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Thromb Res. Author manuscript; available in PMC 2011 January 1.
Published in final edited form as:
PMCID: PMC2826497
NIHMSID: NIHMS136416

Thrombin and activated protein C inhibit the expression of secretory group IIA phospholipase A2 in the TNF-α-activated endothelial cells by EPCR and PAR-1 dependent mechanisms

Abstract

Introduction

Thrombin and tumor necrosis factor (TNF)-α up-regulate the expression of proinflammatory molecules in human umbilical vein endothelial cells (HUVECs). However, activated protein C (APC) down-regulates the expression of the same molecules. The expression level of secretory group IIA phospholipase A2 (sPLA2-IIA) is known to be elevated in inflammatory disorders including in sepsis. Here, we investigated the effects of APC and thrombin on the expression of sPLA2-IIA and extracellular signal-regulated kinase (ERK) in HUVECs.

Materials and methods

The expression level of sPLA2-IIA was quantitatively measured by an enzyme-linked-immunosorbent-assay following stimulation of HUVECs with either thrombin or TNF-α in the absence and presence of the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor LY294002 and the cholesterol-depleting drug methyl-β-cyclodextrin (MβCD).

Results and conclusions

Thrombin had no effect on the expression of sPLA2-IIA in HUVECs, however, TNF-α potently induced its expression. The prior treatment of cells with APC inhibited expression of sPLA2-IIA through the EPCR-dependent cleavage of PAR-1. Further studies revealed that if HUVECs were pretreated with the zymogen protein C to occupy EPCR, thrombin also inhibited the TNF-α-mediated expression of sPLA2-IIA through the cleavage of PAR-1. The EPCR-dependent cleavage of PAR-1 by both APC and thrombin increased the phosphorylation of ERK 1/2. Pretreatment of cells with either LY294002 or MβCD abolished the inhibitory activity of both APC and thrombin against sPLA2-IIA expression, suggesting that the protein C occupancy of EPCR confers a PI3-kinase dependent protective activity for thrombin such that its cleavage of the lipid-raft localized PAR-1 inhibits the TNF-α-mediated expression of sPLA2-IIA in HUVECs.

Keywords: Thrombin, APC, HUVEC, inflammation, PAR-1, PI3 kinase

Introduction

Secretory phospholipase A2 group IIA (sPLA2-IIA) is a member of the secreted phospholipase family of lipid hydrolyzing enzymes that hydrolyses the ester bond at the sn-2 position of phosphoglycerides to release free fatty acid and lysophospholipids [1, 2]. sPLA2-IIA was initially isolated from the rheumatoid arthritic synovial fluid and platelets [3-5]. The biological function of sPLA2-IIA is not well understood, though there are several reports hypothesizing a possible role for sPLA2-IIA in the modulation of coagulation, signal transduction, apoptosis, remodeling of cellular membranes, and host defense systems [6-8]. A high expression level for this enzyme has been observed at inflamed sites and also in the serum of patients with inflammatory disorders such as sepsis, septic shock, polytrauma and atherosclerosis [9-12]. During acute or chronic inflammation, the level of sPLA2-IIA expression has been shown to correlate with the severity of the disease [9-13]. Moreover, it has been demonstrated that sPLA2-IIA promotes inflammation in animal models [14, 15].

It is known that activated protein C (APC), in addition to its critical role in down-regulation of the clotting cascade, plays an important role in down-regulating the inflammatory pathways [16-18]. It is thought that the antiinflammatory activities of APC are responsible [16-18], at least partially, for its beneficial effect in reducing the mortality rate in patients with sever sepsis [19]. The antiinflammatory activities of APC are thought to be mediated through its interaction with endothelial protein C receptor (EPCR) and its subsequent cleavage of protease-activated receptor 1 (PAR-1) on endothelial cells [20]. It is known that the EPCR- and PAR-1-dependent protective signaling of APC results in the activation of the ERK 1/2 signaling pathway [21-23]. However, it is also known that PAR-1 activation by thrombin elicits potent proinflammatory responses in endothelial cells [24, 25]. Recently, we showed that the occupancy of EPCR by the zymogen protein C (PC) changes the PAR-1-dependent signaling specificity of thrombin from a proinflammatory to an antiinflammatory response in cytokine stimulated endothelial cells [26-28]. We demonstrated that the occupancy of EPCR by protein C/APC regulates the interaction of EPCR with caveolin-1 within the membrane lipid-rafts, thereby regulating the signaling specificity of PAR-1 on endothelial cells independent of the protease (thrombin or APC) activating the receptor [28, 29].

