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Nigel Mackman

Publications and source records attributed to Nigel Mackman.

At least 19 recordsLinked to original sources
PubMed2026

A novel G13-RAPGEF2-RAP1 signaling pathway critical for platelet adhesion and aggregation.

Hemostasis and thrombosis are strongly dependent on the unique ability of platelets to rapidly activate integrin receptors and to firmly adhere to sites of injury under shear stress conditions. Central to integrin activation is the small GTPase RAP1, which itself is activated by guanine nucleotide exchange factors (GEFs). CalDAG-GEFI (RASGRP2) is the highest expressed and functionally dominant platelet RAP-GEF. However, a genome-wide association study also suggested a significant role for RAPGEF2 (PDZ-GEFI), a low-expressed RAP-GEF, in human platelet aggregation. Here, we used mice deficient in RAPGEF2 (megakaryocyte-specific, Rapgef2mKO), CalDAG-GEFI (Caldaggef1-/-), or both RAPGEF2 and CalDAG-GEFI (DKO) to characterize the contribution of RAPGEF2 signaling to platelet function, hemostasis, and thrombosis. RAPGEF2 protein was detected in murine and human platelets. Compared with control or Caldaggef1-/- platelets, both RAP1 activation and integrin αIIbβ3-mediated aggregation were significantly diminished in DKO platelets. When compared with controls, Rapgef2mKO platelets exhibited reduced integrin activation, a more reversible aggregation response, and impaired adhesion under conditions of shear stress ex vivo and in vivo. Mechanistic studies strongly suggest that RAPGEF2 operates downstream of receptors coupled to the heterotrimeric G protein G13 (GNA13), such as αIIbβ3 and the thromboxane receptor. Together, our studies provide genetic evidence that RAPGEF2 in platelets operates downstream of G13 as an important regulator of RAP1 signaling and integrin activation, especially under conditions of elevated shear stress. These findings markedly improve our understanding of G protein signaling and integrin function in platelets, with potential implications for the development of improved platelet-targeted therapies for cardiovascular disease.

Animals↗
PubMed2025

TGF-β Receptor-dependent Tissue Factor Release and Proteomic Profiling of Extracellular Vesicles from Mechanically Compressed Human Bronchial Epithelial Cells.

In asthma, tissue factor (TF) concentrations are elevated in the lung. In our previous studies using mechanically compressed human bronchial epithelial (HBE) cells, which are a well-defined in vitro model of bronchoconstriction during asthma exacerbations, we detected TF within extracellular vesicles (EVs) released from compressed HBE cells. Here, to better characterize the potential role of this mechanism in asthma, we tested the extent to which the transcriptional regulation of epithelial cell-derived TF varied between donors with and without asthma. Using RNA in situ hybridization, we detected epithelial expression of F3, the TF protein-encoding gene, in human airways. Next, to determine the role of TGF-β receptor (TGF-βR) in the regulation of TF, we exposed well-differentiated HBE cells to mechanical compression in the presence or absence of a pharmacological inhibitor of TGF-βR. Furthermore, to identify the protein cargo of EVs released from HBE cells, we used tandem mass tag mass spectrometry. Our findings revealed significantly higher F3 expression in the airways of patients with asthma compared with healthy control subjects. However, we observed no differences in F3 expression or TF release between asthmatic and nonasthmatic HBE cells, both at baseline and after compression. Mechanistically, compression-induced F3 expression in HBE cells depended on TGF-βR. Our proteomic analysis identified 22 differentially released proteins in EVs, with higher concentrations in compressed cells compared with controls. Gene Ontology analysis indicates that these proteins are involved in diverse biological processes, highlighting a potential role for epithelial cell-derived EVs during asthma exacerbations.

Humans↗
PubMed2006

Oncogenes, trousseau syndrome, and cancer-related changes in the coagulome of mice and humans.

