Journal of Thrombosis and Haemostasis, 12: 395–408 DOI: 10.1111/jth.12481 ORIGINAL ARTICLE Gas6-induced tissue factor expression in endothelial cells is mediated through caveolin-1–enriched microdomains S. LAURANCE,* M. N. AGHOURIAN,* Z. JIVA LILA,* C. A. LEMARI E* and M . D . B L O S T E I N * † *Lady Davis Institute for Medical Research, McGill University; and †Department of Medicine, Jewish General Hospital, McGill University, Montreal, QC, Canada To cite this article: Laurance S, Aghourian MN, Jiva Lila Z, Lemarie CA, Blostein MD. Gas6-induced tissue factor expression in endothelial cells is mediated through caveolin-1–enriched microdomains. J Thromb Haemost 2014; 12: 395–408. Summary. 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 cSrc 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 cSrc. 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 facCorrespondence: Mark D. Blostein, Jewish General Hospital, 3755 C^ ote-Ste-Catherine Rd., Montreal, QC H3T 1E2, Canada. Tel.: +1 514 340 8222 ext 8797; fax: +1 514 340 8281. E-mail: [email protected] Received 12 June 2013 Manuscript handled by: W. Ruf Final decision: P. H. Reitsma, 1 December 2013 © 2013 International Society on Thrombosis and Haemostasis tor expression in endothelial cells, providing mechanistic insight into how Gas6 exerts its prothrombotic role in the vasculature. Keywords: axl receptor tyrosine kinase; caveolin-1; c-src kinase; growth arrest–specific protein 6; tissue factor. Introduction Gas6 is homologous to the blood coagulation protein protein S and is a member of the vitamin K–dependent family of proteins that includes the procoagulant factors II (FII), FVII, FIX, and FX; the anticoagulant proteins C and S; and protein Z. Despite this structural homology, the role of Gas6 in coagulation was unclear until recently. In vivo, Gas6-deficient mice are protected from lethal thromboembolism, suggesting a prothrombotic role for Gas6 [1]. This phenotype partially results from a loss of platelet signaling with consequent clot instability without excessive blood loss. We recently demonstrated in vivo that Gas6 induces tissue factor (TF) expression in the inferior vena cava, which leads to thrombosis. In vitro, we established that Gas6 is required for thrombininduced TF expression and activity in endothelial cells [2]. However, the signaling pathway involved in Gas6induced TF expression in endothelial cells remains unclear. Gas6 is a ligand for the TAM family of receptors, which is composed of three members: Tyro3, Axl, and Mer [3,4]. Axl has the highest affinity for Gas6, followed by Tyro3 and Mer [5]. These receptors belong to the large family of type I transmembrane receptor tyrosine kinases. Their extracellular domain contains two immunoglobulin-like domains, which are characteristic of adhesion molecules, followed by two fibronectin type III– like motifs. Their cytoplasmic tail contains a tyrosine kinase domain. In platelets, Gas6 receptors were shown to regulate the outside-in signaling via the aIIbb3 integrin and to regulate granule secretion [6]. The binding of 396 S. Laurance et al Enriched A 1 Fraction 2 3 4 5 6 7 8 9 10 Gas 6 (min) 0 5 Cav-1 10 0 5 Axl Axl expression % of total fractions 10 35 * 30 25 20 * 15 10 5 0 1 2 3 4 5 6 7 8 9 10 Fraction number Gas 6 0 min B DAPI Cav-1 Gas 6 5 min Axl Gas 6 10 min Cav-1 + Axl Colocalization (white spot) Gas 6 0 min Gas 6 5 min Gas 6 10 min © 2013 International Society on Thrombosis and Haemostasis Gas6-induced endothelial tissue factor expression 397 Fig. 1. Gas6 induces Axl trafficking into lipid raft/caveolin-1–enriched fractions. (A) Representative Western blots of caveolin-1 and Axl distribution after human umbilical vein endothelial cell (HUVEC) fractionation via sucrose gradient centrifugation. Caveolin-1 protein is mainly found in fractions 3–5, thus identifying the caveolin-1–enriched microdomains of HUVEC membranes. Gas6 treatment has no effect on caveolin-1–enriched microdomains. In resting HUVECs, Axl is mainly found in fractions 6–8. Gas6 treatment induces Axl localization in the lipid raft/caveolin-1–rich fractions (3–5). Axl protein level in each fraction is quantified and expressed relative to the sum of the intensity of the total fraction (n = 5; *P < 0.05 nontreated vs. 5 min of Gas6 or nontreated vs. 10 min of Gas6). (B) Axl and caveolin-1 localization was assessed through confocal microscopy (magnification 960). The colocalization sites are highlighted using ImageJ software and represent pixels where caveolin-1 and Axl are present in the same proportion (n = 5). Gas6 to its receptors activates phosphoinositide 3-kinase (PI3K) and stimulates tyrosine phosphorylation of b3 integrin [6]. In vivo, mice lacking the TAM receptors have impaired thrombosis, partially due to a platelet signaling defect [7]. In endothelial cells, we previously demonstrated that PI3K/Akt is activated downstream of Axl [8]. Gas6/Axl interactions promoted endothelial cell survival through Akt phosphorylation, nuclear factor (NF)jB activation, increased Bcl-2 protein expression, and a reduction in caspase-3 activation [8]. Other signaling molecules are involved in Gas6 signaling, such as the mitogen activating protein (MAP) kinase, ERK1/2, the stressactivated protein kinase/c-Jun NH2-terminal kinase (JNK/SAPK), p38, and the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathways [9–12]. The regulation of various signaling pathways induced by activation of receptor tyrosine kinases is coordinated by colocalization of the receptor and its downstream effectors into lipid raft/caveolin-1–enriched microdomains [13]. Caveolin-1–enriched microdomains are subsets of lipid rafts and are flask-shaped cell membrane invaginations