Noting the enhanced level of sPLA2-IIA in inflammatory disorders and the ability of thrombin to initiate proinflammatory responses in cultured endothelial cells by activating the NF-κB pathway [9-12, 24, 25, 30-32], we initiated this study to determine whether thrombin can induce the expression of sPLA2-IIA in endothelial cells by the same pathway, and if so, whether the APC-EPCR pathway can down-regulate this response. We discovered that TNF-α potently induced the expression of sPLA2-IIA in endothelial cells, however, thrombin had no effect on its expression. APC effectively inhibited the TNF-α-mediated expression of sPLA2-IIA in endothelial cells by a PAR-1-dependent manner. Interestingly, when EPCR was occupied by its ligand, the cleavage of PAR-1 by thrombin also inhibited the expression level of sPLA2-IIA in TNF-α-stimulated endothelial cells. The same results were obtained if a thrombin receptor agonist peptide (TRAP) was used as the PAR-1 activator in endothelial cells. Further studies revealed that the EPCR- and PAR-1-dependent inhibitory effect of both APC and thrombin on endothelial cells is mediated through the activation of the phosphatidylinositol 3-kinase (PI3 kinase) and ERK 1/2 pathways pathways. Based on these results, we conclude that the cleavage of PAR-1 by either APC or thrombin on endothelial cells expressing EPCR would reverse the proinflammatory effects of cytokines if EPCR was occupied by its natural ligand.

Materials and methods

Regents

Tumor necrosis factor-α (TNF-α) was purchased from R&D System (Minneapolis, MN) and used at 10 ng/mL to 200 ng/mL. The cleavage blocking monoclonal anti-PAR-1 antibody was purchased from Santa Cruz Biologics (Santa Cruz, CA) and used at 25 μg/mL. The functional blocking anti-EPCR antibody (Clone RCR-252) was purchased from Cell Sciences (Canton, MA, USA) and used at 25 μg/mL. Thrombin, specific cell permeable phosphatidylinositol 3-kinase inhibitor (LY294002, used at 10 μM), cholesterol-depleting drug methyl-β-cyclodextrin (MβCD, used 10 mM) were obtained from Sigma (St. Louis, MO, USA). PD98059 was purchased from Calbiochem (Schwalbach, Germany) and used at 25 μM. The thrombin receptor agonist peptide (TRAP) agonist peptide (TFLLRN, used at 100 μM) was purchased from Bachem Bioscience (Torrance, CA). Expression and purification of wild type APC, zymogen protein C (PC) and the Ser-195 to Ala substitution mutant of PC (PC-S195A) have been described [28, 29].

Cell culture

Primary HUVECs were obtained from Cambrex Bio Science Inc. (Charles City, IA) and maintained as described as before [27-29]. Immortalized human umbilical vein endothelial cells (EA.hy926) was kindly provided by Dr. C. Edgell from University of North Carolina at Chapel Hill (NC, USA) and maintained as descried before [26-29]. Briefly, Cells were cultured at 37 °C in Dulbecco’s modified Eagle’s medium (Invitrogen, CA, USA) supplemented with 10% fetal bovine serum and antibiotics (penicillin G and streptomycin).

ELISA for sPLA2-IIA and ERK 1/2 phosphorylation

The expression of sPLA2-IIA protein was determined in the cell culture medium and cell lysates by using specific ELISA kit (Cayman Chemical, Ann Arbor, MI, USA) as described previously [33] according to the manufacturer’s instruction. The measurements were performed in duplicate in at least three independent experiments. Briefly, 50 μL of diluted medium or cell lysates containing 5–25 μg of total protein were added to each well of the plate coated with a monoclonal antibody specific for sPLA2-IIA. Then, an acetylcholinesterase-sPLA2-Fab’ conjugate was added to each well after washing. The concentration of the analyte was then determined by measuring the enzymatic activity of the acetylcholinesterase by adding Ellman’s reagent to each well and reading the product of the acetylcholinesterase catalyzed reaction in an ELISA plate reader (Tecan, Mannedorf, Switzerland) at 412 nm. sPLA2-IIA concentrations in the samples were calculated from a standard curve using recombinant sPLA2-IIA as an a standard. The sensitivity of the assay was 5 pg/mL. To avoid possible non-specific reactions due to anti-mouse IgM or IgG heterophilic antibodies, each sample was treated with non-specific mouse serum prior to addition to the well. All values are expressed as pg sPLA2 per mg total protein. Total cell protein concentration was determined by using Bradford protein assay with bovine serum albumin as an internal standard (Sigma, St. Louis, MO, USA).

