Yonago Acta medica 2014;57:119–127

Review Article: Special Contribution

Oxidized Phosphatidylserine: Production and Bioactivities
Tatsuya Matsura
Division of Medical Biochemistry, Department of Pathophysiological and Therapeutic Science, School of Medicine, Tottori University
Faculty of Medicine, Yonago 683-8503, Japan

Production of oxPS
It is well known that non-enzymatic oxidation of phospholipids including PS in membranes can be initiated byfree radicals or non-radical ROS under many pathological conditions.6 Here, selective, but not random oxidation of negatively charged phospholipids such as PS andcardiolipin (CL) is described in detail.

ABSTRACT
Recent development of analytical methods for lipid hydroperoxides and preparation of highly pure lipid hydroperoxides have revealed the important new pathophysiological roles of oxidized phospholipids. Generation ofreactive oxygen species and subsequent oxidative stressleads to random oxidation of membrane phospholipids.
However, recent studies have reported that anionic phospholipid molecules such as phosphatidylserine (PS) andcardiolipin are preferentially oxidized during apoptosis,
resulting in efficient apoptosis execution and apoptotic
cell clearance by phagocytes. This review is exclusively
focused on selective production of oxidized PS (oxPS)
during apoptosis as well as the novel roles of oxPS underpathophysiological conditions.

Asymmetric distribution of phospholipids in plasma membranes

Bretscher 9 has first postulated the concept of “phospholipid asymmetry” suggesting that phospholipids inplasma membrane are distributed asymmetrically between two leaflets. Indeed, in normal cells the cholinecontaining phospholipids, PC and sphingomyelin, residemainly in the outer leaflet of plasma membrane, whereas
the aminophospholipids, PS and, to a lesser extent, phosphatidylethanolamine (PE), are confined to the innerleaflet.10 It seems that at least three lipid translocators
are related to maintain this asymmetry. ATP-dependent
flippases, members of type 4 P-type ATPases, also re-

Key words anti-inflammatory; apoptosis; arteriosclerosis; lipid peroxidation; phosphatidylserine

From the 80s to the 90s, a number of studies on roles oflipid peroxidation in a variety of diseases and/or animal
disease models were carried out.1–3 However, whether
generation of lipid hydroperoxides resulted in or from
diseases was usually argued. It is assumed to be due tothe absence of precise methods for analyzing lipid hydroperoxides at that time. Recent development of analytical methods using mass spectrometry4 as well as preparation of highly pure lipid hydroperoxides5 have revealed
the following novel pathophysiological roles of oxidized
phospholipids: specific epitopes of innate immunity receptors, modification of intracellular signal transduction,pro- and anti-inflammatory activities, enhancement ofreactive oxygen species (ROS) generation, angiogenesis,
calcification of atherosclerotic plaques, inhibition of
acquired immunity and enhancement of blood coagulation.6, 7 These roles of oxidized phospholipids manifested in the above studies were predominantly related
to those of oxidatively modified products of phosphstidylcholine (PC), the most abundant phospholipid in themembrane. Phosphatidylserine (PS) is also known to
be a preferential target of in vivo oxidation.8 The aimof this review is to exclusively focus on the productionof oxidized phosphatidylserine (oxPS) and its biologicalactivities.

