Journal of Thrombosis and Haemostasis, 12: 1918–1920

DOI: 10.1111/jth.12729

COMMENTARY

Secretory group V phospholipase A2: a new player in thrombosis? L. VIJAYA MOHAN RAO Department of Cellular and Molecular Biology, The University of Texas Health Science Center at Tyler, Tyler, TX, USA

To cite this article: Rao LVM. Secretory group V phospholipase A2: a new player in thrombosis? J Thromb Haemost 2014; 12: 1918–20. See also Tamayo I, Velasco SE, Puy C, Esmon CT, Dichiara MG, Montes R, Hermida J. Group V secretory phospholipase A2 impairs endothelial protein C receptor-dependent protein C activation and accelerates thrombosis in vivo. This issue, pp 1921–7.

The protein C anticoagulant system plays a critical role in the regulation of hemostasis and inflammation. Binding of protein C to endothelial cell protein C receptor (EPCR) and the subsequent activation of EPCR-bound protein C by thrombin–thrombomodulin are the key steps in the protein C anticoagulant system. Activated protein C (APC)-mediated cell signaling, supported by its binding to EPCR or independently of EPCR, activates anti-inflammatory and cytoprotective pathways [1]. Therefore, pathophysiologic conditions that impair the interaction of protein C with EPCR have the potential to decrease APC generation. Such reduced binding of protein C to EPCR may not only contribute to thrombotic disorders, but also result in the loss of endogenous APCmediated cytoprotection. EPCR shares a sequence and a three-dimensional homology with the major histocompatibility class 1/CD1 family of proteins, particularly CD1d [2,3]. Similarly to the CD1 family proteins, EPCR has a tightly bound phospholipid in the antigen-presenting groove [3]. This similarity led to an initial belief that EPCR may play a role in presenting protein C/APC or lipid antigen in inflammatory cells [2]. However, at present, there is no evidence that EPCR plays a role in presenting lipid antigens to inflammatory cells. However, the presence of the lipid in EPCR seems to be essential for EPCR binding to protein C, as extraction of the lipid from EPCR abolishes protein C binding [3]. Molecular dynamic simulations of Correspondence: L. Vijaya Mohan Rao, Department of Cellular and Molecular Biology, Center for Biomedical Research, The University of Texas Health Science Center at Tyler, 11937 US Highway 271, Tyler, TX 75708, USA. Tel.: +1 903 877 7332; fax: +1 903 877 7426. E-mail: [email protected] Received 25 August 2014 Manuscript handled by: P. H. Reitsma Final decision: P. H. Reitsma, 29 August 2014

phosphatidylethanolamine-bound and phosphatidylethanolamine-unbound forms of EPCR reveal that the lipid probably maintains the conformation of EPCR that is necessary for the interaction with its ligands [4]. Recently, Hermida et al. showed that phosphatidylcholine (PC) is the major phospholipid bound to human EPCR, and that lipid exchange can occur in EPCR, just as it can in CD1d [5]. They also showed that the exchange of PC in EPCR for lyso-PC or platelet-activating factor (PAF) impaired the ability of EPCR to bind protein C and factor VII, indicating that EPCR function could be modulated by a change in the identity of the phospholipid in the hydrophobic groove of EPCR. Secretory group V phospholipase A2 (sPLA2-V), an enzyme that can be upregulated in a variety of inflammatory conditions and that metabolizes PC to lyso-PC, is capable of modulating both the binding of protein C to EPCR and the generation of APC on endothelial cells [5]. These data have raised the possibility that sPLA2-V may exert prothrombotic and proinflammatory effects through modification of the bound lipid in EPCR (Fig. 1). In a study published in this issue of the Journal of Thrombosis and Haemostasis, Tamayo et al. provide evidence that sPLA2-V does indeed play a thrombogenic role in vivo [6]. The data presented in the article show that overexpression of sPLA2-V in mice by hydrodynamic gene delivery impairs the ability of mice to activate protein C. More importantly, sPLA2-V overexpression accelerates thrombus formation in a carotid artery laser thrombosis model. When EPCR was blocked with a blocking antibody, sPLA2-V overexpression no longer had a significant influence on APC generation or thrombus formation. Furthermore, administration of manoalide, an inhibitor of sPLA2-V, significantly increased APC generation and moderately reduced thrombus formation in wild-type mice. From these data, the authors conclude that sPLA2-V downregulates protein C activation in vivo by encrypting EPCR, and thus promotes thrombus formation. The authors raise the possibility of targeting sPLA2-V activity as an antithrombotic strategy. © 2014 International Society on Thrombosis and Haemostasis

sPLA2-V: a new player in thrombosis? 1919

Protein C

APC

APC Protein C TM

TM

T

T EPCR

PC

sPLA2-V EPCR

Lyso -PC

Fig. 1. A hypothetical model of secretory group V phospholipase A2 (sPLA2-V) impairment of protein C activation. Functional endothelial cell protein C receptor (EPCR) contains a phosphatidylcholine (PC) in its hydrophobic groove. In this conformation, EPCR can bind protein C. Thrombin (T)–thrombomodulin (TM) complex activates the protein C bound to EPCR to generate activated protein C (APC). sPLA2-V hydrolyzes the PC in the EPCR to lyso-PC. The lipid modification in the hydrophobic groove of EPCR leads to a structural rearrangement in the helical region of EPCR, which probably results in narrowing of the pocket of the ligand-binding groove. The structurally rearranged EPCR is unable to bind protein C. The loss of protein C binding to EPCR translates into impaired protein C activation by the T–TM complex.