containing the major structural protein caveolin-1 [13,14]. Lipid rafts compartmentalize various signaling receptors including receptor tyrosine kinases such as the epidermal growth factor receptor (EGFR), the plateletderived growth factor receptor (PDGFR), the insulin receptor (IR), and the vascular endothelial growth factor receptor 2 (VEGFR2) [15,16]. Lipid raft/caveolin-1– enriched microdomains also regulate signaling molecules such as Src-family tyrosine kinases, Ha-Ras, G protein asubunits, endothelial nitric oxide synthase, and protein kinase C, among others [17]. The functional significance of lipid raft/caveolin1–enriched microdomains as a potential platform for Gas6/ Axl signaling is still unexplored. Thus, we hypothesize that the localization of Axl to caveolin-1–enriched microdomains is required for activation of downstream signaling pathways involved in the prothrombotic effects of Gas6. Materials and methods Cell culture and cell transfection Human umbilical vein endothelial cells (HUVECs; Promocell, Heidelberg, Germany) were grown in endothelial cell basal medium (EBM-2) supplemented with an endothelial © 2013 International Society on Thrombosis and Haemostasis cell bullet kit (EGM-2) (Lonza, Basel, Switzerland). HUVECs, between passage 3 and 6, were cultured in tissue culture dishes coated with 0.1% gelatin and maintained at 37 °C in a humidified incubator at 5% CO2. HUVECs were treated for 5 or 10 min or 4 or 6 h with human recombinant Gas6 (100 ng mL 1) produced as previously described [4] or with lipopolysaccharides (LPS) (1 lg mL 1). When indicated, HUVECs were pretreated for 30 min with pharmacological inhibitors PD98059 (ERK1/2 inhibitor, 5 lmol L 1; Selleck Chemicals, Houston, TX, USA) or PP2 (c-Src inhibitor, 10 lmol L 1; Santa Cruz Biotechnology, Dallas, TX, USA). HUVECs were also transfected with a negative control small interfering (si)RNA or siRNA targeting Axl, caveolin-1, c-Src, or Akt (Santa Cruz Biotechnology) following the manufacturer’s instructions. Briefly, HUVECs at 70% of confluence were transfected with 150 pmol of each siRNA mixed with 200 lL of transfection medium (Santa Cruz) and 40 lL of transfection reagent (Santa Cruz). After 48 h, HUVECs were treated by Gas6 as decribed here earlier. Efficiency of the knockdown by siRNA was measured with Western blot analysis. Lipid raft/caveolin-1–enriched microdomain isolation A detergent-free method was used to isolate caveolin-1– enriched microdomains. Gas6-treated HUVECs were lysed in Na2CO3 (500 mmol L 1, pH 11.0). Lysates were homogenized using a dounce homogenizer and a 23-gauge needle. One milliliter of the homogenate was mixed with 1 mL of 90% sucrose in 2-(N-morpholino)ethanesulfonic acid (MES)-buffered saline (MBS) (25 mmol L 1 MES and 150 mmol L 1 NaCl, pH 6.5) and overlayered with 2 mL of 35% sucrose and 1 mL of 5% sucrose in MBS/ Na2CO3 (MBS and 250 mmol L 1 Na2CO3). After centrifugation at 240 000 9 g for 20 h (rotor SW55Ti; Beckman Coulter, Pasadena, CA, USA), 10 fractions of 1 mL were collected. An aliquot of 400 lL from each fraction was subjected to electrophoresis on a 10% polyacrylamide-SDS gel and transferred to nitrocellulose membranes. Membranes were incubated overnight with goat antihuman Axl (R&D Systems, Minneapolis, MN, USA), mouse antihuman c-Src (Santa Cruz Biotechnology), and mouse antihuman caveolin-1 (BD Biosciences, Franklin Lake, NJ, USA) antibodies. An enhanced chemiluminescence system was used as the detection method (Perkin Elmer, Waltham, MA, USA). 398 S. Laurance et al Western blot analysis For Western blot analysis, HUVECs were homogenized in lysis buffer containing 1% Nonidet P40, 0.5% deoxycholic acid (sodium salt), 0.1% SDS, 1% Triton, and antiproteases inhibitor cocktail. Twenty-five micrograms of protein was loaded onto a 10% polyacrylamide-SDS gel, subjected to electrophoresis, and transferred to nitrocellulose membranes. Membranes were incubated overnight with rabbit antihuman phospho-p38, rabbit antihuman p38, rabbit antihuman phospho-ERK1/2, rabbit antihuman ERK1/2, rabbit antihuman phospho-Akt, rabbit antihuman Akt (Cell Signaling Technology, Beverly, MA, USA), mouse antihuman phospho–c-Src, mouse antihuman c-Src (Santa Cruz Biotechnology), goat antihuman Axl (R&D Systems), and mouse antihuman caveolin-1 (BD Biosciences) antibodies. A rabbit antihuman b-actin antibody (Sigma Aldrich, St. Louis, MO, USA) was used to probe the blot as a loading control. An enhanced chemiluminescence system was used as the detection method (Perkin Elmer). RNA isolation, reverse transcription, and quantitative real-time polymerase chain reaction TF expression was evaluated in HUVECs by quantitative real-time reverse transcription polymerase chain reaction (PCR) (qRT-PCR). Total RNA was extracted from cultured cells using a commercial kit following the manufacturer’s instructions (Genaid; Froggabio, North York, Ontario, Canada). One microgram of total RNA was reverse-transcribed per the manufacturer’s instructions (Quanta; VWR, Radnor, PA, USA). The SYBRgreen intercalant was used for amplification detection with the Fast SYBRgreen master mix (Applied Biosystems, Foster City, CA, USA). Primers were designed using the Primer Express Software (Applied Biosystems). The TATA box binding protein (TBP) gene was used for normalization. Fold change were calculated using the DCt method, and results were expressed as fold change SD of five independent experiments. Primer sequences for human TF are: forward TF: 5′-ATTCCCTCCCGAACAGTTAACC-3′ and reverse TF: 5′-GCTCCAATGATGTAGAATATTTC TCTGA-3′. Immunofluorescence microscopy After Gas6 treatment, HUVECs were fixed with 1% paraformaldehyde, permeabilized with 0.5% Triton, and blocked