Corresponding author: Tatsuya Matsura, MD, PhD
tmatsura@med.tottori-u.ac.jp
Received 2014 October 9
Accepted 2014 October 9Abbreviations: 3-AT, 3-amino-1,2,4-triazole; AD, Alzheimer
disease, ALD, alcoholic liver disease; APC, activated protein C;
ATF6, activating transcription factor-6; BAI1, brain angiogenesis
inhibitor 1; Casp-3, caspase-3; Casp-8, caspase-8; CAT, catalase;CL, cardiolipin; COX, cyclooxygenase; cyt c, cytochrome c; EC,
endothelial cell; ER, endoplasmic reticulum; FMLP, formylmethionyl-leucyl-phenylalanine; Gas6, growth-arrest-specific 6;
HPTLC, high-performance thin-layer chromatography; HUVEC,
human umbilical vein endothelial cell; IL, interleukin; IRE1,
inositol-requiring enzyme 1; LBP, LPS-binding protein; LPS, lipopolysaccharide; MD-2, myeloid differentiation 2; Mel, melphalan;
MFG-E8, milk fat globule-EGF factor 8; miR-663, microRNA-663; NF, nuclear factor; NO, nitric oxide; NOX, NADPH oxidase; oxPAPC, oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero3-phosphocholine; oxPAPS, oxidized 1-palmitoyl-2-arachidonoylsn-glycero-3-phosphoserine; oxPS, oxidized phosphatidylserine;
PCI, protein C inhibitor; PC, phosphstidylcholine; PE, phosphatidylethanolamine; PERK, double-stranded RNA-activatedprotein kinase (PKR)-like ER kinase; PI, phosphatidylinositol;
PMA, phorbol myristate acetate; PS, phosphatidylserine; ROS,
reactive oxygen species; sCD14, soluble CD14; TIM-4, T-cell immunoglobulin- and mucin-domain-containing 4; TLR, Toll-likereceptor; TM, thrombomodulin; TMPD, N,N,N’,N’-tetramethylp-phenylenediamine dihydrochloride; TNF-alpha, tumor necrosis
factor-alpha; UPR, unfolded protein response; VEGF, vascularendothelial growth factor

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T. Matsura

ferred to as aminophospholipid translocases, are specificfor aminophospholipids, with a preference for PS overPE, and catalyze the inward translocation of aminophospholipids.11 Another ATP-dependent translocator floppases, members of ATP-binding cassette transporters,
catalyze the efflux of phospholipids from the inner to theouter leaflet with little selectivity for polar head groupof the phospholipids.12 Energy-independent scramblases
are non-selective and catalyze bidirectional transbilayer
movement of phospholipids Ca2+-dependently but ATPindependently.13, 14
PS externalization to the cell surface commonly
found in apoptotic cells is likely to be associated with
the inhibition of flippases as well as the activation ofscramblases in the plasma membrane.15

externalization after Mel exposure. This suggested that
Mel-induced H2O2 indeed plays a pivotal role in implementation of apoptosis (PS oxidation, PS externalizationand cyt c release) as a required messenger.
The presence of oxPS on the apoptotic cell surface

We demonstrated for the first time that oxPS exists bothwithin and on the surface of apoptotic cells.25
To determine PS on the cell surface, Jurkat cells following anti-Fas antibody treatment were labeled withmembrane-impermeable fluorescamine, a probe forvisualizing lipids that contain primary amino groups.
Their total lipids were extracted and subjected to twodimensional high-performance thin-layer chromatography (HPTLC). Thereafter the HPTLC plate was firstsprayed with N,N,N’,N’-tetramethyl-p-phenylenediamine
dihydrochloride (TMPD) to detect the oxidation of PS
and then exposed to UV lights to determine externalized
PS. Since fluorescamine and TMPD interact with a primary amine in the polar head group and hydroperoxidein acyl residue of oxPS, respectively, we can detect both
externalization and oxidation of PS on the same HPTLC
plate, i.e., prove the presence of oxPS on the surface ofthe cells. Additionally, to determine PS unbound to fluorescamine, i.e., PS within the cells, the same HPTLC
plate was sprayed with ninhydrin, another probe thatreacts with primary amino groups. The above methods
enabled direct detection of oxPS both within and on thesurface of cells on the same HPTLC plate. The experiments using the above methods suggested that treatmentof Jurkat cells with anti-Fas antibody increased oxPS
within the cells and caused oxPS to appear on the cellsurface.