For the measurement of ERK 1/2 phosphorylation, HUVECs were cultured in 96 well plates. On the day of the assay, the cell culture medium was replaced with fresh growth medium before treating the monolayer with appropriate reagents. PD98059 was added to HUVECs for 30 min prior to 5 min treatment with the proteases. The phosphorylation of ERK 1/2 was evaluated in the cell lysates by using specific Phospho ERK 1/2 ELISA kit (Thermo Scientific, Rockford, IL, USA) according to the manufacture’s protocols.

Statistical Analysis

Results are expressed as means ± standard deviation (S.D.) of at least three independent experiments. The statistical significance of differences between test groups was used for statistical comparison (SPSS, version 14.0, SPSS Science, Chicago, Il, USA). Statistical relevance was determined using analysis of variance (ANOVA) with p-values less than 0.05 (p < 0.05).

Results and Discussion

TNF-α concentration-dependence of expression of sPLA2-IIA by HUVECs

It is well known that TNF-α and other inflammatory cytokines up-regulate the transcription of sPLA-IIA and its protein level in a variety of cells including endothelial cells [34], vascular smooth muscle cells [35], chondrocytes [36], mesangial cells [37], astrocytes [38, 39], and hepatoma cells [39]. The concentration-dependence of the TNF-α-mediated expression of sPLA2-IIA in endothelial cells is presented in Fig. 1. Analysis of the expression level of sPLA2-IIA by the transformed HUVECs in response to varying concentrations of TNF-α for 4 h indicated that the induction level reaches plateau in both cell culture supernatants (Fig. 1A) and cell lysates (Fig. 1B) at 100 ng/mL TNF-α. Similar expression levels in both supernatants and cell lysates suggest that the TNF-α-mediated enhancement of sPLA2-IIA may not be due to its release from preformed storage vesicles, but rather due to the new synthesis of the molecule by the cytokine stimulated endothelial cells. A similar effect for TNF-α was observed if endothelial cells were cultured in serum free medium containing 0.2% BSA, excluding the possibility that the effect of TNF-α on sPLA2-IIA expression is due to its interference with the function of some unknown factors in the serum. This effect was not specific for the transformed HUVECs since we essentially obtained identical results with primary HUVECs (data not shown). Based on these results, a TNF-α concentration of 100 ng/mL was used to stimulate endothelial cells in all experiments described below.

Figure 1
Inducible effect of TNF-α on the expression of sPLA2-IIA in endothelial cells

Effect of thrombin and APC on the sPLA2-IIA activity

Previous results have indicated that thrombin initiates a proinflammatory response in HUVECs by up-regulating the activation of the NF-κB pathway by a PAR-1-dependent mechanism [9-12, 30-32]. Surprisingly, however, the incubation of HUVECs with thrombin (up to 10 nM) did not have any effect on the expression of sPLA2-IIA (Fig. 2). Under these conditions, thrombin can activate all cell surface PAR-1 [23-25]. Thus, unlike the activation of the NF-κB pathway, the activation of PAR-1 by thrombin does not lead to up-regulation of sPLA2-IIA in HUVECs. A previous study showed that plasma derived APC effectively diminished the expression of sPLA2-IIA by interferon-γ (INF-γ) in human aortic smooth muscle cells [40]. In this study, we investigated this question in HUVECs (both in transformed and primary cells) and found that APC potently inhibits the expression of sPLA2-IIA in TNF-α-stimulated cells (Fig. 2). While thrombin had no effect on the expression of sPLA2-IIA in response to TNF-α, it nevertheless, potently inhibited its expression if endothelial cells were pretreated with wild-type PC or PC-S195A prior to incubation of cells with thrombin and TNF-α (Fig. 2). A lack of activity for either PC or PC-S195A in this assay is not due to the weaker and/or the loss of the affinity of proteins for interaction with EPCR since both wild-type and mutant PC zymogens can bind to the receptor with a dissociation constant of ~30 nM that is essentially identical to the affinity of APC for the receptor [28, 41, 42]. Thus, in agreement with previous results, neither PC nor PC-S195A can elicit PAR-1-dependent cellular response in HUVECs [28, 41, 42].