with 10% BSA. HUVECs were then incubated with goat antihuman Axl, rabbit antihuman c-Src, mouse antihuman TF (Santa Cruz Biotechnology), or mouse antihuman caveolin-1 (BD Biosciences) antibodies. Finally, appropriate Alexa Fluor secondary antibodies were used for detection (antigoat 488, antirabbit 488, antimouse 555, or antimouse 598; Molecular Probes, Eugene, OR, USA). Images were acquired using the Fluoview FV10i micro- scope (Olympus, Shinjuku, Tokyo, Japan). Positive colocalization staining for Axl/Cav-1 or c-Src/Cav-1 or Axl/cSrc was assessed with ImageJ software (National Institutes of Health, Bethesda, MD, USA). The colocalization analysis plugins (http://www.uhnresearch.ca/facilities/wcif/imagej/) were used to highlight (in white) pixels where both signals are present in equal proportion. TF-positive staining was quantified using ImageJ software and normalized by DAPI signal. Statistical analysis Data are presented as mean SE values from multiple independent experiments. Within-group differences were assessed by one-way analysis of variance followed by a post hoc Student–Newman–Keuls test. A value of P < 0.05 was considered statistically significant. Results Gas6 promotes Axl localization into lipid raft/caveolin-1– enriched microdomains We analyzed Axl trafficking within lipid raft/caveolin-1– enriched microdomains after Gas6 treatment. Lipid raft/caveolin-1–enriched microdomains were isolated in HUVECs using sucrose gradient centrifugation. Ten fractions were collected and analyzed by Western blotting. Caveolin-1 was concentrated in fractions 3–5, which correspond to the lipid raft/caveolin-1 fractions (Fig. 1A). Gas6 treatment had no effect on caveolin-1 localization (Fig. 1A). In resting HUVECs, Axl was mainly localized in fractions 6–10 that do not colocalize with caveolin-1. Gas6 treatment induced Axl localization to the caveolin-1 enriched fractions (3–5) at 5 min and to a lesser extent at 10 min (Fig. 1A). The association between Axl and caveolin-1 was also studied using confocal microscopy. Colocalization was analyzed by using ImageJ software and is represented by white spots. In resting cells, Axl and caveolin-1 are localized at different sites of the plasma membrane. Gas6 treatment induced Axl and caveolin-1 colocalization after 5 and 10 min (Fig. 1B). Gas6-induced Akt and ERK1/2 phosphorylation requires Axl and caveolin-1 Gas6 has been shown to induce several signaling pathways, including Akt and the MAP kinases [8,18]. We found that Gas6 induces the phosphorylation of Akt and ERK1/2 but not of p38 (Fig. 2A). HUVECs were then transfected with Axl or caveolin-1 siRNA to establish their respective contribution to Gas6-induced Akt and ERK1/2 phosphorylation. After 48 h of transfection with Axl siRNA, Axl protein levels were reduced by 85% in HUVECs without any changes in caveolin-1 expression © 2013 International Society on Thrombosis and Haemostasis Gas6-induced endothelial tissue factor expression 399 A Control siRNA B Gas6 Gas6 0 0 5 10 5 10 Gas6 Gas6 0 Axl siRNA 5 10 min 0 5 Cav-1 siRNA Gas6 10 0 5 Gas6 10 0 5 10 min Axl PAkt PERK1/2 Pp38 Cav-1 Akt ERK1/2 actin * * 2 * * 1.5 1 0.5 0 0 5 10 5 10 Non-treated C Control siRNA Axl siRNA Gas6 0 5 10 0 0 5 10 min Gas6 Gas6 5 10 0 5 10 1 0.5 0 Control siRNA 0 Akt ERK1/2 2 3.5 ERK1/2 phosphorylation (compared to non-treated) PERK1/2 * 1 0.5 0 0 0 5 10 Control siRNA 5 10 Axl siRNA Non-treated 0 Cav-1 siRNA Gas6 0.5 5 10 0 0 5 10 0 5 10 0 5 10 min Control Axl Cav-1 siRNA siRNA siRNA Cav-1 siRNA Gas6 Gas6 0 5 10 0 0 0 5 10 min 5 10 min * 3 2.5 2 1.5 1 0.5 0 5 10 min 1 Axl siRNA Gas6 min PAkt 1.5 1.5 0 5 10 0 5 10 0 5 10 min Control Axl Cav-1 siRNA siRNA siRNA D Cav-1 siRNA Gas6 1.5 Cav-1 protein level (compared control siRNA) 3 2.5 0 Akt phosphorylation (compared to non-treated) P38 Axl protein level (compared to control siRNA) phosphorylation (compared to non-treated) 4 3.5 0 5 10 Control siRNA 5 10 Axl siRNA Non-treated Cav-1 siRNA Gas6 Fig. 2. Axl and caveolin-1 are required for Gas6-induced Akt and ERK1/2 phosphorylation. (A) In HUVECs treated with Gas6 for 5 and 10 min at 100 ng mL 1, representative Western blots of Akt, ERK1/2, and p38 phosphorylation with densitometric quantification are shown. Gas6 treatment induces Akt and ERK1/2 activation at 5 and 10 min. p38 phosphorylation is not induced by Gas6 (n = 5; *P < 0.05). (B) Representative Western blots of Axl and caveolin-1 protein levels in HUVECs transfected by either control or Axl or caveolin-1 siRNA for 48 h. Axl and caveolin-1 siRNA successfully reduces, respectively, Axl and caveolin-1 protein by 95% (n = 5). Axl and caveolin-1 protein levels were evaluated by densitometric analysis. (C) Akt and (D) ERK1/2 phosphorylation were evaluated by Western blotting and quantified after 48 h in the presence of control, Axl, or caveolin-1 siRNA in HUVECs treated with Gas6 for 5 and 10 min. Akt and ERK1/2 phosphorylation is significantly increased with Gas6 treatment at 10 min in the presence of control siRNA. However, Gas6-induced Akt and ERK1/2 phosphorylation are abolished in the presence of siRNA targeting either Axl or caveolin-1 (n = 5; *P < 0.05 Gas6 vs. vehicle). (Fig. 2B). Caveolin-1 siRNA significantly reduced the caveolin-1 protein level without affecting Axl expression (Fig. 2B). Gas6 significantly increased Akt phosphoryla© 2013 International Society on Thrombosis and Haemostasis tion at 10 min, which was inhibited when Axl expression was downregulated using an Axl siRNA as well as caveolin-1 siRNA (Fig. 2C). ERK1/2 phosphorylation was also 400 S. Laurance et al A Control siRNA Axl siRNA Gas6 0 5 10 Cav-1 siRNA Gas6 0 5 10 Gas6 0 5 10 min P-c-Src c-Src c-Src phoshorylation (compared to non-treated) 1.5 * 