Natural killer cells induce neutrophil extracellular trap formation in venous thrombosis.

Essentials Neutrophil extracellular traps (NETs) are generated during deep vein thrombosis (DVT). The role of interferon γ (IFNγ) and natural killer (NK) cells in NET formation was studied. IFNγ promote venous thrombosis through NET formation. NK cell depletion reduces DVT. SUMMARY: Background Neutrophils contribute to venous thrombosis through the release of neutrophil extracellular traps (NETs), but the mechanism triggering their formation remains unclear. In vitro data show that interferon (IFN)-γ induces the formation of NETs. Objectives To determine whether IFN-γ and the transcription factor T-box expressed on T cells (Tbet) promote venous thrombosis through neutrophil activation. Methods Venous thrombosis was induced by flow restriction in the inferior vena cava in IFN-γ-/- , Tbet-/- or wild-type (WT) mice. After 48 h, thrombus size was measured by the use of high-frequency ultrasound. NET formation was determined by immunofluorescence. Results and Conclusions Thrombus formation was reduced in Tbet-/- and IFN-γ-/- mice, suggesting that Tbet/IFN-γ-expressing cells are required for venous thrombosis. The number of NETs formed during thrombosis was significantly lower in Tbet-/- and IFN-γ-/- mice. NET formation was also decreased in WT mice treated with an IFN-γ-blocking antibody. Injection of recombinant IFN-γ into IFN-γ-/- mice rescued the phenotype. Natural killer (NK) cells were specifically depleted prior to venous thrombosis induction. NK cell depletion results in decreased NET formation and smaller thrombi, suggesting that NK cells are required for thrombus development. In depleted mice, adoptive transfer of WT NK cells induced a similar thrombosis burden as in WT mice. In contrast, adoptive transfer of IFN-γ -/- NK cells resulted in thrombi similar in size to those in depleted mice. In vitro, we showed that WT neutrophils released fewer NETs when they were cocultured with IFN-γ-/- NK cells. This study demonstrates that NK cell-dependent IFN-γ production is crucial for thrombus development by promoting the formation of NETs by neutrophils.

Growth arrest-specific 6 regulates thrombin-induced expression of vascular cell adhesion molecule-1 through forkhead box O1 in endothelial cells.

BACKGROUND: Growth arrest-specific 6 (Gas6)-deficient mice are protected against venous thromboembolism (VTE), suggesting a role for Gas6 in this disorder. We previously demonstrated that Gas6 induces forkhead box O1 (FoxO-1) phosphorylation through the phosphoinositide 3-kinase-Akt pathway. FoxO-1 regulates the expression of vascular cell adhesion molecule-1 (VCAM-1), a molecule that has been implicated in VTE. OBJECTIVES: To assess the role of FoxO-1 in Gas6-dependent VCAM-1 expression. METHODS: Thrombin was used to stimulate endothelial cells (ECs). Wild-type (WT) and Gas6(-/-) ECs were transfected with small interfering RNA targeting Axl or FoxO-1, a luciferase-coupled plasmid containing the FoxO-1 consensus sequence, and a phosphorylation-resistant FoxO-1 mutant, or treated with an Akt inhibitor. VCAM-1 mRNA expression was measured by real time-qPCR. VCAM-1 protein expression and FoxO-1 and Akt phosphorylation were assessed by western blot analysis. FoxO-1 localization was assessed by immunofluorescence. Adhesion of bone marrow mononuclear cells (BM-MCs) on ECs was assessed by fluorescence. RESULTS AND CONCLUSIONS: Thrombin induces both VCAM-1 expression and FoxO-1 phosphorylation and nuclear exclusion in WT ECs only. Silencing of FoxO-1 enhances VCAM-1 expression in both WT and Gas6(-/-) ECs. Inhibition of Akt or FoxO-1 phosphorylation prevents VCAM-1 expression in WT ECs. These data show that Gas6 induces FoxO-1 phosphorylation, leading to derepression of VCAM-1 expression. BM-MC-EC adhesion is increased by thrombin in WT ECs. BM-MC-EC adhesion is further increased when FoxO-1 is silenced, but decreased when FoxO-1 phosphorylation is inhibited. These results demonstrate that the Gas6-FoxO-1 signaling axis plays an important role in VCAM-1 expression in the context of VTE by promoting BM-MC-EC adhesion.