Figure 2
Effect of APC or thrombin on the expression of sPLA2-IIA in HUVECs pretreated with PC-S195A

The expression of sPLA2-IIA is modulated by an EPCR- and PAR-1-dependent mechanism

It is known that both APC and thrombin elicit cellular responses in endothelial cells by activating PAR-1 [17, 43]. To determine whether the effect of APC and thrombin + PC-S195A in inhibition of sPLA2-IIA in response to TNF-α is also mediated through the proteases activating PAR-1, the same studies described above were conducted in the presence of function-blocking antibodies to both PAR-1 and EPCR. As shown in Fig. 3, function-blocking antibodies to both EPCR and PAR-1 eliminated the inhibitory effects of both APC and thrombin on the expression of sPLA2-IIA, suggesting that that the EPCR-dependent cleavage of PAR-1 mediates the cellular effects of both proteases (Fig. 3).

Figure 3
The inhibitory effects of both APC and thrombin on the expression of sPLA2-IIA are mediated through the PAR-1 and EPCR pathways

PI3 kinase modulates the expression of sPLA2-IIA

Previous studies have indicated that the PAR-1-dependent cellular effect of both thrombin and APC is mediated through the activations of the PI3 kinase [25, 44] and ERK 1/2 pathways [21-23]. However, zymogen PC could not alter PI3 kinase pathway [25]. To determine whether PI3 kinase is also involved in the inhibition of sPLA2-IIA expression by APC and thrombin + PC-S195A, endothelial cells were preincubated with specific PI3-kinase inhibitor, LY294002. As presented in Fig. 4, the inhibitor effectively suppressed the sPLA2-IIA modulatory effects of both APC and thrombin in TNF-α-stimulated endothelial cells, suggesting that this effect is mediated through the proteases activating the PI3 kinase pathway by an EPCR- and PAR-1-dependent mechanism. The activation of the PI3 kinase pathway by APC requires an intact active site since the zymogen PC cannot alter this pathway [25].

Figure 4
Effect of the PI3 kinase inhibitor (LY294002) and methyl-β-cyclodextrin (MβCD) on the expression of sPLA2-IIA in endothelial cells

To determine whether, similar to APC, the phosphorylation of ERK 1/2 is involved in the EPCR-dependent protective signaling effect of thrombin, we quantitatively analyzed the extent of phosphorylation of ERK 1/2 by using Phospho ERK 1/2 ELISA kit. As shown in Fig. 5, both APC and thrombin + PC-S195A, but not PC or PC-S195A alone, up-regulated the intracellular levels of phosphorylated ERK 1/2 in transformed HUVECs. Consistent with these results, the ERK 1/2 inhibitor (PD98059) prevented the APC or thrombin + PC-S195A induced ERK 1/2 phosphorylation in HUVECs (Fig. 5). Based on these results, we conclude that the inhibitory effect of both APC and thrombin + PC-S195A on the expression of sPLA2-IIA are mediated through the activation of the ERK 1/2 pathway. As with all other experiments in this manuscript, we confirmed these results are all applicable to primary HUVECs (data not shown).

Figure 5
Effects of APC and thrombin + PC-S195A on the phosphorylation of ERK 1/2

Effect of MβCD on the modulation of sPLA2-IIA activity by APC and thrombin

Recently, we demonstrated that EPCR and PAR-1 are associated with caveolin-1 within the lipid rafts of HUVECs and that the antiinflammatory effects of APC or thrombin + PC-S195A are abolished if endothelial cells are pretreated with cholesterol depleting agent, methyl-β-cyclodextrin (MβCD) [26, 29]. To determine whether similar events are responsible for the EPCR- and PAR-1-dependent inhibition of sPLA2-IIA expression by APC or thrombin, endothelial cells were preincubated with MβCD. As shown in Fig. 4, MβCD eliminated the inhibitory activity of both APC and thrombin on the expression of sPLA2-IIA, suggesting that the cleavage of PAR-1 by either protease outside the lipid rafts has no protective effect against the proinflammatory effect of TNF-α in endothelial cells. However, when EPCR is occupied, the activation of the lipid-raft localized PAR-1 by either thrombin or APC elicits protective responses, thereby inhibiting the expression of proinflammatory molecules by the cytokine-activated endothelial cells. In a previous study we showed that both unoccupied EPCR and PAR-1 are associated with caveolin-1 within lipid rafts of endothelial cells [26, 28]. However, when EPCR is occupied by protein C, the receptor dissociates from caveolin-1, and this process appears to be linked to changing the G-protein coupling specificity of PAR-1 so that its cleavage by either thrombin or APC leads to initiation of protective intracellular responses in endothelial cells [26, 28]. We believe that a similar mechanism accounts for the EPCR- and PAR-1-dependent thrombin suppression of TNF-α-mediated up-regulation of sPLA2-IIA in endothelial cells.

