggestion that the hemostatic effect of pharmacological doses of rFVIIa stems from a TF-independent mechanism.
Subject(s)
Coagulants/pharmacology , Factor VIIa/pharmacology , Hemophilia A/drug therapy , Hemostasis/drug effects , Animals , Antibodies, Monoclonal , Antibodies, Monoclonal, Humanized , Disease Models, Animal , Dose-Response Relationship, Drug , Factor VIII/antagonists & inhibitors , Factor VIII/immunology , Genotype , Hemophilia A/blood , Hemophilia A/genetics , Hemophilia A/immunology , Humans , Mice, Inbred C57BL , Mice, Transgenic , Phenotype , Recombinant Proteins/pharmacology , Thromboplastin/genetics , Thromboplastin/metabolismABSTRACT
BACKGROUND: Recent studies have shown that factor VIIa binds to endothelial cell protein C receptor(EPCR), a cellular receptor for protein C and activated protein C. At present, the physiologic significance of FVIIa interaction with EPCR in vivo remains unclear. OBJECTIVE: To investigate whether exogenously administered FVIIa, by binding to EPCR, induces a barrier protective effect in vivo. METHODS: Lipopolysaccharide(LPS)-induced vascular leakage in the lung and kidney,and vascular endothelial growth factor (VEGF)-induced vascular leakage in the skin, were used to evaluate the FVIIa-induced barrier protective effect. Wild-type, EPCR-deficient, EPCR-overexpressing and hemophilia A mice were used in the studies. RESULTS: Administration ofFVIIa reduced LPS-induced vascular leakage in the lung and kidney; the FVIIa-induced barrier protective effect was attenuated in EPCR-deficient mice. The extent of VEGF-induced vascular leakage in the skin was highly dependent on EPCR expression levels. Therapeutic concentrations of FVIIa attenuated VEGF-induced vascular leakage in control mice but not in EPCR-deficient mice.Blockade of FVIIa binding to EPCR with a blocking mAb completely attenuated the FVIIa-induced barrier protective effect. Similarly, administration of protease activated receptor 1 antagonist blocked the FVIIa induced barrier protective effect. Hemophilic mice showed increased vascular permeability, and administration of therapeutic concentrations of FVIIa improved barrier integrity in these mice. CONCLUSIONS: This is the first study to demonstrate that FVIIa binding to EPCR leads to a barrier protective effect in vivo. This finding may have clinical relevance, as it indicates additional advantages of using FVIIa in treating hemophilic patients.
Subject(s)
Blood Coagulation Factors/metabolism , Factor VIIa/metabolism , Protein C/metabolism , Receptors, Cell Surface/metabolism , Animals , Capillary Permeability , Cells, Cultured , Endothelial Cells/cytology , Factor Xa/metabolism , Female , Genotype , Hemophilia A/metabolism , Humans , Lipopolysaccharides/chemistry , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Permeability , Protein Binding , Thrombin/metabolism , Vascular Endothelial Growth Factor A/metabolismSubject(s)
Genetic Variation , Hemostasis , Plasma , Thromboplastin/genetics , Venous Thrombosis/genetics , Female , Humans , MaleSubject(s)
Coagulants/metabolism , Thromboplastin/metabolism , Animals , Breast Neoplasms/metabolism , Carbohydrates/chemistry , Cell Line , Cell Line, Tumor , Female , Glycoproteins/metabolism , Glycosylation , Human Umbilical Vein Endothelial Cells , Humans , Insecta , Mutation , Phenotype , Placenta/metabolism , PregnancySubject(s)
Blood Coagulation Factors/metabolism , Breast Neoplasms/metabolism , Gene Expression Regulation, Neoplastic , Mammary Neoplasms, Animal/metabolism , Receptors, Cell Surface/metabolism , Animals , Cell Line, Tumor , Disease Progression , Female , Human Umbilical Vein Endothelial Cells , Humans , Mammary Neoplasms, Animal/pathology , Mammary Neoplasms, Experimental/pathology , Mice , Mice, Nude , Neoplasm Transplantation , Platelet Activation , Protein C/metabolism , Thrombin/metabolismABSTRACT
BACKGROUND: Tissue factor (TF) encryption plays an important role in regulating TF coagulant activity. Potential differences in experimental cell model systems and strategies hampered our understanding of the TF encryption mechanisms. OBJECTIVE: To characterize the procoagulant activity status of TF in different cell types, and to determine whether increased TF procoagulant activity following the activation stems from transformation of the cryptic TF to the active form. METHODS: Simultaneous kinetic analyses of TF-FVIIa activation of FX and FVIIa binding to cell surface TF were performed under identical experimental conditions in fibroblast (WI-38), cancer cell (MDA-231), endothelial cell (HUVEC) and monocytic cell (THP-1) model systems. These data were then utilized to estimate TF coagulant-specific activity and percentages of active and cryptic TF present in these cell types. RESULTS: MDA-231 and WI-38 cells express 10 to 100 times more TF on their cell surfaces compared with perturbed HUVEC and THP-1 cells. TF-specific activity on cell surfaces of MDA-231, WI-38 and THP-1 cells was very similar. Nearly 80-90% of the TF in MDA-231, WI-38 and THP-1 cells was cryptic. A plasma concentration of FVII would be sufficient to bind both active and cryptic TF on cell surfaces. Increased TF activity following cell activation stems from decryption of cryptic TF rather than increasing the coagulant activity of the active TF. CONCLUSIONS: Our data demonstrate that TF encryption is not limited to a specific cell type, and unlike previously thought, the majority of the TF expressed in cancer cells is not constitutively procoagulant.