oxPS generation in apoptotic cells

There is accumulating evidence showing that apoptosis
induced by various stimuli in a number of different cell
types is accompanied by preferential oxidation of PS.8,
16–21
Furthermore, it has been reported that oxidationand externalization of PS almost simultaneously occur
during apoptosis.22, 23
As the source of oxidizing equivalents required forredox catalysis of PS oxidation was not identified, we
determined which kind of ROS oxidized PS in the cells
undergoing apoptosis.24 We used H2O2-resistant HP100
cells derived from HL-60 cells, which expressed catalase (CAT) 2.5 times more than HL-60 cells but contained the same levels of primary antioxidant enzymes(glutathione peroxidase and superoxide dismutase) andapoptosis-related proteins (Bcl-2 and Bax) as HL-60
cells. HP100 cells but not HL-60 cells exerted a higher
resistance to apoptosis induced by anti-cancer reagent
melphalan (Mel). This resistance to Mel-induced apoptosis in HP100 cells was abolished by pretreatment with a
CAT inhibitor 3-amino-1,2,4-triazole (3-AT), suggestingthat overexpression of CAT in HP100 cells was mainly
responsible for their resistance to Mel-induced apoptosis.
Treatment of HL-60 cells with Mel induced ROS production in the cells. However, no increase in ROS generation in HL-60 cells co-treated with exogenous CAT
following Mel treatment. On the other hands, there was
no ROS generation in naïve HP100 cells both before andafter Mel treatment although Mel caused ROS production in HP100 cells pretreated with 3-AT as well. Thus,
it was suggested that ROS generated in the cells treatedwith Mel is predominantly H2O2. Mel induced also PS
oxidation and externalization as well as cytochrome c (cytc) from mitochondria into cytosol in HL-60 cells but not
HP100 cells. In addition, CAT inhibition by 3-AT restored the sensitivity of HP100 cells to PS oxidation and

The mechanisms underlying oxidation of PS during
apoptosis

It is considered that a potential mechanism underlying selective oxidation of CL and PS by H2O2 during
apoptosis relies on the specific interactions between
positively charged cyt c (net charge is +8e at neutral pH)
and negatively charged phospholipids such as PS in thecytosolic leaflet of the plasma membrane and CL in the
mitochondria.23, 26 Cyt c is a globular protein containing
heme and its heme iron has 6 coordination bonds. In cytc, heme iron has two axial bonds: one with His17 on theproximal side of heme and one with Met80 on the distalside.23 When cyt c binds to membrane PS or CL withelectrostatic forces, hydrophobic interactions and hydrogen bonding, the protein globule is partially unfolded,
likely leading to disruption of Fe-Met80 coordinationbond followed by enhancement of reactivity of cyt c to
ROS such as H2O2.23, 26 Cyt c seems to get peroxidase120

Production and bioactivities of oxPS

A Cyt cBa

Oxidized PS/externalized PS(fold increase to control)

Relative density to control

6

5

a
4

3

2

b
1

0

Control

Casp-3 Casp-8
inhibitor inhibitor

2

a

a

b
1

0

Control

Casp-3 Casp-8
inhibitor inhibitor
Anti-Fas Ab

Anti-Fas Ab

Fig. 1. Effect of caspase inhibitors on cyt c release into the cytosol (A) as well as oxidation of PS exposed to the cell surface (B) in Jurkat
cells after treatment with anti-Fas antibody.28 aP < 0.01 versus control, bP < 0.01 versus anti-Fas antibody alone.

and the amount of oxPS both within and on the surfaceof the cells after anti-Fas antibody (Fig. 1B) as expected,
although it inhibited activation of Casp-3 and apoptosis. These results strongly suggested that mitochondrial
event, especially cyt c departure from mitochondria
plays a critical role in production of oxPS within thecells and subsequent its appearance on the cell surface
during apoptosis.

activity by such conformational changes. Presumably,highly oxidized heme (compound I or II) and proteinbased tyrosyl radical formed in the presence of H2O2 in
PS- or CL-activated peroxidase forms of cyt c can subtract an electron from unsaturated acyl chains of PS or
CL followed by the addition of oxygen and formation of
oxPS (PSOOH) or oxCL (CLOOH), similar to peroxidase reaction to cyclooxygenase (COX).23 Newly formed
oxPS (PSOOH) or oxCL (CLOOH) may function as
substrates for cyt c peroxidase activity and further propagate lipid peroxidation, even in the absence of H2O2.
In cell-free model experiments,27 cyt c-derived tyrosyl
radical was measured by low-temperature electron paramagnetic resonance spectroscopy in the presence of cytc, phospholipid-containing liposomes and H2O2. The
production of tyrosyl radical depended strongly on thepresence of PS-containing liposomes, supporting theabove hypothesis indirectly.
It has been suggested that peroxidase activity of cyt
c is closely related to PS oxidation and externalization
as mentioned above. Therefore, we determined whether
departure of cyt c from mitochondria is required for production of oxPS as well as its externalization during Fasmediated apoptosis in Jurkat cells using two selective
inhibitors of caspase-8 (Casp-8) and caspase-3 (Casp3) functioning upstream and downstream from mitochondria, respectively.28 As shown in Fig. 1, inhibitionof Casp-8 reduced mitochondrial cyt c release into thecytosol (Fig. 1A), the amount of oxPS not only within
but also on the surface of Jurkat cells (Fig. 1B), Casp3 activation, and apoptotic cell number after treatment
with anti-Fas antibody. In contrast, selective inhibitionof Casp-3 was unable to suppress cyt c release (Fig. 1A),