The present in vivo study builds on the authors’ earlier study in which they investigated the role of lipid and sPLA2-V on modulating protein C binding to EPCR using recombinant human soluble EPCR (sEPCR) and EPCR expressed on endothelial cells. To understand the true significance of the present study and its limitations, one should first know what the earlier study had shown, and, more importantly, what it did not show. The earlier study showed the following: (i) PC is the phospholipid located in the hydrophobic pocket of EPCR; (ii) delipidated sEPCR has reduced ability to interact with its ligands; (iii) lyso-PC and PAF can be located in the hydrophobic pocket of sEPCR; (iv) sEPCR containing lyso-PC or PAF has impaired APC-binding ability; (v) and inhibition of sPLA2-V on endothelial cells, either by treatment of cells with sPLA2-V inhibitor or by silencing the sPLA2-V gene, increases ligand binding to EPCR and enhances APC generation on endothelial cells. The authors strongly infer from these data that sPLA2-V modulates the ability of EPCR to generate APC by hydrolyzing the PC in the EPCR to lyso-PC. It is important to note here that no evidence is presented in this study to show that the inhibition of sPLA2-V activity actually changed the lipid in EPCR on endothelial cells. The data presented in the current report [6] clearly demonstrate that overexpression of sPLA2-V inhibits APC generation, whereas inhibition of endogenous sPLA2-V increases APC generation in vivo. sPLA2-V overexpression did not alter the expression levels of EPCR or thrombomodulin, and appeared to have no significant effect on overall hemostatic balance as measured with whole blood thromboelastometry. However, this study does not provide evidence that sPLA2-V actually modifies the lipid bound to EPCR, or that such lipid © 2014 International Society on Thrombosis and Haemostasis

modification is responsible for the observed decrease in APC generation in mice overexpressing sPLA2-V. With the current existing methodologies, it may not be feasible to show in vivo that sPLA2-V alters the lipid in EPCR on the endothelium. However, lack of direct evidence for such a mechanism, even in the in vitro cell system, makes it difficult to conclude definitively that sPLA2-V-mediated lipid modification in EPCR is responsible for the reduced APC generation that results in accelerated thrombus formation in mice overexpressing sPLA2-V. There are a number of other caveats and limitations to the present study. Some of them are probably unavoidable, but others are avoidable. Lack of a suitable method to effectively measure the plasma levels of sPLA2-V makes it difficult to determine whether any correlation exists between sPLA2-V and APC levels in the above group of mice. There is no information in the present study on whether sPLA2-V overexpression actually impairs protein C binding to EPCR on the endothelium. This could have been easily examined by measuring protein C levels in circulation in wild-type and sPLA2-V-overexpressing mice. This study measured APC levels in mice following PC/phosphatidylserine/FXa challenge, and not in control unchallenged mice. As the reduction in APC levels in sPLA2-Voverexpressing mice is proposed to be responsible for the accelerated thrombus formation following laser injury in mice that are not challenged with PC/phosphatidylserine/ FXa prior to the laser injury, it is more appropriate to measure APC levels in unchallenged mice. To substantiate that EPCR is needed for sPLA2-V to exert its prothrombotic effect, sPLA2-V-overexpressing mice were treated with an anti-EPCR mAb that blocks protein C/APC binding to EPCR. This experiment showed that sPLA2-V overexpression was unable to accelerate thrombus formation when EPCR was blocked with the antibody. However, the EPCR blocking antibody treatment showed a stronger prothrombotic effect than sPLA2-V overexpression (i.e. a greater reduction in APC generation and increased thrombus formation). Thus, blocking protein C binding to EPCR is not an appropriate experiment with which to assess the role of EPCR in the sPLA2-V-mediated prothrombotic effect. Despite the above caveats and limitations, the present study provides valuable and novel information; that is, sPLA2-V plays a thrombogenic role in vivo. sPLA2-V is constitutively expressed by endothelial cells, and its expression is increased by inflammatory stimuli, such as tumor necrosis factor-a and vascular endothelial growth factor [7,8]. sPLA2-V is expressed at all stages of atherosclerosis development, and is thought to play an atherogenic role through degradation of phospholipids [9,10]. Therefore, it is entirely possible that upregulation of sPLA2-V in inflammation and other diseases may contribute, at least partly, to thrombotic disorders associated with these diseases. If this is so, inhibition of sPLA2-V activity in such patients may represent an effective anti-