Cancer is often associated with venous thrombosis, a phenomenon that was first described by Trousseau in 1865 (Trousseau syndrome). Recent studies have begun to explain how oncogenic events may deregulate the hemostatic system. For instance, activated oncogenes (K-ras, EGFR, PML-RARalpha, and MET) or inactivated tumor suppressors (e.g., 53 or PTEN) may increase the risk of thrombosis by inducing the expression of tissue factor, a potent procoagulant molecule, and plasminogen activator inhibitor-1, a fibrinolysis inhibitor. In a more complex clinical reality, transforming genes may often act in concert with numerous epigenetic factors, including hypoxia, inflammation, anticancer therapy, contact between blood and metastatic cancer cells, and emission of procoagulant vesicles from tumors and their stroma into the circulation. To add to mechanistic insights gained from mouse models, which may not fully phenocopy human Trousseau syndrome, we suggest that valuable clues to progression and thrombosis risk may be obtained by monitoring multiple hemostatic variables in cancer patients ("coagulomics").

Animals↗
PubMed2006

Signal-dependent splicing of tissue factor pre-mRNA modulates the thrombogenicity of human platelets.

Tissue factor (TF) is an essential cofactor for the activation of blood coagulation in vivo. We now report that quiescent human platelets express TF pre-mRNA and, in response to activation, splice this intronic-rich message into mature mRNA. Splicing of TF pre-mRNA is associated with increased TF protein expression, procoagulant activity, and accelerated formation of clots. Pre-mRNA splicing is controlled by Cdc2-like kinase (Clk)1, and interruption of Clk1 signaling prevents TF from accumulating in activated platelets. Elevated intravascular TF has been reported in a variety of prothrombotic diseases, but there is debate as to whether anucleate platelets-the key cellular effector of thrombosis-express TF. Our studies demonstrate that human platelets use Clk1-dependent splicing pathways to generate TF protein in response to cellular activation. We propose that platelet-derived TF contributes to the propagation and stabilization of a thrombus.

Blood Coagulation↗
PubMed2006

Tissue factor deficiency and PAR-1 deficiency are protective against renal ischemia reperfusion injury.

Ischemia/reperfusion (IR) injury is a leading cause of acute renal failure and an important contributor to allograft damage. Tissue factor (TF) is up-regulated during IR, and TF inhibition reduces renal injury. However, the underlying mechanisms by which TF contributes to injury have not been elucidated. We postulated that TF contributes to IR injury by production of coagulation proteases and subsequent signaling by protease activated receptor (PARs). We compared renal injury after 25 minutes of bilateral renal ischemia and varying periods of reperfusion in C57BL/6 mice, those expressing low levels of TF (low-TF), hirudin-treated C57BL/6, and mice lacking either PAR-1 or PAR-2. C57BL/6 mice developed severe renal failure and died within 48 hours of reperfusion. In contrast, low-TF, hirudin-treated C57BL/6, and PAR-1-/- mice were protected from renal failure and had reduced mortality, tubular injury, neutrophil accumulation, and lower levels of the chemokines KC and MIP-2. Importantly, PAR-1-/- mice had lower chemokine levels despite up-regulation of TF and fibrin deposition. In addition, treating PAR-1-/- mice with hirudin conferred no additional benefit. Somewhat surprisingly, PAR-2 deficiency did not protect from renal failure. These experiments indicate that increased TF activity after renal IR leads to increased CXC chemokine expression and subsequent neutrophil-mediated injury predominantly by thrombin-dependent PAR-1 signaling.

Animals↗
PubMed2006

Early growth response gene-1 regulates hypoxia-induced expression of tissue factor in glioblastoma multiforme through hypoxia-inducible factor-1-independent mechanisms.