1 0.5 0 5 10 Control siRNA 0 5 Non-treated B DAPI 10 Axl siRNA c-Src 0 5 10 min Cav-1 siRNA Gas6 Axl c-Src + Axl Colocalization (white spot) Gas6 0 min Gas6 5 min Gas6 10 min © 2013 International Society on Thrombosis and Haemostasis Gas6-induced endothelial tissue factor expression 401 Fig. 3. Gas6-induced c-Src phosphorylation and its colocalization with Axl in lipid raft/caveolin-1–enriched microdomains. (A) c-Src phosphorylation was evaluated by Western blot analysis after 48 h of treatment with Gas6 in the presence of control or Axl or caveolin-1 siRNA in HUVECs. Gas6 transiently induces c-Src phosphorylation in the presence of control siRNA at 5 min of treatment, and this upregulation is abolished in the presence of siRNA targeting either Axl or caveolin-1 (n = 5; *P < 0.05). (B) c-Src and Axl localization was assessed by confocal microscopy (magnification 960). The colocalization sites are highlighted using ImageJ software and represent sites where caveolin-1 and cSrc are present in the same proportion (n = 5). (C) Representative Western blots of caveolin-1 and c-Src distribution after sucrose gradient centrifugation. Caveolin-1 protein is most abundantly found in fractions 3–5, thus identifying the lipid raft/caveolin-1–enriched microdomains. In resting HUVECs, c-Src is mainly found in fractions 4–9. Five minutes of Gas6 treatment increases c-Src localization in the lipid raft/caveolin1–rich fractions (3 and 6). After 10 min of Gas6 treatment, c-Src moves out from the lipid raft/caveolin-1–rich fractions. Densitometric quantification show c-Src protein level in each fraction relative to the sum of the intensity of the total fraction (n = 5; *P < 0.05 vehicle vs. 5 min of Gas6 or vehicle vs. 10 min of Gas6). (D) c-Src and caveolin-1 localization was assessed through confocal microscopy (magnification 960). The colocalization sites are highlighted using ImageJ software and represent pixels where caveolin-1 and c-Src are present in the same proportion (n = 5). increased at 10 min of Gas6 treatment, and Axl and caveolin-1 downregulation inhibited ERK1/2 phosphorylation (Fig. 2D). Axl and caveolin-1 are required for Gas6-induced c-Src phosphorylation c-Src is a protein with lipid raft affinity that is known to be recruited by Axl [19]. Thus, we assessed the role of Gas6/Axl on c-Src activation and localization at the plasma membrane. Western blot analysis showed that Gas6 treatment transiently activates c-Src at 5 min. c-Src activation by Gas6 was completely abolished by either Axl or caveolin-1 siRNA (Fig. 3A). Next, confocal microscopy was used to assess Axl and c-Src colocalization in HUVECs. Axl and c-Src staining was colocalized at 5 min of Gas6 treatment but not at 10 min (Fig. 3B). Then, c-Src localization in lipid rafts after Gas6 treatment was analyzed using sucrose gradient centrifugation. In resting HUVECs, c-Src was more abundantly found in fractions 4–9, demonstrating that c-Src can be detected in both the caveolin-1–enriched and –nonenriched fractions. When HUVECs were treated with Gas6 for 5 min, we observed an enrichment of c-Src in fractions 3 and 6, which correspond to the lipid raft/caveolin-1 fractions. Interestingly, after 10 min of Gas6 treatment, c-Src moved out of the lipid raft/caveolin-1 fractions and was mostly localized in fractions 7–10 (Fig. 3C). Confocal microscopy analysis confirmed that after 5 min of treatment with Gas6, c-Src localized with caveolin-1 staining. After 10 min of Gas6 treatment, c-Src and caveolin-1 were no longer colocalized (Fig. 3D), consistent with the data from Western blot analysis. c-Src is required for Gas6-induced Akt phosphorylation We evaluated the role of c-Src in the activation of the downstream targets of Axl, Akt, and ERK1/2. HUVECs were transfected with control or c-Src siRNA. The c-Src siRNA efficiently reduces c-Src protein expression by 90% compared with control siRNA (Fig. 4A). c-Src downregulation inhibited Gas6-induced Akt phosphorylation (Fig. © 2013 International Society on Thrombosis and Haemostasis 4B) but only partially prevented Gas6-induced ERK1/2 phosphorylation (Fig. 4C). The Gas6/Axl/caveolin-1/c-Src/Akt axis is involved in TF expression HUVECs were incubated for 2, 4, or 6 h with Gas6. Akt phosphorylation was increased by Gas6 until 6 h of treatment (Fig. 5A). At 6 h, Gas6-induced Akt activation was associated with a significant increased TF mRNA expression by Gas6 (Fig. 5B). Interestingly, Gas6induced TF expression is similar to that of LPS-induced TF expression (Fig. 5B). Immunofluorescent staining demonstrated that TF protein was also increased by Gas6 at 6 h compared with untreated cells (Fig. 5C). Then, to evaluate the role of the signaling pathways described in the previous series of experiments, HUVECs were transfected with control, Axl, or caveolin-1 siRNA. Both Axl and caveolin-1 downregulation blocked Gas6induced TF mRNA expression compared with the control siRNA (Fig. 6A). HUVECs were pretreated with either an ERK1/2 inhibitor, PD98059, or a c-Src inhibitor, PP2, 30 min before Gas6 treatment. Western blot analysis showed that PP2 and PD098059 prevented Gas6-induced c-Src and ERK1/2 activation, respectively (Figs. S1 and S2). Interestingly, ERK1/2 inhibition did not affect Gas6-induced TF mRNA expression, whereas c-Src inhibition abolished mRNA expression of TF by Gas6 (Fig. 6B). The role of c-Src was confirmed using siRNA. Gas6-induced TF mRNA expression was blocked in HUVECs transfected with c-Src siRNA compared with control siRNA (Fig. 6C). Furthermore, the role of Akt in Gas6-induced TF expression was evaluated by transfecting HUVECs with Akt siRNA, which efficiently reduced Akt expression by 90% (Fig. S3). Gas6-induced TF mRNA expression was blocked in HUVECs transfected with Akt siRNA compared with control siRNA (Fig. 6C). Akt siRNA also blocked Gas6-induced TF protein expression (Fig. 6D). Taken together, these results demonstrate the important role of the Axl/c-Src/Akt pathway in Gas6-induced TF expression in endothelial cells. 