The modulatory effect of proteases on the expression of sPLA2-IIA can be mimicked by TRAP

It is well known that the thrombin receptor agonist peptide (TRAP) TFLLRN specifically activates PAR-1 on endothelial cells [43, 45] and the activation of PAR-1 by the TRAP peptide is known to mimic the proinflammatory effect of thrombin in HUVECs [46, 47]. As presented in Fig. 6, TRAP alone could not inhibit the TNF-α induced sPLA2-IIA expression. However, as observed with thrombin, if endothelial cells were treated with PC-S195A, TRAP inhibited the TNF-α-induced sPLA2-IIA expression, supporting the conclusion that when EPCR is occupied by its ligand, PAR-1 activation reverses the proinflammatory response of TNF-α in endothelial cells (Fig. 6). TRAP alone had no effect on the expression of sPLA2-IIA (Fig. 6). We also tested the effect of function-blocking antibodies against both PAR-1 and EPCR. As expected, the antibody against PAR-1 was not effective because TRAP can activate PAR-1 independent of its cleavage [26, 28]. However, the inhibitory effect of TRAP with PC-S195A on the expression of sPLA2-IIA was abolished by the anti-EPCR antibody, supporting the hypothesis that the occupancy of EPCR modulates the signaling specificity of PAR-1. Consistent with the involvement of the PI3 kinase pathway in the PAR-1-dependent protective response, the PI3 kinase inhibitor, LY294002, inhibited the activity of TRAP in endothelial cells (Fig. 6). These results were reproducible in primary HUVECs (data not shown).

Figure 6
Effect of TRAP on the expression of sPLA2-IIA

Noting that neither thrombin nor TRAP had any effect on the TNF-α-mediated expression of sPLA2-IIA, the results presented above suggest that the activation of PAR-1 on endothelial cells has no modulatory effect on the expression of this molecule unless EPCR is occupied by its physiological ligand. We previously demonstrated that the occupancy of EPCR by protein C switches the PAR-1-dependent signaling specificity of thrombin from a proinflammatory response to an inflammatory response in cultured endothelial cells [26-28]. Thus, consistent with the literature [26-28], in our previous studies we showed that thrombin activates the NF-κB pathway and elevates the expression level of related proinflammatory molecules including the expression of cell adhesion molecules in endothelial cells. PC nor PC-S195A alone could not alter NF-κB pathway [27]. However, we showed that when endothelial cells are pretreated with a catalytically inactive S195A mutant of the zymogen protein C so that to occupy EPCR, the activation of PAR-1 by thrombin can no longer activate the NF-κB pathway, but rather it elicits a protective response thus inhibiting the activation of the proinflammatory pathways in the cytokine stimulated endothelial cells [27]. It is already known that the TNF-α mediated activation of the NF-κB pathway is required for the induction of sPLA2-IIA gene expression [48]. Thus, based on our results here and those published previously [48], we hypothesize that the EPCR and PAR-1-dependent activations of PI3 kinase and ERK 1/2 pathways inhibit the expression of sPLA2-IIA through down-regulation of the TNF-α-mediated NF-κB pathway.

sPLA2-IIA has been hypothesized to play a role in regulating inflammation. Thus, a high level of this molecule has been found in the sera of patients with inflammatory disorders with its level apparently correlating with the severity of inflammation [9-12]. However, the possibility that sPLA2-IIA is only an inflammatory marker rather than a contributor to inflammation, has not been ruled out. Given that the activation of PAR-1 can activate the NF-κB pathway [9-12, 30-32], the observations that neither thrombin nor TRAP induced the expression level of sPLA2-IIA in endothelial cells suggest that, unlike other proinflammatory molecules, the sPLA2-IIA gene may not be modulated through the NF-κB pathway. Thus, further studies will be required to understand how TNF-α up-regulates the expression of sPLA2-IIA in endothelial cells.

