Antiphospholipid Antibody Syndrome: Immunologic and Clinical Aspects By Lisa f?. Sammaritano, Antiphospholipid

antibody

clinical

of vascular

syndrome

bocytopenia,

recurrent

reticularis,

whether

systemic A

lupus

positive

defined by

preceding

similar two

of

lupus

charged

heparin,

INDEX

unknown.

but

by W.B. WORDS:

G, (IgG,)

various

Treatment

remains Saunders

the

the

an-

has not been with

aspirin,

favored.

A role

to be demonstrated. Company.

Antiphospholipid

lupus anticoagulant;

physical alter

for

syndrome

is currently

and

all negatively

Anticoagulation

or warfarin

SLE-

antiphospho-

phospholipids

antibody

defined.

IMMUNOLOGY or

It recognizes

anticardiolipin

antibody:

SLE;

antibody.

A

NTIPHOSPHOLIPID antibody syndrome (APS) has become a well-established clinical entity, and numerous reviews have recently summarized the clinical manifestations of this syndrome’-’ which include arterial and venous thrombosis, thrombocytopenia, fetal loss, cardiac and neurological manifestations, and livedo Abbreviations: aCL, anticardiolipin antibody; ANA, antinuclear antibody; aPL, antiphospholipid antibody; APS, antiphospholipid antibody syndrome; aPTT, activated partial thromboplastin time; BFP-STS, biological false-positive tests for syphilis-serologic tests for syphilis; CL, cardiolipin; dsDNA, double-stranded DNA; ELISA, enzyme-linked immunosorbent assay; IgG, immunoglobulin G; KCT, kaolin clotting time; LAC, lupus anticoagulant; PAI, plasminogen activator inhibitor; PAPS, primary antiphospholipid antibody syndrome; PG, phosphatidylglycerol; RNP, ribonucleoprotein; RVVT, Russell viper venom time; SLE, systemic lupus erythematosus; Sm, Smith antigen; TIA, transient ischemic attack; tPA, tissue plasminogen activator.

SeminarsinArthritisandRheuma~ism,

Vol20, No

2

D. Lockshin

to the

antibody

is as yet

and Michael

reticularis4-7 (Table 1). Antiphospholipid antibodies (aPLs) commonly occur in patients with systemic lupus erythematosus (SLE) but may occur without other evidence of collagen vascular disease in the “primary antiphospholipid antibody syndrome” (PAPS).*-” Recent research has focused on characteristics of the antibody that may determine pathogenicity and possible mechanisms of thrombosis.

associated

antiphospholipid

patterns.

for corticosteroid 0 1990

antibodies.

of

of the

tiphospholipid

or

mechanism

phospholipids,

recognition

itself)

related

has immunoglobulin

characteristics

is as-

The

predominance.

clearly

identical

is less closely

but not infection-induced

lipid antibody IgG,

test

anticoagulant).

and is less regularly

either

of

coexists.

antibody

complications.

a

livedo

antibody

(lupus

but not

anticoagulant

induced

and

immunosorbent

assay

E. Gharavi,

throm-

diagnosis

(SLE)

(antiphospholipid

clinical

action

loss,

antiphospholipid

are

with

fetal

or not a clinical

The test for syphilis

with

thrombosis,

erythematosus

coagulation

These

is associated

by enzyme-linked

say (ELISA)

Azzudin

(October),

1990:

AND

PATHOGENESIS

Wasserman first described a type of aPL in 1906,” the “reagin” associated with syphilis. It later became clear that there were biological false-positive serologic tests for syphilis (BFPSTS) of two types: acute, usually associated with viral or other infection, and chronic, often associated with the presence of collagen vascular disease. I2 So strong is the as sociation with SLE that the American Rheumatism Association criteria for SLE include the BFP-STS.13 The lupus anticoagulant (LAC) was described by Conley and Hartman14 in two patients with hemorrhagic complications; later work showed no association with bleeding complications,‘5,‘6 but rather a paradoxical association of the LAC with thrombosis.‘7-19 The LAC prolongs phospholipid dependent coagulation steps in vitro by competing with coagulation factors for binding to phospholipid.20-22 Because of the numerous