402 S. Laurance et al C Enriched Fraction 1 2 3 4 5 6 7 8 9 10 Gas6 (min) Cav-1 0 0 5 c-Src c-Src expression % of total fractions 10 30 * * 25 * 20 15 * 10 * 5 0 1 2 3 4 5 6 7 8 9 10 Fraction number Gas6 0 min D DAPI Cav-1 Gas6 5 min c-Src Gas6 10 min Cav-1 + c-Src Colocalization (white spot) Gas6 0 min Gas6 5 min Gas6 10 min Fig. 3. (continued) © 2013 International Society on Thrombosis and Haemostasis Gas6-induced endothelial tissue factor expression 403 A Control siRNA Gas6 0 5 B c-Src siRNA Gas6 10 0 5 10 0 min PAkt Actin Akt Gas6 0 5 Gas6 10 0 5 10 min PERK1/2 ERK1/2 3 5 10 Gas6 0 5 10 min 2 c-Src siRNA * Akt phosphorylation (compared to non-treated) Control siRNA c-Src siRNA Gas6 c-Src C ERK1/2 phosphorylation (compared to non-treated) Control siRNA * 1.5 1 0.5 0 0 2.5 5 10 Control siRNA 2 * 1.5 Non-treated 0 5 10 min c-Src siRNA Gas6 1 0.5 0 0 5 10 Control siRNA Non-treated 0 5 10 min c-Src siRNA Gas6 Fig. 4. c-Src is required for Akt, but not ERK1/2, activation by Gas6. (A) Representative Western blot of c-Src protein level in HUVECs with control or c-Src siRNA for 48 h (n = 5). (B) Akt and (C) ERK1/2 phosphorylation were evaluated by Western blot in presence of control or cSrc siRNA in HUVECs treated by Gas6 for 5 and 10 min. Akt and ERK1/2 phosphorylation are significantly increased after 10 min of Gas6 treatment in the presence of control siRNA. However, Gas6-induced Akt but not ERK1/2 phosphorylation is abolished in the presence of an siRNA targeting c-Src (n = 5;*P < 0.05, Gas6 vs. vehicle). Discussion We demonstrate, for the first time, that (1) Gas6 induces Axl and c-Src localization in lipid raft/caveolin-1– enriched microdomains, (2) Axl and c-Src localization in lipid raft/caveolin-1–enriched microdomains is required for Akt and ERK1/2 phosphorylation, and (3) the activation of the c-Src/Akt signaling pathway by Gas6 leads to expression of TF in endothelial cells. Under physiological conditions, the endothelium provides an anti-inflammatory and an antithrombotic surface. However, following a physical (e.g. trauma, injury) or pathological (e.g. infection) insult, the endothelium adopts a proinflammatory and/or procoagulant phenotype. The endothelium has a number of properties that directly or indirectly affect the hemostatic balance. © 2013 International Society on Thrombosis and Haemostasis Upregulation of procoagulant factors promotes activation of the coagulation cascade and platelets, which, in turn, can lead to further activation of the endothelium. This is characterized by the expression of procoagulant mediators (e.g. von Willebrand factor, TF), and other soluble factors (e.g. cytokines, vasomodulators). Among the soluble factors, Gas6 has been shown to be involved in several pathological conditions such as atherosclerosis, cancer, and thrombosis [11]. We and others have demonstrated that Gas6 is involved in vascular homeostasis by regulating cellular and molecular processes such as proliferation, survival, efferocytosis, leukocyte migration, and platelet aggregation [1,6,8,19–23]. Interestingly, Gas6 null mice are protected against lethal thromboembolism without any excessive bleeding [1]. The mechanisms supporting this protection remain unclear. To date, an effect on platelets 404 S. Laurance et al Gas6 A 2 0 B 4 6 h 2 * TF mRNA fold change (conpared to non-treated) PAkt Akt Akt phosphorylation (compared to non-treated) 2.5 * 2 * * 1.5 0.5 * 4 6 1.5 1 0.5 0 1 * 0 4 0 6 Non-treated h LPS Gas6 0 0 2 4 6 Non-treated C DAPI h Gas6 TF Merge Gas6 0 hr Gas6 6 hr TF expression (%area/dapi, A.U.) 1.5 * 1 0.5 0 0 Non-treated 6 h Gas6 Fig. 5. Gas6-induced activation of Akt and TF expression in HUVECs. (A) Representative Western blots of Akt phosphorylation in HUVECs treated with Gas6 for 2, 4, and 6 h. Akt phosphorylation is maintained by Gas6 for 6 h (n = 5; *P < 0.05). (B) TF expression was evaluated by qRT-PCR in HUVECs treated with Gas6 or LPS for 4 and 6 h. Gas6 and LPS significantly increase tissue factor (TF) mRNA expression (n = 5; *P < 0.05). (C) Immunofluorescent staining shows that Gas6 increases TF protein expression at 6 h (n = 3; *P < 0.05). Fig. 6. Gas6-induced TF expression in HUVECs is mediated by Axl/Cav-1/c-Src/Akt signaling. (A) Tissue factor (TF) expression was evaluated by qRT-PCR in HUVECs transfected with control, Axl or caveolin-1 siRNA and treated with Gas6 for 6 h. Axl and caveolin-1 downregulation abolish Gas6-induced TF expression (n = 5; *P < 0.05). (B) TF expression was evaluated by qRT-PCR in HUVECs pretreated with either PD98059 or PP2 and incubated with Gas6 for 6 h. Pharmacological inhibition of c-Src, but not ERK1/2, inhibits Gas6-induced TF expression (n = 5; *P < 0.05). (C) TF expression was analyzed by qRT-PCR in HUVECs transfected with control siRNA, c-Src siRNA, or Akt siRNA and treated with Gas6 for 6 h. Downregulation of c-Src and Akt blocks Gas6-induced TF expression (n = 5; *P < 0.05). (D) TF immunofluorescent staining demonstrates that Gas6 treatment induces TF expression in the presence of control siRNA, whereas Akt siRNA abolishes this induction (n = 3; *P < 0.05, Gas6 vs. nontreated). © 2013 International Society on Thrombosis and Haemostasis Gas6-induced endothelial tissue factor expression 405 B 2.5 TF mRNA fold change (conpared to non-treated) TF mRNA fold change (conpared to non-treated) A * 2 1.5 1 0.5 0 0 6 Control siRNA 0 6 0 Axl siRNA TF mRNA fold change (conpared