Acknowledgements

We would like to thank Korea National Herbal Cosmeceutical Material Bank supported by KOSEF for providing some materials for these studies. These studies were supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MEST, 2009-0065105) to Bae JS and by grants awarded by the National Heart, Lung, and Blood Institute of the National Institute of Health HL 68571 and HL 62565 to ARR.

Abbreviations

sPLA2-IIA
secretory phospholipase A2 of group IIA
TNF-α
tumor necrosis factor alpha
PC
protein C
APC
activated protein C
PAR-1
protease-activated receptor-1
ELISA
enzyme-linked immunosorbent assay
EPCR
endothelial protein C receptor
HUVEC
human umbilical vein endothelial cell
TRAP
thrombin receptor agonist peptide
BSA
bovine serum albumin
Th
thrombin
PI3
phosphatidylinositol 3
PC-S195A
Ser-195 to Ala substitution mutant of protein C
ERK 1/2
extracellular signal-regulated kinase

Footnotes

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References

[1] Six DA, Dennis EA. The expanding superfamily of phospholipase A(2) enzymes: classification and characterization. Biochim Biophys Acta. 2000;1488:1–19. [PubMed]
[2] Kudo I, Murakami M. Phospholipase A2 enzymes. Prostaglandins Other Lipid Mediat. 2002;68-69:3–58. [PubMed]
[3] Chang HW, Kudo I, Tomita M, Inoue K. Purification and characterization of extracellular phospholipase A2 from peritoneal cavity of caseinate-treated rat. J Biochem. 1987;102:147–54. [PubMed]
[4] Kramer RM, Hession C, Johansen B, Hayes G, McGray P, Chow EP, et al. Structure and properties of a human non-pancreatic phospholipase A2. J Biol Chem. 1989;264:5768–75. [PubMed]
[5] Seilhamer JJ, Pruzanski W, Vadas P, Plant S, Miller JA, Kloss J, et al. Cloning and recombinant expression of phospholipase A2 present in rheumatoid arthritic synovial fluid. J Biol Chem. 1989;264:5335–8. [PubMed]
[6] Murakami M, Shimbara S, Kambe T, Kuwata H, Winstead MV, Tischfield JA, et al. The functions of five distinct mammalian phospholipase A2S in regulating arachidonic acid release. Type IIa and type V secretory phospholipase A2S are functionally redundant and act in concert with cytosolic phospholipase A2. J Biol Chem. 1998;273:14411–23. [PubMed]
[7] Menschikowski M, Hagelgans A, Siegert G. Secretory phospholipase A2 of group IIA: is it an offensive or a defensive player during atherosclerosis and other inflammatory diseases? Prostaglandins Other Lipid Mediat. 2006;79:1–33. [PubMed]
[8] Mounier CM, Hackeng TM, Schaeffer F, Faure G, Bon C, Griffin JH. Inhibition of prothrombinase by human secretory phospholipase A2 involves binding to factor Xa. J Biol Chem. 1998;273:23764–72. [PubMed]
[9] Vadas P, Pruzanski W. Role of secretory phospholipases A2 in the pathobiology of disease. Lab Invest. 1986;55:391–404. [PubMed]
[10] Pruzanski W, Vadas P. Phospholipase A2--a mediator between proximal and distal effectors of inflammation. Immunol Today. 1991;12:143–6. [PubMed]
[11] Waydhas C, Nast-Kolb D, Duswald KH, Lehnert P, Schweiberer L. Prognostic value of serum phospholipase A in the multitraumatized patient. Klin Wochenschr. 1989;67:203–6. [PubMed]
[12] Pruzanski W, Vadas P, Stefanski E, Urowitz MB. Phospholipase A2 activity in sera and synovial fluids in rheumatoid arthritis and osteoarthritis. Its possible role as a proinflammatory enzyme. J Rheumatol. 1985;12:211–6. [PubMed]
[13] Vadas P, Browning J, Edelson J, Pruzanski W. Extracellular phospholipase A2 expression and inflammation: the relationship with associated disease states. J Lipid Mediat. 1993;8:1–30. [PubMed]
[14] Tanaka K, Kato T, Matsumoto K, Yoshida T. Antiinflammatory action of thielocin A1 beta, a group II phospholipase A2 specific inhibitor, in rat carrageenan-induced pleurisy. Inflammation. 1993;17:107–19. [PubMed]