From the Division of Rheumatic Diseases, Hospital for Special Surgery, Cornell University Medical Center, New York, NY. Supported in part by NIH grant AR32929 and b,y a grant from the New York Chapter, .4rthritis Foundation. Lisa R. Sammaritano, MD: Hospiialfor Special Surgery, Cornell University Medical Center, New York, NY; Azzudin E. Gharavi, MD: Hospital for Special Surgery, Cornell Medical Center, New York, NY; Michael D. Lockshin, MD: National Institutes of Health, National Institute of Arthritis and Musculoskeletal and Skin Diseases, Bethesda, MD. Address reprint requests to Michael D. Lockshin, MD, NIH, National Institute of Arthritis and Musculoskeletal and Skin Diseases, Bldg 31. Rm IC-32. Bethesda, MD 20892. D 1990 b_vW.B. Saunders Company. 0049-0172/90/2002-0003$5.00/O pp El-96

81

82

SAMMARITANO,

Table

1:

tions

Commonly

Agreed

of Antiphospholipid 1. Arterial occlusion, Extremity

Clinical

Antibody

Manifesta Syndrome

including

gangrene

Stroke Myocardial

infarct

Other visceral infarct Aortic occlusion 2.

Venous occlusion, Peripheral

including

venous

Visceral venous (Budd-Chiari,

portal vein,

etc) 3.

Recurrent

4.

Thrombocytopenia

fetal loss

5.

Coombs’

6.

Livedo reticularis

positive hemolysis

7.

Neurological

abnormalities

Chorea Multiple sclerosislike Nonstroke

syndrome

ischemia syndrome

8.

Valvular heart disease

9.

Sudden multisystem

arterial occlusion

assays used for diagnosis, difficulty with definition of the LAC arose. This lack of standardization, and increasing interest in aPLs and their association with various types of thrombosis, prompted Harris et al to develop a solid phase radioimmunoassay, then later an enzyme-linked immunesorbent assay (ELISA), for aPLs using cardiolipin (CL) as the antigen.’ Concordance/Discordance

of LAC and aCL

Most authors conceive of aPLs as representing a spectrum of antibodies including the BFP-STS, the LAC, and anticardiolipin antibody (aCL). Technical differences in performance are listed in Table 2. aCL binding in ELISA and VDRL titer are not correlated in patients with SLE23-25; Table

2:

Relations

Among

Antiphospholipid

Antibodies

GHARAVI, LOCKSHIN

McNeil et a126found that LAC activity and aCL binding also do not necessarily correlate. LAC and aCL often are present in the same patients, but many studies demonstrate a lack of concordance between these two antibodies.27’31 A major problem is the lack of standard definition of the LAC. Numerous clotting tests exist, with varying degrees of sensitivity, for the LAC. Because antibody binding to phospholipid is necessary for certain coagulation steps to occur, limiting the quantity of phospholipid within the coagulation assay improves the sensitivity of the assay for LAC.32e34 The most sensitive assays for LAC seem to be the kaolin clotting time (KCT) and the Russell viper venom time (RVVT).32v37 The activated partial thromboplastin time (aPTT), probably the most commonly used test in clinical studies, is less sensitive than the KCT or RVVT.32e37 Exner et al have shown separation of aCL and LAC using a cardiolipin-coated polystyrene column, thus identifying these as two separate populations of antibodies. 38 McNeil et al also have been able to achieve column separation of these two activities3’ Fleck et al postulated that discordance between LAC and aCL was due to a population of LAC that functions as antiprothrombin antibodies. 4o However, Harris et a14’ and others42 have shown LAC activity in affinitypurified aCL using a sensitive dilute kaolincephalin clotting time. We evaluated 12 immunoglobulin G (IgG) preparations from aCL positive sera, not all of which were believed to have LAC by aPTT. Using a KCT with platelet-free plasma as described by Exner34 we found prolongation with all samples.43 In 1986, Harris et al provided guidelines for a standardized aCL assay with reference sera available to all participating laboratories.44 As a in Rheumatic

Disease

Patients

Usual

aPL

ELISA

Titer

Antigen

Assay Type

Any negatively

charged

phospho-

>1:100

Other Isotype,

light chain can be sepa-

rately determined.

