cell damage at origin antiphospholipid antibodies their...

Infectious Diseases in Obstetrics and Gynecology 5:176-180 (1997)(C) 1997 Wiley-Liss, Inc.

Cell Damage at the Origin of AntiphospholipidAntibodies and Their Pathogenic Potential in

Recurrent Pregnancy Loss

V6ronique Piroux, Val6rie Eschwge, and Jean-Marie FreyssinetInstitut d’Hgmatologie et d’Immunologie, Facultg de Mgdecine, Universitg Louis Pasteur,

Strasbourg (V.P., J.-M.F.); Unitg 143 INSERM, Hapital de Bicgtre,Le Kremlin-Bicgtre; France (V.E., J.-M.F.)

ABSTRACT

Antiphospholipid antibodies (APA) are associated with thrombosis, thrombocytopenia and fetalloss but they occur in a variety of diseases. Despite many efforts, a correlation between the speci-ficity of particular subgroups of APA and particular clinical situations remains to be established.The antigens at the origin of APA remain to be identified. We discuss here the possible linksbetween cell apoptosis or necrosis, leading to plasma membrane alterations, and the occurrence ofAPA in response to sustained stimulation. The pathogenic potential ofAPA is also considered withrespect to recurrent pregnancy loss. Infec. Dis. Obstet. Gynecol. 5:176-180, 1997.(C) 1997 Wiley-Liss, Inc.

KEY WORDS

Apoptosis, necrosis, membrane phospholipid asymmetry, annexin V, procoagulant lipid exposure

ROUTINE DETECTION OFANTIPHOSPHOLIPID ANTIBODIES

Antiphospholipid antibodies (APA) are frequentlydetected in routine laboratory practice. Various tar-

gets are used in ELISA. However their reactivity is

highly heterogeneous. The different phospholipidsused are either negatively-charged, cardiolipin andphosphatidylserine, or neutral like phosphatidyl-ethanolamine, or mixtures. Concerning the proteincomponents of complex antigens, the importanceof [32-glycoprotein I and prothrombin is now widelyaccepted. Some other phospholipid-binding pro-teins like kininogens, protein C, protein S or an-

nexin V could also expose neoepitopes whenbound to phospholipids. 1,z Under certain in vitroconditions, the presence of phospholipid is not

even required for APA binding to protein cofac-tors.3 In vivo, it is certainly conceivable that phos-

pholipids are needed to present the proteins. APAprobably result from the reactivity of the immune

system to membrane components that are normallysequestered in the absence of cell stimulation and/or to plasma proteins showing a certain affinity forsuch components when accessible. The differentmodes of phospholipid exposure combined withthe binding of different proteins could explain theheterogeneity of APA.

EXPOSURE OF SEQUESTEREDPHOSPHOLIPIDS

Cardiolipin (diphosphatidylglycerol), phosphati-dylserine and phosphatidylethanolamine are themost frequent phospholipids used as targets forAPA. Phosphatidylserine and phosphatidylethanol-amine are ubiquitous aminophospholipid compo-nents of eukaryotic cell plasma membranes. In

Correspondence to: J.-M. Freyssinet, Institut d’H6matologie et d’Immunologie, Facult6 de M6decine, 4, rue Kirschleger,67085 Strasbourg, France; e-mail: [email protected]

Received October 1997Accepted 21 October 1997

ANTIPHOSPHOLIPID AND PREGNANCY PIROUX ETAL.

resting cells, they are mostly restricted to the inner

leaflet of the membrane.4 The ability of phospha-tidylethanolamine to adopt hexagonal phase orga-nization could explain its immunogenicity, but thephysiopathological conditions allowing this confor-mation are probably not frequent. Cardiolipin is

mainly localized in the inner membrane of the mi-tochondria. A drastic lesion, due to mechanicalstresss or necrosis, is necessary to expose this phos-pholipid. Apoptosis and necrosis, as well as inflam-mation and infection, are cell death pathways andclinical situations during which the loss of mem-brane asymmetry is observed. Resting platelets,monocytes, endothelial cells and red blood cellspresent an asymmetric distribution of the phospho-lipid constituents of their plasma membrane.Stimulated cells, apoptotic ceils and bodies, shedmicroparticles and necrotic fragments presentmembrane rearrangements.