Production of oxPS under pathological conditions(in vivo)

Pulmonary phospholipid peroxidation after inhalation
exposure of mice to single-walled carbon nanotubes was
identified in three relatively minor classes of anionicphospholipids, PS, CL and phosphatidylinositol (PI).29
This non-random peroxidation of phospholipids coincided with the accumulation of apoptotic cells in thelung. In another animal models of lung injury (hyperoxic
acute lung injury30 and gamma-radiation-induced lunginjury31), the preferential formation of oxPS and oxCL in
the lung was also detected. These lung injuries were allaccompanied by apoptotic cell accumulation in the lung.
In mouse model of Alzheimer disease (AD), whichshowed aberrant expression of human myeloperoxidase
in astrocytes, there was selective accumulation of twoanionic phospholipid hydroperoxides, oxPS (PSOOH)
and oxPI (PIOOH) in the brains.32 Furthermore, in postmortem brain samples from AD patients, accumulationof the same individual molecular species of oxPS andoxPI was observed.32 In addition to the above brain diseases, it has been demonstrated that selective oxidationof CL and PS in rat cortical neurons is triggered during
staurosporine-induced apoptosis.33
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T. Matsura

Total body irradiation to mice induced intestinal
injury accompanied by apoptosis as well as selective accumulation of oxCL and oxPS in small intestines.34
It has been reported that anti-phospholipid antibodies in sera from patients with alcoholic liver disease(ALD) target apoptotic cells by specifically recognizing
oxPS, suggesting the production of oxPS in ALD.35

hance the clearance of apoptotic cells by phagocytes. In
addition, it has been reported that oxPS acts as a “nonenzymatic scramblase” to facilitate translocation of both
PS and oxPS molecules into the cell surface.41
Anti-inflammatory activities

Protection against endothelial barrier dysfunction
and acute lung injury
The tight intercellular barrier of endothelial cell (EC)
maintaining low permeability is adequately regulated bya counterbalance of barrier-protective and barrier-disruptive bioactive molecules in the circulation.42 Birukova
et al. showed that oxidized 1-palmitoyl-2-arachidonoylsn-glycero-3-phosphoserine (oxPAPS) potently protectedpulmonary EC barrier function and induced the remodeling of pulmonary EC actin cytoskeleton.42 In thatstudy, oxPAPS inhibited the increase in permeability inhuman pulmonary arterial EC induced by lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria. oxPAPS also attenuated neutrophil accumulation and myeloperoxidase activation in bronchoalveolar lavage fluid in mice treated withLPS (in vivo model of acute lung injury), and suppressedlung barrier dysfunction in the mice. Furthermore, oxPAPS diminished pulmonary EC barrier dysfunction
induced by interleukin (IL)-6, an inflammatory mediatoror thrombin, an edemagenic mediator. The protection
by oxPAPS against thrombin-induced pulmonary EC
barrier dysfunction was attributed to both attenuationof Rho-dependent pathway of endothelial contraction
leading to hyperpermeability and stimulation of Racmediated EC barrier recovery. These results suggestthat oxPAPS potently suppresses endothelial barrier
dysfunction induced by inflammatory and edemagenic
agents in vitro and in vivo potentially due to attenuationof Rho signaling as well as stimulation of Rac signaling.
Since the protective effect of oxPAPS is much more thanthat of oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero3-phosphocholine (oxPAPC), it seems that negatively
charged polar head group but not oxidized sn-2 residueof oxPAPS may play some important role in its protective ability although PAPS, non-oxidized form, showsno protective effects.