Hypoxia strongly up-regulates tissue factor and promotes plasma clotting by glioblastoma multiforme, but transcriptional mechanisms remain undefined. Here, we investigated the potential roles of early growth response gene-1 (Egr-1), Sp1, nuclear factor-kappaB (NF-kappaB), activator protein-1 (AP-1), and hypoxia-inducible factor-1 (HIF-1) in the hypoxic regulation of tissue factor by glioblastoma multiforme cells in vitro. Hypoxia (1% O2) strongly induced Egr-1 mRNA within 1 hour and led to nuclear localization of Egr-1 protein. Using luciferase reporter plasmids in glioma cells, we found that hypoxia dramatically increased luciferase activity in cells with constructs containing Egr-1-binding sites but not in cells with constructs containing AP-1- or NF-kappaB-binding sites. Electrophoretic mobility shift assays revealed hypoxia-induced Egr-1, but not Sp1, binding to oligonucleotides containing the Egr-1/Sp1 motif of tissue factor gene promoter. Using an expression vector containing the minimal tissue factor promoter (-111 to +14 bp) and small interfering RNA (siRNA) directed at Egr-1 and Sp1 mRNAs, we found that Egr-1 was required for maximal hypoxic induction of promoter activity. Forced overexpression of Egr-1 but not Sp1 by cDNA transfection caused up-regulation of tissue factor in glioma cells under normoxia (21% O2), whereas siRNA directed at Egr-1 strongly attenuated hypoxia-induced tissue factor expression. To examine the effects of HIF-1alpha on tissue factor expression, we used glioma cells stably transfected with a HIF-1alpha siRNA expression vector and found that HIF-1alpha mRNA silencing did not affect tissue factor expression under hypoxia. We conclude that hypoxic up-regulation of tissue factor in glioblastoma multiforme cells depends largely on Egr-1 and is independent of HIF-1.

Cell Hypoxia↗
PubMed2006

Role of tissue factor in hemostasis and thrombosis.

Tissue factor (TF) is a transmembrane glycoprotein that functions as the primary cellular initiator of blood coagulation. Perivascular cells express TF and provide a hemostatic barrier to limit hemorrhage after vessel injury. In addition, TF is expressed in a tissue-specific manner with high levels in vital organs, such as the heart and lung. TF expression in these tissues may provide additional hemostatic protection from mechanical injury to blood vessels. Recent studies have also detected TF in the blood. This circulating TF is present in the form of microparticles (MPs), which are membrane vesicles shed from cells, and possibly platelets. At present, the cell types that contribute to this pool of TF-positive MPs have not been fully defined. Monocytes, endothelial cells and platelets are the most likely sources of this circulating TF. However, TF-positive MPs represent only a minor subset of circulating MPs. Importantly, TF-negative MPs also possess procoagulant activity. In various diseases, such as sepsis and cancer, TF is expressed by vascular cells and this leads to thrombosis. Levels of circulating TF are also elevated in these diseases and may contribute to thrombosis. Recent studies have analyzed the role of TF-positive MPs in thrombus propagation using different in vivo models. Circulating TF was found to contribute to thrombosis in some models but not others. Inhibition of TF activity in patients with TF expression in vascular cells and with elevated levels of circulating TF may decrease thrombosis associated with a variety of diseases.

Animals↗
PubMed2006

Genetic susceptibility to thrombosis.

Thrombosis is associated with atherosclerosis, sepsis, cancer, and numerous other inflammatory diseases. Complications of thrombosis, such as myocardial infarction, stroke, and venous thromboembolism, contribute significantly to morbidity and mortality. Susceptibility to thrombosis is conferred by both genetic and environmental factors. Tissue factor is the primary cellular initiator of blood coagulation and is a major contributor to thrombosis. In this review, we discuss the association between various polymorphisms and the risk for thrombosis.

Cytokines↗
PubMed2006

Tissue factor in hemostasis and thrombosis.

Tissue factor (TF) plays an essential role in hemostasis. The tissue-specific pattern of TF expression is consistent with additional hemostatic protection in vital organs. An aberrant expression of TF within the vasculature occurs in a variety of diseases, including atherosclerosis, cancer, and sepsis. TF expression in these diseases is associated with thrombotic events. Future therapeutic strategies may prove beneficial in the treatment of thrombosis. However, these strategies should be designed to avoid compromising hemostasis.

Animals↗
PubMed2006

A non-anticoagulant synthetic pentasaccharide reduces inflammation in a murine model of kidney ischemia-reperfusion injury.