Aminophospholipids become exposed at theouter surface of the plasma membrane of stimu-lated cells and that of shed microparticles. Themost frequent example is that of platelets,4 butmonocytes6 and endothelial cells7 are also able to

elicit a similar membrane response. Once acces-

sible to plasma vitamin K-dependent clotting fac-tors, phosphatidylserine, in cooperation with phos-phatidylethanolamine, acquires the ability to pro-mote the assembly of the characteristic enzymecomplexes of the coagulation cascade.4 Differentclinical situations are associated with increased cir-

culating platelet microparticles.8-13 One of them,idiopathic thrombocytopenic purpura, is furthercharacterized by the three following observations:APA positivity is a common feature in patients with

idiopathic thrombocytopenic purpura, 14 APA are

able to bind to platelet microparticles in idiopathicthrombocytopenic purpura, is and platelet micro-

particles in idiopathic thrombocytopenic purpuraare enriched in [32-glycoprotein I. 6 This is cer-

tainly suggestive of the generation of APA in re-

sponse to an increase of platelet microparticles.Inflammation is a common pathological situa-

tion in which increased aminophospholipid inside-out migration and microparticle shedding are likelyto occur. Proinflammatory mediators, interleukin

1-13 and tumor necrosis factor-a, induce procoagu-lant phospholipid exposure, increased thrombin

generation and subsequent or concomitant cell-cellinteractions. When combined, these effects result

in a dramatic augmentation of the thrombotic risk.

APA have been reported to inhibit procoagulantactivity of activated platelets and platelct-dcrivedmicroparticles. 17 Hence, two questions arise fromthese considerations: do APA occur in response to

sustained exposure of thrombogenic phospholipidsurfaces?, and the corollary: are APA first synthe-sized as ’protecting antibodies’?The APA associated with infection have been

claimed to be 132-glycoprotein I-independent anti-bodies and probably not related to thrombosis.Nevertheless, it is not an easy task to discriminatebetween these two different types of antibodies. Inbacteria, different membrane components such as

lipid A and cardiolipin are candidate targets forAPA. Concerning parasites, phosphatidylscrine ex-

pression at the surface of malaria-parasitized eryth-rocytes is now well described. More data are avail-

able regarding viruses. Membrane rearrangementsoccur during fusion and viral maturation of envel-

oped viruses. The envelope is a modified host cellplasma membrane, is In all cases of infection,complement-mediated cell lysis leads to inflamma-tion and increased circulating cell fragments. Theassociation between hepatitis C virus and APA hasbeen studied in patients with hemophilia19 andHIV infection,e Furthermore, the increased rate ofthrombin generation in hepatitis C virus cirrhotic

patients,zl combined with the increased prevalenceof anticardiolipin antibodies in chronic hepatitisC,zz suggests an implication of this virus in the as-

sociated antiphospholipid syndrome.Exposure of phosphatidylserine at the surface of

apoptotic cells and derived fragments is an earlyfeature of apoptosis,e3,e4 Fadok et al. and other

groups demonstrated that phosphatidylserine trig-gers recognition and removal by phagocyte.2s-27

The identity of phosphatidylserine receptor(s) re-

mains to be established but [32-glycoprotein I couldbe involved, as it has been detected on rapidly-cleared liposomes. 28 Apoptotic cells and bodieshave comparable procoagulant properties as acti-vated platelets and shed microparticles which can

be counteracted by purified immunoglobulin Gfrom patients with APA syndrome. The procoagu-lant potential of apoptotic cells and derived frag-ments could lead to pathologic coagulation andtheir immunologic potential could be responsiblefor the generation of APA.29 Hence, APA are ex-

pected to occur in diseases known to be associated

INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 177

ANTIPHOSPHOLIPID AND PREGNANCY PIR0UX ET AL.