to non-treated) h * * 2 1.5 1 0.5 0 0 6 Cav-1 siRNA 0 6 0 6 PD98059 Gas6 Non-treated C 6 2.5 h PP2 Gas6 Non-treated 2.5 * 2 1.5 1 0.5 0 0 6 Control siRNA 0 6 0 c-Src siRNA Non-treated D DAPI 6 h Akt siRNA Gas6 TF Merge Gas6 0h Control siRNA Gas6 6h Akt siRNA * TF expression (%area/dapi, A.U.) Gas6 0h 1 0.5 0 0 6 Control siRNA Gas6 6h © 2013 International Society on Thrombosis and Haemostasis 0 6 Akt siRNA Non-treated Gas6 h 406 S. Laurance et al in Gas6 null mice has been demonstrated, but the impact on platelet aggregation is mild compared with the dramatic phenotype observed in these mice. The impaired platelet aggregate formation in Gas6 null mice suggests that Gas6 plays a role in thrombus stabilization, probably through the regulation of aIIbb3 integrin ‘outside-in’ signaling and platelet degranulation via a PI3K-dependent mechanism [6]. More recently, Cosemans et al. [24] have shown a synergistic effect of Gas6 on the ADP pathway leading to aIIbb3 integrin activation. Taken together, these studies emphasize the role of Gas6 and its receptors in platelet biology. We recently established that Gas6 from vascular cells is involved in thrombus generation [2]. We demonstrated that Gas6 positively regulates TF expression in murine endothelial cells and could, in part, explain how Gas6 contributes to thrombus formation in vivo [2]. On the endothelial surface, Axl is the major Gas6 receptor expressed [21]. Axl belongs to a large family of type I transmembrane receptor tyrosine kinases. This family of receptors has been shown to interact with lipid raft/caveolin-1–enriched microdomains. Caveolin-1–enriched microdomains have been identified as pivotal sites for the initiation and the regulation of cell signaling. Lipid raft/ caveolin-1–enriched microdomains include or exclude proteins creating a restricted and integrative microenvironment, allowing efficacy and rapid coupling of receptors with downstream signaling partners. Caveolin-1–enriched microdomains are present at different densities in different cell types but are mainly found in endothelial cells, adipocytes, fibroblasts, and epithelial cells [13]. Several studies have shown that activation of Akt is required for the antiapoptotic function of Gas6/Axl signaling in different cell types such as endothelial cells, vascular smooth muscle cells, and fibroblasts [8,19,25,26]. c-Src kinase seems to be also important in Gas6/Axl signaling since a c-Src binding site has been identified on Axl [19,27]. In addition, ERK1/2 activation seems to be necessary for Gas6/Axl-mediated mitogenic activity [18,27]. However, the signaling pathways involved in the endothelial cell procoagulant phenotype induced by Gas6 have not been elucidated. Thus, we hypothesize that Gas6 promotes the interaction of Axl with lipid raft/caveolin-1 microdomains, leading to a procoagulant phenotype in endothelial cells. First, using siRNA-mediated knockdown experiments, we show that, in HUVECs, Gas6 induces Akt, ERK1/2, and c-Src phosphorylation through the Axl receptor. We and others have previously shown that, in HUVECs, Gas6 induces Akt phosphorylation [8]. However, ERK1/2 and c-Src activation by Gas6 has not previously been demonstrated in endothelial cells. Second, we show that Gas6 treatment induces Axl and c-Src segregation into caveolin-1–enriched microdomains. Several studies have highlighted the crucial role of lipid raft/caveolin-1– enriched microdomains in vascular cell signaling mediated through receptor tyrosine kinases (e.g. PDGFR or VEGFR2), G protein-coupled receptors, and transforming growth factor-b type I and II receptors as well as certain steroid receptors [13,28]. Interestingly, Banfi et al. [29,30] have shown that lipid raft/caveolin-1–enriched microdomains are associated with the receptor non–tyrosine kinase PAR-1 and are required for PAR-1 downstream signaling pathways. However, this is the first time that an association between Axl and lipid raft/caveolin-1–enriched microdomains is demonstrated. c-Src has been found in lipid raft/caveolin-1–enriched microdomains, which lead to the activation of signaling mediators such as RasMAPK or PI3K/Akt [31,32]. We found, in the present study, that c-Src is divided into two clusters between the caveolin-1–enriched fractions and the non–caveolin-1– enriched fractions. After 5 min of Gas6 treatment, c-Src localizes into lipid raft/caveolin-1–enriched microdomains. However, after 10 min of treatment, c-Src seems to be completely excluded from these domains, suggesting a negative feedback mechanism. More importantly, we demonstrate that caveolin-1 is required for Gas6/Axl induction of Akt, ERK1/2, and c-Src phosphorylation. By using siRNA-mediated knockdown experiments, we show that c-Src is a molecular intermediate between Axl and Akt activation but is not involved in Gas6-mediated ERK1/2 activation. Similarly, c-Src has been recently identified upstream Akt in androgen receptor activation of Akt in endothelial cells [33]. PAR-1 signaling pathways were shown to be mediated through c-Src in endothelial cells [30], thus reinforcing the role of c-Src as a central regulator of several signaling pathways. Finally, we demonstrate the ability of Gas6 to induce a procoagulant phenotype by measuring the expression of TF in HUVECs treated with Gas6. LPS was also showed to induce TF expression in different cell types in vitro and in a model of endotoxemia [34]. As a comparison, we also evaluated the expression of TF in response to LPS. Interestingly, the expression of TF was comparable after Gas6 or LPS stimulation. We show that Gas6 induces TF expression in HUVECs through an Axl/c-Src/Akt pathway, independently of ERK1/2 activation, and requires the presence