[15] Murakami M, Nakatani Y, Atsumi G, Inoue K, Kudo I. Regulatory functions of phospholipase A2. Crit Rev Immunol. 1997;17:225–83. [PubMed]
[16] Joyce DE, Gelbert L, Ciaccia A, DeHoff B, Grinnell BW. Gene expression profile of antithrombotic protein c defines new mechanisms modulating inflammation and apoptosis. J Biol Chem. 2001;276:11199–203. [PubMed]
[17] Mosnier LO, Zlokovic BV, Griffin JH. The cytoprotective protein C pathway. Blood. 2007;109:3161–72. [PubMed]
[18] Esmon CT. Molecular events that control the protein C anticoagulant pathway. Thromb Haemost. 1993;70:29–35. [PubMed]
[19] Bernard GR, Vincent JL, Laterre PF, LaRosa SP, Dhainaut JF, Lopez-Rodriguez A, et al. Efficacy and safety of recombinant human activated protein C for severe sepsis. N Engl J Med. 2001;344:699–709. [PubMed]
[20] Riewald M, Petrovan RJ, Donner A, Mueller BM, Ruf W. Activation of endothelial cell protease activated receptor 1 by the protein C pathway. Science. 2002;296:1880–2. [PubMed]
[21] Uchiba M, Okajima K, Oike Y, Ito Y, Fukudome K, Isobe H, et al. Activated protein C induces endothelial cell proliferation by mitogen-activated protein kinase activation in vitro and angiogenesis in vivo. Circ Res. 2004;95:34–41. [PubMed]
[22] Xue M, Campbell D, Sambrook PN, Fukudome K, Jackson CJ. Endothelial protein C receptor and protease-activated receptor-1 mediate induction of a wound-healing phenotype in human keratinocytes by activated protein C. J Invest Dermatol. 2005;125:1279–85. [PubMed]
[23] Bretschneider E, Uzonyi B, Weber AA, Fischer JW, Pape R, Lotzer K, et al. Human vascular smooth muscle cells express functionally active endothelial cell protein C receptor. Circ Res. 2007;100:255–62. [PubMed]
[24] Garcia JG, Pavalko FM, Patterson CE. Vascular endothelial cell activation and permeability responses to thrombin. Blood Coagul Fibrinolysis. 1995;6:609–26. [PubMed]
[25] Finigan JH, Dudek SM, Singleton PA, Chiang ET, Jacobson JR, Camp SM, et al. Activated protein C mediates novel lung endothelial barrier enhancement: role of sphingosine 1-phosphate receptor transactivation. J Biol Chem. 2005;280:17286–93. [PubMed]
[26] Bae JS, Rezaie AR. Protease activated receptor 1 (PAR-1) activation by thrombin is protective in human pulmonary artery endothelial cells if endothelial protein C receptor is occupied by its natural ligand. Thromb Haemost. 2008;100:101–9. [PMC free article] [PubMed]
[27] Bae JS, Rezaie AR. Thrombin inhibits nuclear factor kappaB and RhoA pathways in cytokine-stimulated vascular endothelial cells when EPCR is occupied by protein C. Thromb Haemost. 2009;101:513–20. [PMC free article] [PubMed]
[28] Bae JS, Yang L, Manithody C, Rezaie AR. The ligand occupancy of endothelial protein C receptor switches the protease-activated receptor 1-dependent signaling specificity of thrombin from a permeability-enhancing to a barrier-protective response in endothelial cells. Blood. 2007;110:3909–16. [PubMed]
[29] Bae JS, Yang L, Rezaie AR. Receptors of the protein C activation and activated protein C signaling pathways are colocalized in lipid rafts of endothelial cells. Proc Natl Acad Sci U S A. 2007;104:2867–72. [PubMed]
[30] Joyce DE, Chen Y, Erger RA, Koretzky GA, Lentz SR. Functional interactions between the thrombin receptor and the T-cell antigen receptor in human T-cell lines. Blood. 1997;90:1893–901. [PubMed]
[31] Lawrence T, Gilroy DW, Colville-Nash PR, Willoughby DA. Possible new role for NF-kappaB in the resolution of inflammation. Nat Med. 2001;7:1291–7. [PubMed]
[32] Joyce DE, Grinnell BW. Recombinant human activated protein C attenuates the inflammatory response in endothelium and monocytes by modulating nuclear factor-kappaB. Crit Care Med. 2002;30:S288–93. [PubMed]