Bioactivities of oxPS
Role in apoptotic cell clearance

It has been established that PS serves as a recognizable“eat-me” signal for phagocytes through its translocationfrom the inner to the outer leaflet of the plasma membrane during apoptosis.36 However, oxPS also appearson the surface of apoptotic cells during apoptosis.25
Therefore, it is important to determine whether oxPS
on the surface of apoptotic cells enhances the recognition and engulfment of apoptotic cells by phagocytes.
Several receptors for apoptotic cell uptake have been
identified on the surface of the phagocytes. They include
the lectins that bind altered sugars on apoptotic cells,
CD36 (in conjunction with integrins alphaVbeta3 andalphaVbeta5) that binds thrombospondin, LRP1/CD91(in conjunction with calreticulin) that binds complement
C1q, CD14 that binds intercellular adhesion molecule 3(ICAM3), and the scavenger receptors that bind oxidized
LDL.37 Furthermore, PS is recognized either directly via
receptors such as brain angiogenesis inhibitor 1 (BAI1),
T-cell immunoglobulin- and mucin-domain-containing
4 (TIM-4), TIM-1, and Stabilin-2 or indirectly via bridging molecules such as milk fat globule-EGF factor 8(MFG-E8), growth-arrest-specific 6 (Gas6), or protein
S.37 MFG-E8 is expressed and secreted by professional
phagocytes, associates with integrins alphaVbeta3/alphaVbeta5 on phagocytes, and binds PS on apoptotic
cells.38
As for oxPS, it has been reported that the oxPS, but
not non-oxidized PS, serves as a preferred ligand forclass B scavenger receptor CD36-mediated phagocytosis by macrophages.39 Moreover, it has been shown that
MFG-E8 preferentially interacts with oxPS, and to alesser extent, with non-oxidized PS.40 In another study,8liposomes containing oxPS inhibited phagocytosis of
apoptotic cells more potently than non-oxidized PS.
Furthermore, non-apoptotic cells treated with liposomes
containing both oxPS and non-oxidized PS were more
efficiently phagocytosed than cells treated with nonoxidized PS alone.8
Taken together, these findings indicate that oxPS
may act in combination with naïve PS as an importantsignal on the cell surface to facilitate the recognitionof apoptotic cells. In other words, oxPS is likely to en-

Inhibition of LPS recognition by TLR4
LPS is a potent activator of macrophages and a causal
agent of endotoxin shock.43 LPS is well known to induce production of pro-inflammatory mediators suchas tumor necrosis factor-alpha (TNF-alpha), IL-1beta,
IL-6, ROS and nitric oxide (NO), leading to death fromendotoxin shock in animal models.44, 45 Moreover, it has
been shown that LPS enhances cytokine and/or NO pro122

Production and bioactivities of oxPS

duction in macrophages,46 microglial cells47 and ECs.48
The process of these cellular responses involves recognition of LPS by Toll-like receptor (TLR) 4 on the surfaceof the host cells, followed by activation of nuclear factor(NF)-kappa B.49 TLR is a receptor family protein related to innate immunity and to date, 12 functional TLRs
have been identified in mice and 10 in humans.50 TLR4
has now been established as the receptor for LPS. The
activation of innate immunity response by LPS starts
from the interaction of LPS with LPS-binding protein(LBP). LBP in the serum removes LPS from the outermembrane of the bacteria and delivers LPS to soluble
CD14 (sCD14) and/or membrane-bound CD14 and then
LPS bound to CD14 is transferred to TLR4-myeloid differentiation 2 (MD-2) complex. The formation of dimeric LPS/MD-2/TLR4 complex initiates the intracellularsignaling.51
According to a recent study,52 oxPAPS inhibited
LPS-induced elevation of E-selectin mRNA in humanumbilical vein endothelial cells (HUVECs), indicating that it acted as an antagonist on induction of TLR4
downstream genes. oxPAPS prevented binding of LPS to
LBP in an in vitro competitive assay. Moreover, oxPAPS
formed complexes with sCD14 and prevented interactionof LPS with sCD14 in vitro.
Taken together, it was suggested that oxPAPS binds
to LBP and sCD14, thus preventing recognition of LPS
via MD-2/TLR4 complex, leading to inhibition of LPSinduced inflammatory reaction.