Gas6-induced tissue factor expression in endothelial cells is mediated through caveolin-1-enriched microdomains.

BACKGROUND: Gas6 has been shown to interact with Axl in endothelial cells and to induce several signaling pathways involved in cell survival and proliferation. However, the interaction of Gas6/Axl with lipid raft/caveolin-1 in endothelial cells and its role in thrombosis are unknown. OBJECTIVES: We tested whether Axl and/or caveolin-1 is involved in Gas6-induced Akt, ERK1/2, and c-Src activation leading to altered tissue factor expression in endothelial cells. METHODS: Gas6-treated endothelial cells were transfected with small interfering RNA (siRNA) for Axl, caveolin-1, c-Src, and Akt or treated with pharmacological inhibitors of c-Src and ERK1/2. Sucrose gradient centrifugation and confocal microscopy were used to study lipid raft/caveolin-1-enriched fractions. Akt, ERK1/2, p38, and c-Src activation was analyzed by Western blot analysis. Tissue factor expression was assessed by real-time quantitative polymerase chain reaction and immunofluorescence. RESULTS AND CONCLUSION: Gas6 induced Axl and c-Src localization into lipid raft/caveolin-1-enriched fractions. Gas6 increased the phosphorylation of Akt, ERK1/2, and c-Src but not p38. Using siRNA, we demonstrated that Axl is required for Akt, ERK1/2, and c-Src activation after Gas6 stimulation. siRNA for caveolin-1 blocked Gas6-induced phosphorylation of Akt, ERK1/2, and c-Src. c-Src downregulation inhibited Gas6-induced Akt but not ERK1/2 phosphorylation. Finally, Gas6 increased tissue factor mRNA and protein expression in endothelial cells. Tissue factor expression was blocked by siRNA for Axl, caveolin-1, or Akt as well as c-Src inhibition. These data demonstrate that the signaling pathway Gas6/Axl/caveolin-1/c-Src/Akt is required for tissue factor expression in endothelial cells, providing mechanistic insight into how Gas6 exerts its prothrombotic role in the vasculature.

In vivo monitoring of venous thrombosis in mice.

BACKGROUND: Venous thrombosis (VT) is an important cause of morbidity and mortality in clinical medicine. Animal models studying venous thrombosis are scarce and, in most cases, very crude and rely on sacrificing the animals to excise formed thrombi. Developing an in vivo murine model of venous thrombosis can be a powerful tool for studying venous thrombosis. OBJECTIVES: We sought to use a high-frequency ultrasound system (HFUS) to dynamically and non-invasively monitor thrombus formation in the inferior vena cava (IVC) of mice. METHODS: We developed a murine model of venous thrombosis using, for detection, the Vevo 770(®), a micro-imaging HFUS. Two different thrombosis models were used to generate thrombi in the IVC of C57Bl/6NCr mice: (i) ligation and (ii) application of ferric chloride (FeCl(3)). We then assessed venous thrombosis by HFUS. RESULTS: In both models, measurements of the clot pathologically correlated favorably with measurements acquired with HFUS. Thrombus develops less than an hour after ligation or FeCl(3) -induced injury of the IVC and the size of the clot increases over time for up to 24 h. Importantly, we demonstrate that HFUS can be used to monitor the effect of an anticoagulant such as dalteparin until complete resolution of the thrombus. CONCLUSIONS: These data show that HFUS assesses venous thrombosis in mice reliably and non-invasively. Developing a murine model of thrombosis using more accurate, and clinically more relevant, techniques such as ultrasonography, is a step towards a better understanding of the pathophysiology of venous thromboembolism.

Amphipathic peptides can act as an anticoagulant by competing with phospholipid membranes for blood coagulation factors.