Apoptosis

Central cell death signal(s)

Survival factor deprivation

Necrosis

Acute cell injury

Endonuclease activation

Cell surface alterations,Phosphatidylserine exsure

Cytoskeletal reorganization

Cell & organellefragmentation

Leakage ofcytoplasmic content

. Phagocytosis

Inflammatory response

Apoptosis sequesteredphospholipid APA generation

Necrosis

Thrombotic risk

Fig. I. The possible contribution of programmed cell death (apoptosis) and necrosis in the occurrence of antiphospholipidantibodies (APA). Phosphatidylserine and phosphatidylethanolamine are sequestered in the inner leaflet of the plasma mem-brane of unstimulated cells. Cardiolipin is mainly found in the inner membrane of mitochondria. Hence, an apoptotic processis expected to lead to phosphatidylserine and ’phosphatidylethanolamine externalization. In addition to these two lipids,cardiolipin becomes accessible after cell necrosis.

with increased apoptosis such as AIDS, myocardialinfarction and stroke.3 Another situation of in-creased apoptosis is alcoholic intoxication.3 Che-did et al. found that the prevalence of APA in-creases dramatically in parallel with liver function

impairment.31

Finally, it is important to emphasize the majordifferences between apoptosis and necrosis. Apop-tosis is induced by central cell death signal(s) or

survival factor deprivation and is characterized byendonuclease activation, surface alterations and cy-toskeletal reorganization. The different cell surfacealterations are as many triggers for removal byphagocytes and, except when the reticuloendothe-lial system is saturated, cell death and eliminationoccur rapidly without inflammation. At the oppo-site, necrosis is an acute cell injury accompanied byleakage of cytoplasmic content and by the disinte-

gration of lysosomes and mitochondria which are

responsible for an inflammatory response.3 Vari-ous mechanisms including apoptosis and necrosiscould be responsible for APA generation, but, be-cause of the associated inflammatory response, ne-

crosis could represent a true thrombotic risk. Thisdoes not rule out the possible pathogenicity ofAPAwhen synthesized in large excess (Figure 1).

A POSSIBLE THROMBOGENIC MECHANISMAT THE ORIGIN OF PREGNANCY LOSS

Several pathogenic mechanisms have been pro-posed to explain the association of APA with an

increased thrombotic risk,3z but none regarding a

possible interference of APA in pregnancy.A recent contribution deserves particular atten-

tion regarding a possible mechanism of recurrent

fetal loss.33 These authors have evidenced thepresence of annexin V on placental villi and con-

firmed their observation on cultured trophoblastsand endothelial cells. Owing to its strong affinityfor phosphatidylserine, annexin V, a protein whosephysiologic function remains to be established, ex-

erts a potent in vitro anticoagulant effect. Its asso-

ciation with the outer leaflet of the plasma mem-

brane of tissues fulfilling a barrier function mayoriginate from the active membrane remodeling oc-

curring in these cells. Aminophospholipid exposureand microvesicle shedding result in the develop-ment of procoagulant catalytic surfaces which haveto be neutralized. Annexin V, also translocated dur-ing these membrane events, could preciselyachieve such an anticoagulant control. The inves-

tigators have observed that annexin V levels are

markedly reduced on placental villi from women

178 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY

ANTIPHOSPHOLIPID AND PREGNANCY PIROUX ET AL.

presenting the antiphospholipid-antibody syn-drome. In addition, they have shown that APAfrom three patients who experienced repeated fetalloss are able to displace annexin V from culturedtrophoblasts or endothelial cells. The reduction ofsurface annexin V resulted in increased cell surfaceprocoagulant potential. This provides a rationalefor the recurrence of pregnancy loss.

CONCLUSIONPERSPECTIVES

The antiphospholipid-antibody syndrome may re-

flect cell damage at the origin of the so-called APA,which in turn may acquire pathogenic potential de-pending on special circumstances. The cellular al-terations may lead to a thrombogenic phenotypewhich has to be counteracted. Nevertheless, we are

left with an immunologic problem. Solutionsshould be found in the light of the possible corre-

lation(s) between the presence of particular subsetsof APA and the occurrence of documented clinicalmanifestations. This implies that APA are bettercharacterized at a molecular/gene level whichshould help to explore their specificity towards bet-ter defined antigens.

Because programmed cell death is of fundamen-tal significance in embryogenesis and develop-ment,3 a challenging demonstration would be thatof an interference of APA in an apoptotic processresulting in pregnancy loss or fetal growth retarda-tion.

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