Fondaparinux is a synthetic pentasaccharide that selectively inhibits factor Xa (FXa) in an antithrombin-dependent fashion. This newly developed anticoagulant is used in the prevention and treatment of venous thromboembolism. Recently, we showed that fondaparinux reduces inflammation and protects the kidney from ischemia-reperfusion (I/R) injury. However, the relative contributions of the anticoagulant and anti-inflammatory activities of fondaparinux to the observed protection is unknown. To address this, we chemically modified fondaparinux to abolish its affinity for antithrombin and analyzed the effect of this non-anticoagulant (NAC)-pentasaccharide on binding of U937 cells to P-selectin in vitro and on inflammation in a murine model of kidney I/R injury. NAC-pentasaccharide was as effective as fondaparinux at inhibiting the binding of U937 cells to P-selectin. In addition, NAC-pentasaccharide significantly reduced IL-6 and MIP-2 expression and injury in the kidney I/R model. These findings indicate that the anti-inflammatory activity of fondaparinux can be dissociated from its anticoagulant activity and that NAC-pentasaccharide is protective in kidney I/R injury.

Animals↗
PubMed2005

Atherosclerosis in mice is not affected by a reduction in tissue factor expression.

OBJECTIVE: To determine whether tissue factor (TF) contributes to the progression of atherosclerotic lesions in mice. METHODS AND RESULTS: We determined the effect of a 50% reduction of TF levels in all cells on atherosclerosis in apolipoprotein E-deficient (apoE(-/-)) mice. No differences were observed in the extent of atherosclerosis in apoE(-/-)/TF(+/+) and apoE(-/-)/TF(+/-) mice fed regular chow for 34 weeks. Atherosclerosis could not be analyzed in apoE(-/-) mice expressing low levels of TF because of premature death of these mice. Macrophages are a major source of TF in atherosclerotic plaques. Therefore, in a second series of experiments, we investigated the effect on atherosclerosis of selectively reducing hematopoietic cell-derived TF by transplanting bone marrow from mice expressing low levels of TF into low-density lipoprotein receptor deficient (LDLR(-/-)) mice. Atherosclerosis within the arterial tree and aortic root were similar in LDLR(-/-) mice with low-TF bone marrow compared with control bone marrow (TF(+/+) or TF(+/-)) after 4 and 16 weeks on an atherogenic diet. Furthermore, the cellular composition of the aortic root lesions was similar between the 2 groups. CONCLUSIONS: Our data indicate that either a 50% reduction of TF in all cells or a selective reduction in hematopoietic cell-derived TF does not affect the development of atherosclerotic lesions in mice.

Animals↗
PubMed2005

Lipopolysaccharide-induced heme oxygenase-1 expression in human monocytic cells is mediated via Nrf2 and protein kinase C.

Monocytes play a key role in mobilization of the immune response during sepsis. In response to LPS, monocytes produce both proinflammatory mediators and regulatory proteins that counteract the inflammation and oxidative stress. In murine macrophages, LPS stimulates expression of heme oxygenase 1 (HO-1), a cytoprotective enzyme that catalyzes the degradation of heme. The HO-1 5'-untranslated region, similarly to other cytoprotective genes, contains antioxidant-response elements (AREs) that can bind the transcription factor NF-E2-related factor 2 (Nrf2). At present, the role of Nrf2 in LPS-induced HO-1 expression in monocytic cells has not been investigated. In this study, LPS induced HO-1 mRNA and protein expression in human monocytes and THP-1 cells. Nrf2 translocated from the cytosol to the nucleus in response to LPS and bound to the ARE site in the human HO-1 promoter. In addition, a dominant negative Nrf2 mutant inhibited LPS-induced HO-1 mRNA expression but not TNF-alpha mRNA expression in THP-1 cells. Ro-31-8220, a pan-protein kinase C (PKC) inhibitor, and Go6976, a classical PKC inhibitor, blunted LPS-induced HO-1 mRNA expression in monocytes and THP-1 cells. Both PKC inhibitors also blocked LPS-induced Nrf2 binding to the ARE. These results indicate that LPS-induced HO-1 expression in human monocytic cells requires Nrf2 and PKC.