An ideal amphipathic peptide (IAP), composed of simply lysine and leucine residues in a 1:2 ratio (K(7)L(15)), specifically prolongs in vitro coagulation assays that use phospholipids, such as the activated partial thromboplastin time (APTT). The main hypothesis of the present work is that IAP's anticoagulant effect occurs by competing with phospholipid membranes in in vitro coagulation reactions. We verified this hypothesis by employing different phospholipid-dependent coagulation assays, such as the APTT, the dilute prothrombin time (dPT) and the dilute Russell viper venom time (dRVVT) with both low and high amounts of phospholipids. We show that coagulation times are prolonged by IAP in a concentration-dependent manner, and that this prolongation is abrogated by adding excess phospholipid, demonstrating a phospholipid dependence for this inhibition. Using an ELISA-based binding assay, we show IAP inhibits the binding of one of the vitamin K-dependent coagulation factors, factor X, to phospholipid membranes. This is further confirmed with fluorescence spectroscopy, where the interaction of IAP and factor X is inhibited by phospholipid. In summary, this work demonstrates that IAP can act as an anticoagulant by impairing the interaction of coagulation factors with phospholipid membranes and provides a paradigm for the development of novel anticoagulants.

GAS6-induced signaling in human endothelial cells is mediated by FOXO1a.

BACKGROUND: Growth Arrest Specific gene product 6 (gas6) is a gamma-carboxylated protein that protects endothelial cells against apoptosis. Gas6 has previously been shown to induce phospatidyl-3-inositol-kinase (PI3K)/Akt signaling. Other studies have demonstrated a link between PI3K/Akt signaling and forkhead transcription factors in endothelial cells. OBJECTIVE: To test the hypothesis that gas6 promotes cell survival via a forkhead-dependent pathway. RESULTS AND CONCLUSIONS: Treatment of serum-starved human umbilical vein endothelial cells (HUVECs) with gas6 induced time-dependent phosphorylation and nuclear exclusion of FOXO1a. This effect was suppressed by the PI3K inhibitor wortmannin, demonstrating that FOXO1a phosphorylation is PI3-kinase dependent. Transduction of HUVECs with a phosphorylation-resistant form of FOXO1a [triple mutant (TM)-FOXO1a] abrogated the pro-survival effect of gas6 on serum-starved endothelial cells. Finally, treatment of serum-starved HUVECs with gas6 resulted in a reduction of FOXO1a transcriptional activity and downregulation of the pro-apoptotic gene, p27(kip1). Taken together, these findings suggest that gas6 protects endothelial cells from apoptosis by a mechanism that involves PI3K-Akt-dependent inactivation of FOXO1a.

Amphipathic helices support function of blood coagulation factor IXa.

Blood coagulation factor IXa gains proteolytic efficiency upon binding to a phospholipid membrane. We have found that an amphipathic, membrane-binding peptide from the C2 domain of factor VIII, fVIII(2303)(-23), enhances proteolytic efficiency of factor IXa in the absence of phospholipid membranes. This enhancement is the result of a reduction in the K(M) for the substrate, factor X, with little effect on the k(cat). Enhanced function requires interaction of the gamma-carboxyglutamic acid (Gla) domains of factor IXa and factor X since (i) a synthetic peptide comprising the Gla domain of factor IXa and antibodies directed to the Gla domain of factor IXa inhibit this acceleration, (ii) the acceleration is Ca(II) dependent, and (iii) conversion of Gla-domainless factor X is not affected by the presence of fVIII(2303)(-23). The effect of fVIII(2303)(-23) on factor IXa parallels the enhanced function produced by phosphatidylserine-containing bilayers, and fVIII(2303)(-23) does not further enhance function of factor IXa when phospholipid vesicles are present. The critical feature of fVIII(2303)(-23) is apparently its amphipathic helix-forming structure [Gilbert, G. E., and Baleja, J. D. (1995) Biochemistry 34, 3022-3031] because other alpha-helical peptides such as a homologous peptide from the C2 domain of factor V and melittin have similar effects. Diastereomeric analogues of fVIII(2303)(-23) and melittin, which have reduced helical content, do not support factor IXa activity. A truncated peptide of fVIII(2303)(-23) with three C-terminal residues deleted retains alpha-helical content but loses capacity to enhance factor X cleavage, suggesting that a minimum length of alpha-helix is required. Although these results probably do not illuminate the physiologic function of the factor VIII peptide corresponding to fVIII(2303)(-23), they demonstrate a novel, membrane-mimetic role of amphipathic helical peptides in supporting function of factor IXa.

The Gla domain of human prothrombin has a binding site for factor Va.