Subject(s)
Blood Coagulation , Breast Neoplasms/metabolism , Fibroblasts/metabolism , Human Umbilical Vein Endothelial Cells/metabolism , Monocytes/metabolism , Signal Transduction , Thromboplastin/metabolism , Cell Line, Tumor , Cell Membrane/metabolism , Factor VIIa/metabolism , Factor Xa/metabolism , Female , Humans , KineticsABSTRACT
Tissue factor (TF) is a transmembrane glycoprotein and an essential component of the factor VIIa-TF enzymatic complex that triggers activation of the coagulation cascade. Formation of TF-FVIIa complexes on cell surfaces not only trigger the coagulation cascade but also transduce cell signaling via activation of protease-activated receptors. Tissue factor is expressed constitutively on cell surfaces of a variety of extravascular cell types, including fibroblasts and pericytes in and surrounding blood vessel walls and epithelial cells, but is generally absent on cells that come into contact with blood directly. However, TF expression could be induced in some blood cells, such as monocytes and endothelial cells, following an injury or pathological stimuli. Tissue factor is essential for hemostasis, but aberrant expression of TF leads to thrombosis. Therefore, a proper regulation of TF activity is critical for the maintenance of hemostatic balance and health in general. TF-FVIIa coagulant activity at the cell surface is influenced not only by TF protein expression levels but also independently by a variety of mechanisms, including alterations in membrane phospholipid composition and cholesterol content, thiol-dependent modifications of TF allosteric disulfide bonds, and other post-translational modifications of TF. In this article, we critically review the key literature on mechanisms by which TF coagulant activity is regulated at the cell surface in the absence of changes in TF protein levels with specific emphasis on recently published data and provide the authors' perspective on the subject.
Subject(s)
Cell Membrane/metabolism , Coagulants/chemistry , Gene Expression Regulation , Thromboplastin/chemistry , Allosteric Site , Animals , Caveolae/chemistry , Cholesterol/chemistry , Disulfides/chemistry , Fibroblasts/metabolism , Glycosylation , Hemostasis , Humans , Membrane Microdomains/chemistry , Pericytes/cytology , Phospholipids/chemistry , Protein Disulfide-Isomerases/chemistry , Protein Processing, Post-Translational , Sulfhydryl Compounds/chemistryABSTRACT
BACKGROUND: Recent studies show that activated factor VII (FVIIa) binds to the endothelial cell protein C receptor (EPCR) on the vascular endothelium; however, the importance of this interaction in hemostasis or pathophysiology is unknown. OBJECTIVE: The aim of the present study was to investigate the role of the FVIIa interaction with EPCR on the endothelium in mediating FVIIa transport from the circulation to extravascular tissues. METHODS: Wild-type, EPCR-deficient or ECPR-over-expressing mice were injected with human recombinant (r)FVIIa (120 µg kg(-1) body weight) via the tail vein. At varying time intervals after rFVIIa administration, blood and various tissues were collected to measure FVIIa antigen and activity levels. Tissue sections were analyzed by immunohistochemistry for FVIIa and EPCR. RESULTS: The data reveal that, after intravenous (i.v.) injection, rFVIIa rapidly disappears from the blood and associates with the endothelium in an EPCR-dependent manner. Immunohistochemical analyses revealed that the association of FVIIa with the endothelium was maximal at 30 min and thereafter progressively declined. The FVIIa association with the endothelium was undetectable at time points exceeding 24 h post-FVIIa administration. The levels of rFVIIa accumulated in tissue correlate with expression levels of EPCR in mice and FVIIa associated with tissues remained functionally active for periods of at least 7 days. CONCLUSIONS: The observation that an EPCR-dependent association of FVIIa with the endothelium is most pronounced soon after rFVIIa administration and subsequently declines temporally, combined with the retention of functionally active FVIIa in tissue homogenates for extended periods, indicates that FVIIa binding to EPCR on the endothelium facilitates the transport of FVIIa from circulation to extravascular tissues where TF resides.