of caveolin-1. This is in accordance with the study from Banfi et al. [30], in which it is shown that intact lipid raft/ caveolin-1–enriched microdomains are required for TF biosynthesis. However, TF expression is not sufficient to trigger the activation of the coagulation cascade, suggesting that its activity is also regulated. It has been recently shown in in vitro models that TF is primarily in an inactive or a cryptic form. After stimulation, TF can be decrypted and activated. In fact, it seems that an increase in TF activity results from TF decryption rather than an increase in TF activity per se. The decryption mechanism may involve a thiol/disulfide switch and exposure of phosphatidylserine. However, the molecular mechanism of protein disulfide action in TF encryption/decryption remains to be determined, and the lack of specific tools to differentiate cryptic and uncryptic TF is a strong limitation and beyond the scope of the current study [35,36]. © 2013 International Society on Thrombosis and Haemostasis Gas6-induced endothelial tissue factor expression 407 The present study demonstrates the crucial role of Akt in Gas6-induced TF expression in endothelial cells. However, the PI3K/Akt pathway has been found to inhibit TF expression in different cell types, including endothelial cells, in response to several stimuli [37–39]. Gas6 is a strong inducer of the PI3K/Akt pathway. Thus, it was expected that PI3K/Akt activation would lead to TF expression after Gas6 stimulation. However, our data strongly support the role of PI3K/Akt in Gas6-induced TF expression. Together with our recently published in vivo study, we have identified a procoagulant pathway for Gas6 in endothelial cells [2]. The present study demonstrates that lipid raft/caveolin-1–enriched microdomains are essential for the transduction of Gas6 signaling through Axl and provides significant molecular insights in the procoagulant role of Gas6. Addendum S. Laurance designed the experiments, performed research, analyzed the data and wrote the manuscript. M. N. Aghourian and Z. J. Lila performed research. C. A. Lemarie designed experiments, analyzed the data and wrote the manuscript. M. D. Blostein conceived and financed the study and wrote the manuscript. Acknowledgements This work was supported by a grant from the Canadian Institutes for Health Research. Disclosure of Conflicts of Interest The authors state that they have no conflicts of interest. Supporting Information Additional Supporting Information may be found in the online version of this article: Fig. S1. Representative Western blots of c-Src phosphorylation in human umbilical vein endothelial cells pretreated with PP2 and incubated with Gas6. Fig. S2. Representative Western blots of PERK1/2 phosphorylation in human umbilical vein endothelial cells pretreated with PD98059 and incubated with Gas6. Fig. S3. Representative Western blots of Akt protein expression in human umbilical vein endothelial cells transfected with control or Akt siRNA for 48 h (n = 4). References 1 Angelillo-Scherrer A, de Frutos P, Aparicio C, Melis E, Savi P, Lupu F, Arnout J, Dewerchin M, Hoylaerts M, Herbert J, Collen D, Dahlback B, Carmeliet P. Deficiency or inhibition of Gas6 causes platelet dysfunction and protects mice against thrombosis. Nat Med 2001; 7: 215–21. © 2013 International Society on Thrombosis and Haemostasis 2 Robins RS, Lemarie CA, Laurance S, Aghourian MN, Wu J, Blostein MD. Vascular Gas6 contributes to thrombogenesis and promotes tissue factor up-regulation after vessel injury in mice. Blood 2013; 121: 692–9. 3 Stitt TN, Conn G, Goret M, Lai C, Bruno J, Radzlejewski C, Mattsson K, Fisher J, Gies DR, Jones PF, Masiakowski P, Ryan TE, Tobkes NJ, Chen DH, DiStefano PS, Long GL, Basilico C, Goldfarb MP, Lemke G, Glass DJ, et al. The anticoagulation factor protein S and its relative, Gas6, are ligands for the Tyro 3/Axl family of receptor tyrosine kinases. Cell 1995; 80: 661–70. 4 Varnum BC, Young C, Elliott G, Garcia A, Bartley TD, Fridell Y-W, Hunt RW, Trail G, Clogston C, Toso RJ, Yanagihara D, Bennett L, Sylber M, Merewether LA, Tseng A, Escobar E, Liu ET, Yamane HK. Axl receptor tyrosine kinase stimulated by the vitamin K-dependent protein encoded by growth-arrest-specific gene 6. Nature 1995; 373: 623–6. 5 Nagata K, Ohashi K, Nakano T, Arita H, Zong C, Hanafusa H, Mizuno K. Identification of the product of growth arrest-specific gene 6 as a common ligand for Axl, Sky, and Mer receptor tyrosine kinases. J Biol Chem 1996; 271: 30022–7. 6 Angelillo-Scherrer A, Burnier L, Flores N, Savi P, DeMol M, Schaeffer P, Herbert JM, Lemke G, Goff SP, Matsushima GK, Earp HS, Vesin C, Hoylaerts MF, Plaisance S, Collen D, Conway EM, Wehrle-Haller B, Carmeliet P. Role of Gas6 receptors in platelet signaling during thrombus stabilization and implications for antithrombotic therapy. J Clin Invest 2005; 115: 237–46. 7 Lu Q, Gore M, Zhang Q, Camenisch T, Boast S, Casagranda F, Lai C, Skinner MK, Klein R, Matsushima GK, Earp HS, Goff SP, Lemke G. Tyro-3 family receptors are essential regulators of mammalian spermatogenesis. Nature 1999; 398: 723–8. 8 Hasanbasic I, Cuerquis J, Varnum B, Blostein MD. Intracellular signaling pathways involved in Gas6-Axl-mediated survival of endothelial cells. Am J Physiol Heart Circ Physiol 2004; 287: H1207–13. 9 Bellosta P, Zhang Q, Goff SP, Basilico C. Signaling through the ARK tyrosine kinase receptor protects from apoptosis in the absence of growth stimulation. Oncogene 1997; 15: 2387–97. 10 Goruppi S, Ruaro E, Varnum B, Schneider C. Gas6-mediated survival in NIH3T3 cells activates stress signalling cascade and is independent of Ras. Oncogene 1999; 18: 4224–36. 