[33] Menschikowski M, Hagelgans A, Heyne B, Hempel U, Neumeister V, Goez P, et al. Statins potentiate the IFN-gamma-induced upregulation of group IIA phospholipase A2 in human aortic smooth muscle cells and HepG2 hepatoma cells. Biochim Biophys Acta. 2005;1733:157–71. [PubMed]
[34] Murakami M, Kudo I, Inoue K. Molecular nature of phospholipases A2 involved in prostaglandin I2 synthesis in human umbilical vein endothelial cells. Possible participation of cytosolic and extracellular type II phospholipases A2. J Biol Chem. 1993;268:839–44. [PubMed]
[35] Nakano T, Ohara O, Teraoka H, Arita H. Group II phospholipase A2 mRNA synthesis is stimulated by two distinct mechanisms in rat vascular smooth muscle cells. FEBS Lett. 1990;261:171–4. [PubMed]
[36] Kerr JS, Stevens TM, Davis GL, McLaughlin JA, Harris RR. Effects of recombinant interleukin-1 beta on phospholipase A2 activity, phospholipase A2 mRNA levels, and eicosanoid formation in rabbit chondrocytes. Biochem Biophys Res Commun. 1989;165:1079–84. [PubMed]
[37] Schalkwijk C, Pfeilschifter J, Marki F, van den Bosch H. Interleukin-1 beta, tumor necrosis factor and forskolin stimulate the synthesis and secretion of group II phospholipase A2 in rat mesangial cells. Biochem Biophys Res Commun. 1991;174:268–75. [PubMed]
[38] Oka S, Arita H. Inflammatory factors stimulate expression of group II phospholipase A2 in rat cultured astrocytes. Two distinct pathways of the gene expression. J Biol Chem. 1991;266:9956–60. [PubMed]
[39] Crowl RM, Stoller TJ, Conroy RR, Stoner CR. Induction of phospholipase A2 gene expression in human hepatoma cells by mediators of the acute phase response. J Biol Chem. 1991;266:2647–51. [PubMed]
[40] Menschikowski M, Hagelgans A, Hempel U, Lattke P, Ismailov I, Siegert G. On interaction of activated protein C with human aortic smooth muscle cells attenuating the secretory group IIA phospholipase A2 expression. Thromb Res. 2008;122:69–76. [PubMed]
[41] Bae JS, Yang L, Manithody C, Rezaie AR. Engineering a disulfide bond to stabilize the calcium-binding loop of activated protein C eliminates its anticoagulant but not its protective signaling properties. J Biol Chem. 2007;282:9251–9. [PubMed]
[42] Mosnier LO, Griffin JH. Inhibition of staurosporine-induced apoptosis of endothelial cells by activated protein C requires protease-activated receptor-1 and endothelial cell protein C receptor. Biochem J. 2003;373:65–70. [PubMed]
[43] Ossovskaya VS, Bunnett NW, de Garavilla L, Vergnolle N, Young SH, Ennes H, et al. Protease-activated receptors: contribution to physiology and disease. Physiol Rev. 2004;84:579–621. [PubMed]
[44] Bae JS, Kim YU, Park MK, Rezaie AR. Concentration dependent dual effect of thrombin in endothelial cells via Par-1 and Pi3 Kinase. J Cell Physiol. 2009;219:744–51. [PMC free article] [PubMed]
[45] Steinhoff M, Buddenkotte J, Shpacovitch V, Rattenholl A, Moormann C, Vergnolle N, et al. Proteinase-activated receptors: transducers of proteinase-mediated signaling in inflammation and immune response. Endocr Rev. 2005;26:1–43. [PubMed]
[46] Shankar R, de la Motte CA, Poptic EJ, DiCorleto PE. Thrombin receptor-activating peptides differentially stimulate platelet-derived growth factor production, monocytic cell adhesion, and E-selectin expression in human umbilical vein endothelial cells. J Biol Chem. 1994;269:13936–41. [PubMed]
[47] Feistritzer C, Riewald M. Endothelial barrier protection by activated protein C through PAR1-dependent sphingosine 1-phosphate receptor-1 crossactivation. Blood. 2005;105:3178–84. [PubMed]
[48] Alaoui-El-Azher M, Wu Y, Havet N, Israel A, Lilienbaum A, Touqui L. Arachidonic acid differentially affects basal and lipopolysaccharide-induced sPLA(2)-IIA expression in alveolar macrophages through NF-kappaB and PPAR-gamma-dependent pathways. Mol Pharmacol. 2002;61:786–94. [PubMed]