Subject(s)
Blood Coagulation Factors/metabolism , Endothelial Cells/cytology , Factor VIIa/chemistry , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , Thromboplastin/metabolism , Animals , Biological Transport , Endothelial Protein C Receptor , Endothelium, Vascular/metabolism , Hemophilia A/metabolism , Hemostasis , Humans , Mice , Mice, Inbred C57BL , Mice, Transgenic , Protein Binding , Recombinant Proteins/blood , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Signal Transduction , Time Factors , Tissue DistributionSubject(s)
Factor VIIa/pharmacokinetics , Glycoproteins/blood , Animals , Biological Availability , Endothelial Protein C Receptor , Factor VIIa/administration & dosage , Genotype , Glycoproteins/deficiency , Glycoproteins/genetics , Half-Life , Humans , Injections, Intravenous , Mice , Mice, Knockout , Mice, Transgenic , Models, Biological , Phenotype , Receptors, Cell Surface , Recombinant Proteins/blood , Recombinant Proteins/pharmacokineticsABSTRACT
BACKGROUND: Glycosylation plays an important role in protein function. The importance of glycosylation for tissue factor (TF) function is unclear. OBJECTIVE: The aim of the present study is to investigate the importance of TF glycosylation in transport to the cell surface and its coagulant and signaling functions. METHODS: Endothelial cells and peripheral blood mononuclear cells (PBMC) were treated with tunicamycin to inhibit N-linked glycosylation. Site-specific mutagenesis of one or more potential N-linked glycosylation sites in TF was used to generate TF mutants lacking glycans. TF expression at the cell surface was determined in binding assays using (125)I-FVIIa or (125)I-TF mAb and confocal microscopy. TF coagulant activity was measured by factor (F) Xa generation assay, and TF signaling function was assessed by measuring cleavage of protease activated receptor 2 (PAR2) and activation of p44/42 MAPK. RESULTS: Tunicamycin treatment reduced TF activity at the endothelial cell surface; however, this reduction was found to be the result of decreased TF protein production in tunicamycin-treated cells. Tunicamycin treatment had no significant effect on TF activity or antigen levels in PBMC. No significant differences were observed in TF protein expression and procoagulant activity among cells transfected to express either wild-type TF or TF mutants. A fully non-glycosylated TF is shown to bind FVIIa and interact with FX with the same efficiency as that of wild-type TF. Non-glycosylated TF is also capable of supporting FVIIa cleavage of PAR2 and PAR2-dependent p44/42 MAPK activation. CONCLUSIONS: Glycosylation is not essential for TF transport and coagulant or signaling functions.