1920 L. Vijaya Mohan Rao

thrombotic therapeutic strategy. To further investigate this possibility, it is important to first determine whether sPLA2-V levels correlate with thrombotic risk in specific patient groups. This requires the development of a suitable assay with which to measure sPLA2-V levels in plasma. APC plays an influential role in many disease processes, including thrombosis, sepsis, inflammation, diabetes, and cancer [1,11–13]. Protein C binding to EPCR is critical for the generation of APC. APC-mediated antiinflammatory and cytoprotective effects most frequently require APC binding to EPCR. In addition to protein C and APC, many other ligands bind EPCR. They include FVIIa, proteinase-3, b2-integrin Mac-1, Plasmodium falciparum erythrocyte membrane protein 1, and cd T-cell antigen receptor [1]. Recent studies elucidating the importance of interactions between these novel ligands and EPCR indicate new roles for EPCR in hemostasis, the pathogenesis of severe malaria, innate immunity, and cancer [1]. Interestingly, most of the ligands that bind EPCR do so at sites that overlap with the binding site for protein C. It will therefore be very interesting to ascertain whether EPCR lipid modification also influences the binding of other ligands to EPCR. If the proposed mechanism of sPLA2-V-mediated impairment of protein C activation is proven to be correct, i.e. if sPLA2-V-mediated lipid modification in EPCR results in the loss of ligand binding to EPCR, it opens an exciting new area of research investigation related to regulation of EPCR function through lipid modifications within EPCR. If the present findings are confirmed and extended, the use of sPLA2-V inhibitors to treat various thrombotic, inflammatory and vascular diseases may become a viable option in the future. For this, we have to build a stronger basic foundation by convincingly demonstrating that sPLA2-V modifies the lipid in EPCR in vivo, and that this lipid modification is responsible for EPCR encryption. Disclosure of Conflict of Interests This work was supported by National Institutes of Health grant HL1074830. The author states that he has no conflict of interest. For the record, the author wants to disclose that he has ongoing collaboration with Esmon, who was one of the coauthors of the article that was com-

mented on here, on projects that are not related to the present study. References 1 Rao LV, Esmon CT, Pendurthi UR. Endothelial cell protein C receptor: a multi-liganded and multi-functional receptor. Blood 2014; 124: 1553–62. 2 Fukudome K, Esmon CT. Identification, cloning, and regulation of a novel endothelial cell protein C/activated protein C receptor. J Biol Chem 1994; 269: 26486–91. 3 Oganesyan V, Oganesyan N, Terzyan S, Qu D, Dauter Z, Esmon NL, Esmon CT. The crystal structure of the endothelial protein C receptor and a bound phospholipid. J Biol Chem 2002; 277: 24851–4. 4 Chiappori F, Merelli I, Milanesi L, Rovida E. Exploring the role of the phospholipid ligand in endothelial protein C receptor: a molecular dynamics study. Proteins 2010; 78: 2679–90. 5 Lopez-Sagaseta J, Puy C, Tamayo I, Allende M, Cervero J, Velasco SE, Esmon CT, Montes R, Hermida J. sPLA2-V inhibits EPCR anticoagulant and antiapoptotic properties by accommodating lysophosphatidylcholine or PAF in the hydrophobic groove. Blood 2012; 119: 2914–21. 6 Tamayo I, Velasco SE, Puy C, Esmon CT, Dichiara MG, Montes R, Hermida J. sPLA2-V impairs EPCR-dependent protein C activation and accelerates thrombosis in vivo. J Thromb Haemost 2014; 12: 1921–27. 7 Sawada H, Murakami M, Enomoto A, Shimbara S, Kudo I. Regulation of type V phospholipase A2 expression and function by proinflammatory stimuli. Eur J Biochem 1999; 263: 826–35. 8 Sonoki K, Iwase M, Ohdo S, Ieiri I, Matsuyama N, Takata Y, Kitazono T. Telmisartan and N-acetylcysteine suppress group V secretory phospholipase A2 expression in TNFalpha-stimulated human endothelial cells and reduce associated atherogenicity. J Cardiovasc Pharmacol 2012; 60: 367–74. 9 Kimura-Matsumoto M, Ishikawa Y, Komiyama K, Tsuruta T, Murakami M, Masuda S, Akasaka Y, Ito K, Ishiguro S, Morita H, Sato S, Ishii T. Expression of secretory phospholipase A2s in human atherosclerosis development. Atherosclerosis 2008; 196: 81–91. 10 Murakami M, Kudo I. New phospholipase A(2) isozymes with a potential role in atherosclerosis. Curr Opin Lipidol 2003; 14: 431– 6. 11 Mosnier LO, Zlokovic BV, Griffin JH. The cytoprotective protein C pathway. Blood 2007; 109: 3161–72. 12 Rezaie AR. Regulation of the protein C anticoagulant and antiinflammatory pathways. Curr Med Chem 2010; 17: 2059– 69. 13 Esmon CT. Protein C anticoagulant system – anti-inflammatory effects. Semin Immunopathol 2012; 34: 127–32.

© 2014 International Society on Thrombosis and Haemostasis

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Secretory group V phospholipase A2 : a new player in thrombosis?

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