The role of the Gla domain of human prothrombin in interaction with the prothrombinase complex was studied using a peptide with the sequence of the first 46 residues of human prothrombin, PT-(1-46). Intrinsic fluorescence measurements showed that PT-(1-46) undergoes a conformational alteration upon binding calcium; this conclusion is supported by one-dimensional (1)H NMR spectroscopy, which identifies a change in the chemical environment of tryptophan 41. PT-(1-46) binds phospholipid membranes in a calcium-dependent manner with a K(d) of 0.5 microm and inhibits thrombin generation by the prothrombinase complex with a K(i) of 0.8 microm. In the absence of phospholipid membranes, PT-(1-46) inhibits thrombin generation by factor Xa in the presence but not absence of factor Va, suggesting that PT-(1-46) inhibits prothrombin-factor Va binding. The addition of factor Va to PT-(1-46) labeled with the fluorophore sulfosuccinimidyl-7-amino-4-methylcoumarin-3-acetic acid (PT-(1-46)AMCA) caused a concentration-dependent quenching of AMCA fluorescence, providing direct evidence of a PT-(1-46)-factor Va interaction. The K(d) for this interaction was 1.3 microm. These results indicate that the N-terminal Gla domain of human prothrombin is a functional unit that has a binding site for factor Va. The prothrombin Gla domain is important for interaction of the substrate with the prothrombinase complex.

Identification of the phospholipid binding site in the vitamin K-dependent blood coagulation protein factor IX.

The blood coagulation and regulatory proteins that contain gamma-carboxyglutamic acid are a part of a unique class of membrane binding proteins that require calcium for their interaction with cell membranes. Following protein biosynthesis, glutamic acids on these proteins are converted to gamma-carboxyglutamic acid (Gla) in a reaction that requires vitamin K as a cofactor. The vitamin K-dependent proteins undergo a conformational transition upon metal ion binding, but only calcium ions mediate protein-phospholipid interaction. To identify the site on Factor IX that is required for phospholipid binding, we have determined the three-dimensional structure of the Factor IX Gla domain bound to magnesium ions by NMR spectroscopy. By comparison of this structure to that of the Gla domain bound to calcium ions, we localize the membrane binding site to a highly ordered structure including residues 1-11 of the Gla domain. In the presence of Ca2+, Factor IX Gla domain peptides that contain the photoactivatable amino acid p-benzoyl-L-phenylalanine at positions 6 or 9 cross-link to phospholipid following irradiation, while peptides lacking this amino acid analog or with this analog at position 46 did not cross-link. These results indicate that the NH2 terminus of the Gla domain, specifically including leucine 6 and phenylalanine 9 in the hydrophobic patch, is the contact surface on Factor IX that interacts with the phospholipid bilayer.

A comparison of clinical criteria for the diagnosis of veno-occlusive disease of the liver after bone marrow transplantation.

Two major studies have established clinical criteria for the diagnosis of veno-occlusive disease of the liver (VOD) after bone marrow transplantation (BMT). McDonald and co-workers defined VOD as the onset of two of the following occurring before day 30 post-BMT: (a) jaundice (bilirubin > 27 mmol/l), (b) tender hepatomegaly, and (c) ascites or weight gain. In contrast, Jones and co-workers defined VOD as the onset, before day 21 post-BMT, of hyperbilirubinemia (bilirubin > 34 mmol/l) as well as two of the following: (a) hepatomegaly, (b) ascites, and (c) weight gain. We retrospectively reviewed the occurrence of VOD in 101 patients transplanted primarily for hematologic malignancies between 1979 and 1990, applying both sets of criteria. Of the 101 patients, eight (7.9%) fulfilled the Jones criteria whereas 32 (31.7%) had VOD according to the McDonald criteria (p < 0.001). Early mortality (prior to 50 days post-BMT) was 75% (6/8) in patients who fulfilled the Jones criteria but only 28.1% (9/32) in the McDonald group (p < 0.005). Overall, mortality in each group was 75% (6/8) and 65.6% (21/32), respectively. All of the six patients with VOD according to the Jones criteria who died had evidence of hepatic failure. Of the 32 patients who fulfilled the McDonald criteria, eight have also fulfilled the Jones criteria and are described above. Of the remaining 24 patients, 22 had complete resolution of VOD as defined by these criteria within 50 days of BMT, none developed hepatic failure, and 15 died.(ABSTRACT TRUNCATED AT 250 WORDS)