Active Transport, Cell Nucleus↗
PubMed2005

Tissue-specific hemostasis in mice.

Blood coagulation is essential to maintain hemostasis in organisms with a vascular network. Formation of a fibrin-rich clot at a site of vessel injury is a highly complex process that is orchestrated by the coagulation protease cascade. This cascade is regulated by 3 major anticoagulant pathways. Removal of a clot is mediated by the fibrinolytic system. Defects in the regulation of clot formation lead to either hemorrhage or thrombosis. Tissue factor, the primary cellular initiator of blood coagulation, is a transmembrane receptor that is expressed in a tissue-specific manner. The 3 major anticoagulants are tissue factor pathway inhibitor, antithrombin, and protein C, the latter requiring a transmembrane receptor called thrombomodulin for its activation. Tissue factor pathway inhibitor and thrombomodulin are expressed by endothelial cells in a tissue-specific manner, whereas antithrombin and protein C circulate in the plasma. Fibrinolysis requires the activation of plasminogen to plasmin, which is mediated by tissue-type plasminogen activator and urokinase-type plasminogen activator. Interestingly, tissue-type plasminogen activator is expressed by a subset of endothelial cells of discrete size and location. These observations, together with the phenotypes of mice that have defects in the procoagulant, anticoagulant, and fibrinolytic pathways, indicate that hemostasis is regulated in a tissue-specific manner.

Animals↗
PubMed2005

Interleukin-10 suppresses tissue factor expression in lipopolysaccharide-stimulated macrophages via inhibition of Egr-1 and a serum response element/MEK-ERK1/2 pathway.

Atherosclerosis is considered to be an inflammatory disease. Tissue factor (TF), a prothrombotic molecule expressed by various cell types within atherosclerotic plaques, is thought to play an essential role in thrombus formation after atherosclerotic plaque rupture. Recent studies suggest that the antiinflammatory cytokine interleukin-10 (IL-10) has many antiatherosclerotic properties. Therefore, the effects of IL-10 on TF expression in response to inflammation were investigated. Mouse macrophages were stimulated with lipopolysaccharide (LPS) in the presence or absence of IL-10. Pretreatment with IL-10 resulted in a 50% decrease in TF mRNA expression and TF promoter activity. Binding of early growth response gene-1 (Egr-1) to the consensus DNA sequence, a key transcriptional activator of TF expression in response to inflammation, and the expression of Egr-1 mRNA were also inhibited by IL-10. This inhibition was independent of the induction of suppressor of cytokine signaling protein-3 by IL-10. Macrophages that had been transfected with luciferase reporter constructs containing the murine Egr-1 5'-flanking sequence exhibited reduced reporter gene activity in response to LPS stimulation with IL-10 pretreatment. Studies with deletion constructs of the Egr-1 promoter identified the proximal serum response element SRE3 as a potential regulatory site for the IL-10 mediated suppression of Egr-1 expression. Furthermore, activation of the upstream signal-transduction elements, such as mitogen-activated protein kinase kinase (MEK) 1/2, extracellular signal-regulated kinase 1/2, and Elk-1 were also inhibited by IL-10 pretreatment. Taken together, these results demonstrate a pathway for the IL-10 mediated inhibition of TF expression during inflammation and may explain the antiatherosclerotic effects of IL-10.

Animals↗
PubMed2005

Regulation of tissue factor--mediated initiation of the coagulation cascade by cell surface grp78.