11 Laurance S, Lemarie CA, Blostein MD. Growth arrest-specific gene 6 (gas6) and vascular hemostasis. Adv Nutr 2012; 3: 196–203. 12 Yanagita M, Arai H, Ishii K, Nakano T, Ohashi K, Mizuno K, Varnum B, Fukatsu A, Doi T, Kita T. Gas6 regulates mesangial cell proliferation through Axl in experimental glomerulonephritis. Am J Pathol 2001; 158: 1423–32. 13 Gratton JP, Bernatchez P, Sessa WC. Caveolae and caveolins in the cardiovascular system. Circ Res 2004; 94: 1408–17. 14 Razani B, Woodman SE, Lisanti MP. Caveolae: from cell biology to animal physiology. Pharmacol Rev 2002; 54: 431–67. 15 Pike LJ. Lipid rafts: bringing order to chaos. J Lipid Res 2003; 44: 655–67. 16 Labrecque L, Royal I, Surprenant DS, Patterson C, Gingras D, Beliveau R. Regulation of vascular endothelial growth factor receptor-2 activity by caveolin-1 and plasma membrane cholesterol. Mol Biol Cell 2003; 14: 334–47. 17 Anderson RG. The caveolae membrane system. Annu Rev Biochem 1998; 67: 199–225. 18 Fridell YW, Jin Y, Quilliam LA, Burchert A, McCloskey P, Spizz G, Varnum B, Der C, Liu ET. Differential activation of the Ras/extracellular-signal-regulated protein kinase pathway is responsible for the biological consequences induced by the Axl receptor tyrosine kinase. Mol Cell Biol 1996; 16: 135–45. 19 Goruppi S, Ruaro E, Varnum B, Schneider C. Requirement of phosphatidylinositol 3-kinase-dependent pathway and Src for 408 S. Laurance et al 20 21 22 23 24 25 26 27 28 29 Gas6-Axl mitogenic and survival activities in NIH 3T3 fibroblasts. Mol Cell Biol 1997; 17: 4442–53. Goruppi S, Ruaro E, Schneider C. Gas6, the ligand of Axl tyrosine kinase receptor, has mitogenic and survival activities for serum starved NIH3T3 fibroblasts. Oncogene 1996; 12: 471–80. Tjwa M, Bellido-Martin L, Lin Y, Lutgens E, Plaisance S, Bono F, Delesque-Touchard N, Herve C, Moura R, Billiau AD, Aparicio C, Levi M, Daemen M, Dewerchin M, Lupu F, Arnout J, Herbert JM, Waer M, Garcia de Frutos P, Dahlback B, et al. Gas6 promotes inflammation by enhancing interactions between endothelial cells, platelets, and leukocytes. Blood 2008; 111: 4096–105. Friggeri A, Banerjee S, Xie N, Cui H, De Freitas A, Zerfaoui M, Dupont H, Abraham E, Liu G. Extracellular histones inhibit efferocytosis. Mol Med 2012; 18: 825–33. Lemke G, Burstyn-Cohen T. TAM receptors and the clearance of apoptotic cells. Ann N Y Acad Sci 2010; 1209: 23–9. Cosemans JM, van Kruchten R, Olieslagers S, Schurgers LJ, Verheyen FK, Munnix IC, Waltenberger J, Angelillo-Scherrer A, Hoylaerts MF, Carmeliet P, Heemskerk JW. Potentiating role of Gas6 and Tyro3, Axl and Mer (TAM) receptors in human and murine platelet activation and thrombus stabilization. J Thromb Haemost 2010; 8: 1797–808. Melaragno MG, Cavet ME, Yan C, Tai LK, Jin ZG, Haendeler J, Berk BC. Gas6 inhibits apoptosis in vascular smooth muscle: role of Axl kinase and Akt. J Mol Cell Cardiol 2004; 37: 881–7. O’Donnell K, Harkes IC, Dougherty L, Wicks IP. Expression of receptor tyrosine kinase Axl and its ligand Gas6 in rheumatoid arthritis: evidence for a novel endothelial cell survival pathway. Am J Pathol 1999; 154: 1171–80. Braunger J, Schleithoff L, Schulz AS, Kessler H, Lammers R, Ullrich A, Bartram CR, Janssen JW. Intracellular signaling of the Ufo/Axl receptor tyrosine kinase is mediated mainly by a multi-substrate docking-site. Oncogene 1997; 14: 2619–31. Sowa G. Caveolae, caveolins, cavins, and endothelial cell function: new insights. Front Physiol 2012; 2: 120. Banfi C, Brioschi M, Barcella S, Pignieri A, Parolari A, Biglioli P, Tremoli E, Mussoni L. Tissue factor induction by protease- 30 31 32 33 34 35 36 37 38 39 activated receptor 1 requires intact caveolin-enriched membrane microdomains in human endothelial cells. J Thromb Haemost 2007; 5: 2437–44. Banfi C, Brioschi M, Lento S, Pirillo A, Galli S, Cosentino S, Tremoli E, Mussoni L. Statins prevent tissue factor induction by protease-activated receptors 1 and 2 in human umbilical vein endothelial cells in vitro. J Thromb Haemost 2011; 9: 1608–19. Tiruppathi C, Song W, Bergenfeldt M, Sass P, Malik AB. Gp60 activation mediates albumin transcytosis in endothelial cells by tyrosine kinase-dependent pathway. J Biol Chem 1997; 272: 25968–75. Thomas SM, Brugge JS. Cellular functions regulated by Src family kinases. Annu Rev Cell Dev Biol 1997; 13: 513–609. Yu J, Akishita M, Eto M, Koizumi H, Hashimoto R, Ogawa S, Tanaka K, Ouchi Y, Okabe T. Src kinase-mediates androgen receptor-dependent non-genomic activation of signaling cascade leading to endothelial nitric oxide synthase. Biochem Biophys Res Commun 2012; 424: 538–43. Pawlinski R, Mackman N. Cellular sources of tissue factor in endotoxemia and sepsis. Thromb Res 2010; 125(Suppl 1): S70–3. Kothari H, Pendurthi UR, Rao LV. Analysis of tissue factor expression in various cell model systems: cryptic vs. active. J Thromb Haemost 2013; 11: 1353–63. Chen VM, Hogg PJ. Encryption and decryption of tissue factor. J Thromb Haemost 2013; 11(Suppl 1): 277–84. Blum S, Issbruker K, Willuweit A, Hehlgans S, Lucerna M, Mechtcheriakova D, Walsh K, von der Ahe D, Hofer E, Clauss M. An inhibitory role of the phosphatidylinositol 3-kinase-signaling pathway in vascular endothelial growth factor-induced tissue factor expression. J Biol Chem 2001; 276: 33428–34. Eto M, Kozai T, Cosentino F, Joch H, Luscher TF. Statin prevents tissue factor expression in human endothelial cells: role of Rho/Rho-kinase and Akt pathways. Circulation 2002; 105: 1756–9. Schabbauer G, Tencati M, Pedersen B, Pawlinski R, Mackman N. PI3K-Akt pathway suppresses coagulation and inflammation in endotoxemic mice. Arterioscler Thromb Vasc Biol 2004; 24: 1963–9. © 2013 International Society on Thrombosis and Haemostasis
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