It has been shown that ROS produced by activatedneutrophils promotes oxPS production.18 Blüml et al.
reported that oxPAPS inhibited ROS production inphorbol myristate acetate (PMA)- or formyl-methionylleucyl-phenylalanine (FMLP)-stimulated neutrophils
in a dose dependent manner.55 In contrast, unoxidized
PAPS dose-dependently increased the production of
ROS in neutrophils, suggesting that there is an oxidation
state-dependent regulatory role of oxPS on neutrophil
respiratory burst formation. oxPAPS did not inhibit upregulation of CD11b which is involved in adhesion of activated neutrophil to the endothelium, or phagocytosis. It
has been known that MAPK (ERK1/2 and p38) are activated during respiratory burst and there is involvementof these kinases in the production of ROS.56 However,
oxPAPS did not inhibit activation of ERK1/2 and p38 in
neutrophils stimulated with either PMA or FMLP.
Taken together, it was shown that oxPAPS inhibited respiratory burst in neutrophils induced by various
stimuli without inhibiting MAPK activation.
Atherogenic activities

Induction of VEGF via UPR
Endoplasmic reticulum (ER) is the site for proper folding of newly synthesized proteins and formation of
three-dimensional conformation of proteins, and only
proteins assured of quality in ER are released into thesecretary pathway. ER stress induced by hypoxia, nutrient deprivation, acidosis and certain chemicals disturbs
the protein quality control leading to accumulation of
the incorrect folded proteins in ER, and this triggers
the activation of the following three ER transmembraneproteins, which generate an adaptive response called theunfolded protein response (UPR): inositol-requiring enzyme 1 (IRE1), double-stranded RNA-activated protein
kinase (PKR)-like ER kinase (PERK) and activatingtranscription factor-6 (ATF6).57 The activation of thesetransducer proteins triggers signaling cascades, which
induce downstream an adaptive UPR via protein kinases
and transcription factors. The PERK/eIF2alpha/ATF4
and IRE1/ASK1/JNK cascades can trigger the inductionof vascular endothelial growth factor (VEGF).58
It has been shown that oxPAPS stimulates gene
expression of well-characterized angiogenesis inducers (VEGF, IL-8 and COX-2) in HUVECs.59 oxPAPS
induced VEGF via activation of ATF4 branch of UPR
in HUVECs.60 According to a recent report, VEGF
induction by oxPAPS in ECs depended on microRNA-663 (miR-663).61 Given that miR-663 is required for
oxPAPC-induced elevation of ATF4 and its target gene
TRIB (tribbles pseudokinase),61 it is suggested that oxPAPS may also induce ATF4 and its downstream gene

Inhibition of respiratory burst
Neutrophils are recruited to inflamed sites upon infections and exert microbicidal activities. The assembly
and activity of NADPH oxidase (NOX) are essential forneutrophil microbicidal activities. Neutrophils engulf invading microbes and kill them by ROS such as hydroxyl
radicals and hypochlorous acids derived from superoxide anions generated by NOX together with microbicidal
peptides and proteases in phagolysosomes.53, 54 Neutrophil NOX (NOX2), also referred to as respiratory burst
oxidase is a multicomponent enzyme system composedof membrane proteins (p22phox and gp91phox, which
form cytochrome b558) and cytosolic proteins (p47phox,
p67phox, p40phox and Rac2), which assemble at membrane sites upon cell activation. The importance of
this enzyme in host defenses is highlighted by the factthat loss of function mutations of NOX subunits cause
chronic granulomatous disease in which the phagocyte
enzyme is dysfunctional, leading to life-threatening bacterial and fungal infections. In contrast, excessive ROS
generation can damage surrounding tissues. Thus, NOX
activation and ROS production have to be tightly regulated.
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T. Matsura

Extrinsic apoptosis

FasL

Intrinsic apoptosis
Stress (ROS, UV, drugs, radiation)