OBJECTIVE: To test the hypothesis that Grp78 negatively regulates cell surface tissue factor (TF) procoagulant activity and whether this is mediated by physical interaction. METHODS AND RESULTS: Biopanning with phage-displayed peptidyl libraries has identified peptide probes that bind selectively in vivo to the surface of atherosclerotic plaque endothelium. The highest affinity peptide, EKO130, binds 78-kDa glucose regulated protein (Grp78). Grp78 participates in numerous pathological processes, including the regulation of the coagulation cascade, but the mechanism of Grp78 regulation of coagulation is unknown. To characterize this function, we analyzed the effect of Grp78 on TF-mediated procoagulant activity on murine brain endothelial cells (bEND.3) and macrophage-like (RAW) cells, which are relevant in mediation of atherothrombosis. We show that Grp78 is present on the surface of endothelium and monocyte/macrophage-like cells in atherosclerotic lesions. Inhibition of Grp78 resulted in increased procoagulant activity. We demonstrate that Grp78 negatively regulates procoagulant activity by interacting physically with the TF extracellular domain on the cell surface. CONCLUSIONS: The evidence indicates that Grp78 negatively regulates TF functional activity via direct binding to and functional inhibition of TF. Identification of the mechanism by which Grp78 regulates TF function may advance insight into the pathobiology of atherosclerosis and associated arterial thrombosis.

Animals↗
PubMed2005

Potent inhibition of thrombin with a monoclonal antibody against tissue factor (Sunol-cH36): results of the PROXIMATE-TIMI 27 trial.

AIMS: Exposure of tissue factor (TF) is a critical proximal step in the pathogenesis of acute coronary syndromes. Sunol-cH36, a chimaeric monoclonal antibody to TF, blocks binding of factor X to the TF:VIIa complex. This report describes the first completed trial of Sunol-cH36 in humans. METHODS AND RESULTS: We assessed the safety and pharmacokinetics of Sunol-cH36 in an open-label, dose-escalating trial among subjects with stable coronary artery disease. The safety analysis included all adverse events with a focus on overt or occult bleeding. Five doses of Sunol-cH36 (0.03, 0.06, 0.08, 0.1, 0.3 mg/kg) were administered as a single intravenous bolus to 26 subjects (three to eight subjects per dose tier). No major bleeding (> or =2 g/dL haemoglobin decline) occurred. Spontaneous minor bleeding was observed with a dose-related pattern. Notably, the majority of spontaneous bleeding episodes were clinically consistent with platelet-mediated bleeding (e.g. gum, tongue) without thrombocytopenia. The median terminal half-life was 72.2 (25th, 75th: 28.4, 72.5) h. CONCLUSION: Sunol-cH36 exhibited dose-dependent anticoagulant effects. We postulate that the mucosal bleeding observed with this potent inhibitor of thrombin generation may reflect antiplatelet effects resulting from networking between the coagulation cascade and platelet pathways that could prove clinically relevant with this novel class of anticoagulants.

Adult↗
PubMed2005

Postprandial elevation of tissue factor antigen in the blood of healthy adults.

Atherosclerosis is a dynamic disease involving lipid metabolism, inflammation and thrombosis. A key factor in thrombosis is tissue factor, a small transmembrane glycoprotein. Tissue factor binds FactorVIIa, and this complex converts Factor X to Factor Xa, leading to thrombin generation and fibrin formation. Inhibition of this pathway is by tissue factor pathway inhibitor (TFPI). Tissue factor is found sequestered within atherosclerotic plaques, and plaque rupture allows tissue factor exposure to the circulation, leading to formation of a thrombus. Tissue factor is also associated with membrane microparticles in the circulation, most likely released from monocytes activated by an inflammatory event. We hypothesize that consumption of a typical western diet that is moderate in fat content leads to elevated levels of circulating tissue factor that may act as a marker of a prothrombotic state. Healthy volunteers, aged 18-55, consumed a moderate (40%) fat meal, with blood taken before and 3.5 and 6 h after the meal. Plasma was isolated and assayed for plasma triglycerides, tissue factor, thrombin antithrombin (TAT) complexes, TFPI and TNFalpha. The levels of circulating tissue factor increased 56% (from 78 pg/ml to 120 pg/ml) 3.5 h after the meal. Levels decreased, but had not returned to baseline 6 h postprandially. No significant differences in TAT, TFPI and TNFa levels were observed postprandially. These results demonstrate increased tissue factor levels in individuals who consumed a moderate fat diet. This suggests that the typical western diet may play a larger role in cardiovascular disease than merely altering lipid profiles.

Adolescent↗