Subject(s)
Human Umbilical Vein Endothelial Cells/metabolism , Leukocytes, Mononuclear/metabolism , Protein Processing, Post-Translational , Thromboplastin/metabolism , Animals , Blood Coagulation , CHO Cells , Cricetinae , Cricetulus , Dose-Response Relationship, Drug , Enzyme Activation , Factor Xa/metabolism , Glycosylation , Human Umbilical Vein Endothelial Cells/drug effects , Humans , Interleukin-1beta/metabolism , Leukocytes, Mononuclear/drug effects , Microscopy, Confocal , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Mutagenesis, Site-Directed , Mutation , Protein Processing, Post-Translational/drug effects , Protein Transport , Receptor, PAR-2/metabolism , Recombinant Fusion Proteins/metabolism , Signal Transduction , Thromboplastin/genetics , Transfection , Tumor Necrosis Factor-alpha/metabolism , Tunicamycin/pharmacologySubject(s)
Factor VIIa/therapeutic use , Animals , Dogs , Genetic Therapy , Hemorrhage , Humans , Immunohistochemistry/methods , Mice , Mice, Inbred C57BL , Rabbits , Recombinant Proteins/blood , Recombinant Proteins/chemistry , Recombinant Proteins/therapeutic use , Time Factors , Tissue DistributionABSTRACT
BACKGROUND: Recent clinical studies suggest that the prophylactic use of recombinant factor VIIa (rFVIIa) markedly reduces the number of bleeding episodes in hemophilic patients with inhibitors. Given the short biological half-life of rFVIIa, it is unclear how rFVIIa could be effective in prophylactic treatment. OBJECTIVES: To examine the extravascular distribution of pharmacologically administered rFVIIa to obtain clues on how rFVIIa could work in prophylaxis. METHODS: Recombinant mouse FVIIa tagged with AF488 fluorophore (AF488-FVIIa) was administered into mice via the tail vein. At different time intervals following the administration, mice were exsanguinated and various tissues were collected. The tissue sections were processed for immunohistochemistry to evaluate distribution of rFVIIa. RESULTS: rFVIIa, immediately following the administration, associated with the endothelium lining of large blood vessels. Within 1 h, rFVIIa bound to endothelial cells was transferred to the perivascular tissue surrounding the blood vessels and thereafter diffused throughout the tissue. In the liver, rFVIIa was localized to sinusoidal capillaries and accumulated in hepatocytes. In bone, rFVIIa was accumulated in the zone of calcified cartilage and some of it was retained there for a week. The common finding of the present study is that rFVIIa in extravascular spaces was mostly localized to regions that contain TF expressing cells. CONCLUSIONS: The present study demonstrates that pharmacologically administered rFVIIa readily associates with the vascular endothelium and subsequently enters into extravascular spaces where it is likely to bind to TF and is retained for extended time periods. This may explain the prolonged pharmacological effect of rFVIIa.
Subject(s)
Coagulants/administration & dosage , Coagulants/pharmacokinetics , Factor VIIa/administration & dosage , Factor VIIa/pharmacokinetics , Animals , Bone and Bones/metabolism , Diffusion , Endothelium, Vascular/metabolism , Fluorescent Dyes/administration & dosage , Fluorescent Dyes/pharmacokinetics , Immunohistochemistry , Injections, Intravenous , Liver/metabolism , Male , Mice , Mice, Inbred C57BL , Protein Binding , Recombinant Proteins/administration & dosage , Recombinant Proteins/pharmacokinetics , Thromboplastin/metabolism , Tissue DistributionABSTRACT
BACKGROUND: Mesothelial cells that line the thoracic cavity play an important role in maintaining the local balance between procoagulant and fibrinolytic activity, a role akin to the endothelial cells in blood vessels. The mechanism(s) responsible for increased tissue factor (TF) expression in mesothelial cells in response to injury are at present unclear. OBJECTIVE: To investigate whether plasmin or thrombin, two major proteases that may be generated on the pleural surface upon injury, induce TF expression in human pleural mesothelial cells (HMC) and elucidate the underlying mechanism(s). METHODS: Confluent monolayers of HMC and human umbilical vein endothelial cells (HUVEC) were exposed to plasmin or thrombin for varying time periods and TF expression was analyzed by measuring its activity in a factor Xa generation assay, TF antigen levels by immunoblot analysis and TF mRNA by Northern blot analysis. RESULTS: Both plasmin and thrombin treatments increased cell surface TF activity in HMC by 3- to 4-fold. In contrast to thrombin, plasmin-induced TF activity is not dependent on the de novo synthesis of TF. In HUVEC, plasmin had a minimal effect on unperturbed HUVEC whereas it markedly increased TF activity of activated HUVEC. Plasmin treatment neither affected anionic phospholipid levels at the cell surface nor released protein disulfide isomerase, an oxidoreductase protein that was newly described to play a role in TF activation. Plasmin cleaved cell-associated TFPI. CONCLUSION: Thrombin up-regulates TF activity in HMC through the transcriptional activation of TF whereas plasmin increases TF activity by inactivating the cell-associated TFPI by a limited proteolysis.