Fas

M
-OOH

HOO-

FADD

PS

oxPS

Caspase-8
Procaspase-8

Bid

oxCL

tBid

Caspase-9

Procaspase-3

cyt c
ROS
H2O2

-OOH

CL

oxCL

Procaspase-9-OOH

Apaf-1
Bcl-2

Bak Bax

Caspase-3
Apoptosis

IAP

cyt c

Smac/DIABLO

Fig. 2. Selective oxidation of PS and externalization of oxPS in apoptotic cells as well as recognition of oxPS on the cell surface by macrophages. Its proposed mechanisms are indicated by bold arrows.

VEGF in a miR-663-dependent manner, playing a rolein angiogenesis especially in atherosclerotic plaques.

teases including blood coagulation factors, fibrinolytic
enzymes, tissue kallikrein, and the sperm protease acrosin.64
It has been shown that oxPAPS, PAPS and oxPAPE
directly bind PCI and stimulate its inhibitory activity
toward APC.64 Positively charged amino acids in the Hhelix of PCI were involved not only in heparin binding,but also in the binding of oxPAPS, PAPS and oxPAPE.
Given that PCI and annexin V, a protein binding to
the head group of PS, were found to be endogenously
colocalized in atherosclerotic plaques, it was suggested
that oxPAPS in vivo could promote blood coagulationand thrombus formation as well as inflammation at sitesof tissue injury by stimulating the inhibition of APC by
PCI.

Procoagulation
Protein C is a vitamin-K dependent anti-coagulant
serine protease serving as a negative feedback regulator against coagulation.62 When thrombin generated byblood coagulation binds to thrombomodulin (TM) on
the EC membrane, protein C is activated by thrombinTM complex to be an activated protein C (APC). This
conversion is augmented by a specific receptor for protein C, endothelial cell protein C receptor. APC converts
activated coagulation factors V (Va) and VIII (VIIIa) to
the inactive forms by proteolysis with an APC cofactor
protein S, resulting in inhibition of blood coagulation.
In addition to its role in coagulation, APC decreases inflammatory responses by inhibition of nuclear translocation of NF-kappa B.62
Protein C inhibitor (PCI) is a serine protease inhibitor belonging to the class of serpins.63 PCI, originallyan inhibitor of APC, inactivates lots of other serine pro-

Immunosuppressive activities

oxPAPS inhibited the proliferation of peripheral blood T
cells induced by anti-CD3/CD28 monoclonal antibody,demonstrating a novel immunosuppressive molecule foradaptive immunity.65 In that study, oxPAPC prevented
124

Production and bioactivities of oxPS

the development of Th1-type responses, T cell proliferation and the induction as well as the effector phase of
CD8+ effector cytotoxic T lymphocytes induced throughstimulation via TCR/CD3 complex, although there wereno data for oxPAPS. Furthermore, T cells activated in
the presence of oxPAPC failed to proliferate in responseto restimulation, a phenomenon called anergy. If oxPS
functions similarly to oxPC, oxPS may also induce T
cell anergy, resulting in avoiding overwhelming Th1driven immune responses at the inflammation sites. Further examinations for oxPS will be required to resolve
the above issues.

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Concluding remarks

The oxidative stress associated with apoptosis results inthe selective oxidation of PS in the cytoplasmic layer ofthe plasma membranes, followed by egress of oxPS tothe surface of the apoptotic cells and effective recognition of oxPS by phagocytes. This is no longer than amere non-specific oxidation of phospholipids, but seems
to be a finely tuned system via non-random oxidation of
PS for clearance of apoptotic cells by phagocytes to prevent the inflammation (Fig. 2). oxPS has some biologicalactivities such as anti-inflammatory activities, atherogenic activities, and immunosuppressive activities otherthan the role in apoptotic cell clearance as mentioned
above.
In contrast, oxPC has more novel functions and isrecognized as a lipid mediator. In the future, we have to
examine using highly pure oxPS whether oxPS has thesame functions as oxPC and furthermore detect undiscovered biological activities of oxPS. In addition, it isalso important to detect the specific receptor for oxPS in
the phagocytes.
The author declares no conflict of interest.
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