Subject(s)
Endothelial Cells/metabolism , Epithelium/metabolism , Fibrinolysin/pharmacology , Thromboplastin/analysis , Thromboplastin/genetics , Cells, Cultured , Endothelium, Vascular/cytology , Humans , Lipoproteins/antagonists & inhibitors , Peptide Hydrolases/metabolism , RNA, Messenger/analysis , Thrombin/pharmacology , Transcription, GeneticABSTRACT
BACKGROUND: Recombinant coagulation factor VIIa (rFVIIa) has proven to be a safe and effective drug for treatment of bleeding episodes in hemophilic patients with inhibitors. However, rFVIIa is cleared from the circulation relatively quickly. Protein modification with poly(ethylene glycol) (PEG) can prolong the circulatory lifetime of proteins but it could also impair protein function by molecular shielding of the protein surface. OBJECTIVES: To characterize the interaction of glycoPEGylated rFVIIa - rFVIIa-10K PEG and rFVIIa-40K PEG - with tissue factor (TF), factor X (FX) and plasma inhibitors, tissue factor pathway inhibitor (TFPI) and antithrombin (AT). METHODS: The amidolytic and FX activation assays were employed to investigate the interaction of glycoPEGylated rFVIIa with its macromolecular substrate and inhibitors. RESULTS: Both the glycoPEGylated rFVIIa analogs exhibited similar amidolytic activity as that of rFVIIa in the absence or the presence of relipidated TF. The analogs were as effective as rFVIIa in activating FX in the absence of TF. In the presence of TF, the glycoPEGylated rFVIIa variants, relative to rFVIIa, were slightly less effective at lower concentrations, but no significant differences were found among them in activating FX at saturating concentrations. Both AT/heparin and TFPI effectively inhibited the glycoPEGylated rFVIIa bound to relipidated TF or TF on stimulated endothelial cells. In contrast to their normal interaction with TF, the glycoPEGylated rFVIIa variants appeared to interact poorly with phospholipids. CONCLUSIONS: The glycoPEGylated rFVIIa variants retained their catalytic activity and interacted efficiently with TF, FX and the plasma inhibitors. Further work with appropriate in vitro and in vivo model systems is needed to determine the feasibility of using glycoPEGylated rFVIIa to improve therapeutic options for bleeding disorders.
Subject(s)
Factor VIIa/pharmacology , Polyethylene Glycols/chemistry , Antithrombins/pharmacology , Catalysis , Cell-Free System , Cells, Cultured , Factor VIIa/antagonists & inhibitors , Factor VIIa/chemistry , Glycosylation , Humans , Phospholipids/chemistry , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/chemistry , Recombinant Proteins/pharmacology , Thromboplastin/chemistryABSTRACT
BACKGROUND: Factor VIIa (FVIIa) binding to tissue factor (TF) induces cell signaling via the protease activity of FVIIa and protease-activated receptor 2 (PAR2). OBJECTIVE: We examined how the gene-expression profile induced by FVIIa corresponds to the profiles induced by protease-activated receptor 1 (PAR1) or PAR2 agonists using MDA-MB-231 breast carcinoma cells that constitutively express TF, PAR1 and PAR2. RESULTS AND CONCLUSIONS: Out of 8500 genes, FVIIa stimulation induced differential regulation of 39 genes most of which were not previously recognized as FVIIa regulated. All genes regulated by FVIIa were similarly regulated by a PAR2 agonist peptide confirming FVIIa signaling via PAR2. An appreciable fraction of the PAR2-regulated genes was also regulated by a PAR1 agonist peptide suggesting extensive redundancy between FVIIa/PAR2 signaling and thrombin/PAR1 signaling. The FVIIa regulated genes encode cytokines, chemokines and growth factors, and the gene repertoire induced by FVIIa in MDA-MB-231 cells is consistent with a role for TF-FVIIa signaling in regulation of a wound healing type of response. Interestingly, a number of genes regulated exclusively by FVIIa/PAR2-mediated cell signaling in MDA-MB-231 cells were regulated by thrombin and a PAR1 agonist, but not by FVIIa, in the TF-expressing glioblastoma U373 cell line.
Subject(s)
Factor VIIa/metabolism , Gene Expression Regulation, Neoplastic , Neoplasms/metabolism , Receptor, PAR-1/metabolism , Receptor, PAR-2/metabolism , Thromboplastin/metabolism , Transcription, Genetic , Binding Sites , Cell Line, Tumor , Gene Expression Profiling , Humans , Oligonucleotide Array Sequence Analysis , Peptides/chemistry , Signal TransductionABSTRACT
BACKGROUND: Elevation of C-reactive protein (CRP) levels in blood was recognized as one of the cardiac disease risk factors. Consumption of wine is shown to reduce the risk from heart disease and improve longevity. OBJECTIVES: In the present study, we evaluated the effect of various wine polyphenolic compounds and several active synthetic derivatives of resveratrol on the inflammatory cytokines (IL-1beta + IL-6)-induced CRP expression in Hep3B cells. RESULTS: Among the wine phenolics tested, quercetin and resveratrol, in a dose-dependent manner, suppressed cytokine-induced CRP expression. Two of the synthetic derivatives of resveratrol, R3 and 7b, elicited a fiftyfold higher suppressive effect compared with resveratrol. The inhibitory effects of resveratrol and its derivatives on CRP expression were at the level of mRNA production. Investigation of signaling pathways showed that the cytokines induced the phosphorylation of p38 and p44/42 MAP kinases. Inhibitors of p38 and p44/42 mitogen-activated protein kinase (MAPK) activation inhibited CRP expression, implicating the involvement of both pathways in cytokine-induced CRP expression. These data revealed a previously unrecognized role of the p44/42 MAPK signaling pathway in CRP expression. Wine polyphenolics or the synthetic compounds of resveratrol did not affect cytokine-activated phosphorylation of these MAPKs. CONCLUSIONS: Wine phenolics inhibit CRP expression; however, to do so, they do not utilize the MAPK pathways.
Subject(s)
C-Reactive Protein/genetics , Phenols/isolation & purification , Phenols/pharmacology , Wine/analysis , Cell Line , Cytokines/pharmacology , Gene Expression/drug effects , Humans , MAP Kinase Signaling System/drug effects , Quercetin/isolation & purification , Quercetin/pharmacology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Resveratrol , Stilbenes/isolation & purification , Stilbenes/pharmacologyABSTRACT
BACKGROUND: Variants of recombinant factor VIIa (rFVIIa) with increased intrinsic activity have been developed to improve efficacy in the treatment of bleeding disorders in the future. The increased potency of FVIIa variants was demonstrated in limited in vitro and in vivo studies. However, further characterization of FVIIa variants is needed to evaluate their potential clinical use. METHODS: In the present study, we investigated the interactions of two FVIIa variants, FVIIa(Q) and FVIIa(DVQ), with plasma inhibitors, tissue factor pathway inhibitor (TFPI) and antithrombin (AT), and vascular endothelium. TF-FVIIa activity or its inhibition was measured directly in an amidolytic activity assay or for its ability to activate factor X. RESULTS: Both TFPI and AT/heparin inhibited the FVIIa variants more rapidly than the wild-type (WT) FVIIa in the absence of tissue factor (TF). In the presence of TF, TFPI, TFPI-Xa, and AT/heparin inhibited FVIIa and FVIIa variants at similar rates. Although the WT FVIIa failed to generate significant amounts of FXa on unperturbed endothelial cells, FVIIa variants, particularly FVIIa(DVQ), generated a substantial amount of FXa on unperturbed endothelium. Annexin V fully attenuated the FVIIa-mediated activation of FX on unperturbed endothelial cells. On stimulated human umbilical vein endothelial cells, FVIIa and FVIIa variants activated FX at similar rates, and annexin V blocked the activation only partly. AT/heparin and TFPI-Xa inhibited the activity of FVIIa and FVIIa variants bound to endothelial cell TF in a similar fashion. Interestingly, despite significant differences observed in FXa generation on unperturbed endothelium exposed to FVIIa and FVIIa analogs, no differences were found in thrombin generation when cells were exposed to FVIIa or FVIIa analogs under plasma mimicking conditions. CONCLUSION: Overall, the present data suggest that although FVIIa variants generate substantial amounts of FXa, they do not generate excessive thrombin on the surface of endothelium.
Subject(s)
Endothelium, Vascular/drug effects , Factor VII/chemistry , Factor VII/pharmacology , Annexin A5/pharmacology , Antithrombin III/pharmacology , Cells, Cultured , Endothelial Cells/drug effects , Endothelium, Vascular/cytology , Factor VII/antagonists & inhibitors , Factor VIIa , Factor Xa/biosynthesis , Humans , Lipoproteins/pharmacology , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/chemistry , Recombinant Proteins/pharmacology , Structure-Activity Relationship , Thrombin/biosynthesis , Thromboplastin/pharmacologyABSTRACT
The liver is believed to be the primary clearance organ for coagulation proteases, including factor VIIa (FVIIa). However, at present, clearance mechanisms for FVIIa in liver are unknown. To obtain information on the FVIIa clearance mechanism, we investigated the binding and internalization of FVIIa in liver cells using a human hepatoma cell line (HEPG2), and primary rat and human hepatocytes as cell models. 125I-FVIIa bound to HEPG2 cells in a time- and dose-dependent manner. Anti-tissue factor antibodies reduced the binding by about 25%, whereas 50-fold molar excess of unlabeled FVIIa had no effect. HEPG2 cells internalized FVIIa with a rate of 10 fmol 10(-5) cells h(-1). In contrast to HEPG2 cells, FVIIa binding to primary rat hepatocytes was completely independent of TF, and excess unlabeled FVIIa partly reduced the binding of 125I-FVIIa to rat hepatocytes. Further, compared with HEPG2 cells, three- to fourfold more FVIIa bound to rat primary hepatocytes, and the bound FVIIa was internalized at a faster rate. Similar FVIIa binding and internalization profiles were observed in primary human hepatocytes. Plasma inhibitors had no effect on FVIIa binding and internalization in hepatocytes. In contrast, annexin V, which binds to phosphatidylserine, blocked the binding and internalization. Consistent with this, binding of gla-domain-deleted FVIIa to hepatocytes was markedly diminished. In summary, the data presented herein reveal differences between HEPG2 cells and primary liver cells in FVIIa binding and internalization, and suggest that the rapid turnover of membrane and not a receptor-mediated endocytosis may be responsible for internalization of FVII/FVIIa in primary hepatocytes.
Subject(s)
Factor VIIa/metabolism , Hepatocytes/metabolism , Animals , Annexin A5/pharmacology , Cell Line , Cell Membrane/metabolism , Cells, Cultured , Hepatocytes/physiology , Humans , Phosphatidylserines , Protein Binding , Protein Transport , Rats , Thromboplastin/physiologyABSTRACT
The plasminogen activator/plasmin system is believed to play an important role in diverse pathophysiological processes, including wound healing, vascular remodeling and pulmonary fibrosis. Our recent studies show that plasmin upregulates the expression of Cyr61, a growth factor-like gene that has been implicated in cell proliferation and migration. In the present study, we investigated whether plasmin promotes fibroblast proliferation and, if so, determine the role of Cyr61 in the plasmin-induced response. Human lung fibroblasts were exposed to varying concentrations of plasmin and DNA synthesis was monitored by measuring the incorporation of 3H-thymidine into DNA. Plasmin increased DNA synthesis of fibroblasts in a dose-dependent manner. Protease-activated receptor-1 (PAR-1)-specific antibodies, but not PAR-2-specific antibodies, reduced the plasmin-induced DNA synthesis. Consistent with this, plasmin had no substantial effect on the DNA synthesis in PAR-1-deficient mouse fibroblasts. Plasmin activated both p38 and p44/42 MAPKs and specific inhibitors of these pathways inhibited the plasmin-induced DNA synthesis. Plasmin-induced increase in the DNA synthesis was completely abrogated by anti-Cyr61 antibodies. Interestingly, thrombin, which is a potent inducer of Cyr61, had only a minimal effect on fibroblast proliferation. Additional experiments suggested that plasmin cleaved cell/extracellular matrix-associated Cyr61 and the conditioned media from plasmin-treated cells could support the cell proliferation. Overall, these data suggest that plasmin promotes fibroblast proliferation by a novel pathway, involving two independent steps. In the first step, plasmin induces Cyr61 expression via activation of PAR-1, and in the second step, plasmin releases Cyr61 deposited in the extracellular matrix, thus making it accessible to act on cells.