lipid

May require

serum cofactor LAC

Clotting

Various thromboplastins

tl:lO

Requires all coagulation be normal

STS

Agglutination

CL + cholesterol choline

+ phosphatidyl-

tl:lO

Emphasizes

IgM

factors

to

83

ANTIPHOSPHOLIPID ANTIBODY SYNDROME

result, recent aCL literature reflects this uniformity, allowing better comparison between studies. Controversy continues, however, as to which test provides greater predictive value for thrombosis. Both Derksen et al45 and Petri et a146 evaluated patients with SLE for aCL and for various clotting tests to detect the LAC; both found their LAC assays to offer greater specificity for thrombosis in comparison to the greater sensitivity of the aCL assay. Lockshin et aL4’ however, found aCL to have both greater sensitivity and greater specificity in predicting fetal death, and Alving et al” found that despite a close correlation between LAC and aCL, high titer aCL better predicted thrombotic complications. Although the close association of aPLs with clinical complications suggests a pathogenic role for the antibody, evidence for the mechanism of action remains controversial. In an effort to understand both the antibody’s mechanism of action and why some, but not all, patients with the antibody experience clinical complications, many groups have studied antibody characteristics. C’huracteristics

of aPL

The aPL common in syphilis patients are not associated with clinical complications. Much research has focused on syphilis aPL in addition to SLE aPL in an effort to understand what differences may be important. It has been suggested that the absence of clinical complications in syphilis and drug-induced aPL, and the presence of these complications in SLE-associated aPL, are related to differences in isotype,48 antibody avidity,49,50 IgG subclass distribution,s0*5’ and phospholipid specificity.23*S2 Isotype is believed to have clinical importance: many, but not Table

3:

Comparison

of SLE and Syphilis

all, authors have found that IgG associates most strongly with clinical complications,4*6*31,53 and IgG titer appears to be significant.6~31~53 aPL IgG subclasses also may reflect this nature of the inciting antigen and potential for pathogenicity. In SLE, IgG subclasses for antidouble-stranded DNA (anti-dsDNA), anti-Sm, and antiribonucleic protein anti-RNP) are largely IgGl and IgG354-56; anti-Ro, La, and Ul RNP are predominantly IgGl.” In contrast, SLE aPLs encompass all four IgG subclasses, with IgG2 and IgG4 predominating.50,5’ One group’* found IgGl, IgG2, and IgG3 in SLE aPL using an ELISA, which does not meet 1986 anticardiolipin workshop standards.44 Syphilis-induced aPLs are largely IgGl and IgG3.50 Differences between SLE and syphilis aPLs are shown in Table 3. The difference in subclass distribution between SLE and syphilis aPL implies variation in autoantibody induction and gene rearrangement and suggests a difference in inciting antigen or antigen presentation. The presence of IgG4 in SLE aPL raises further questions about chronicity of inciting antigen, as IgG4 has been described as a response to prolonged immunization and may reflect chronic antigenic stimulation.59’60 Light chain type in SLE aPL is predominantly Aso; normal light chain distribution is approximately 2/3 K and iis x,6’ and light chains of other autoantibodies54.55 and syphilis-induced5’ aPL are mainly K. Cross-reactivity of aPL with anti-dsDN.4 antibodies had been suggested by some investigators,24,62,64largely on the basis of reactivities of monoclonal antibodies.62,64 The polar phosphate head group of the phospholipid was felt to mimic the phosphodiester backbone of DNA. Subsequent work has largely disproved this theory, with the apparent explanation that low-avidity

aPL SLE

r

Syphilis

Usual titer

>l:lOO

IgM

IgM > IgG

High

Low

CL

CL-cholesterol-phosphatidylcho-

Avidity Preferred

antigen

line IgG Subclass

I I

All, especially

Light Chain Anticoagulant

activity

lgG2 and lgG4

IgG 1 and lgG3

A

K

Yes

No

a4

antibodies-monoclonals-may cross-react, while high-avidity antibodies-those derived from patientsao not.25@ SLE aPL appear to have higher avidity compared with syphilis aPLSo when avidity is estimated using ELISA-based methods.4g366 However, whether this relates to possible pathogenicity is unclear. aPL bind to anionic phospholipids CL, phosphatidylserine, phosphatidylinositol, phosphatidic acid) in general, rather than to CL in particular20*48750*67;some disagree.23 Most investigators have found that aPL does not bind to the nonanionic phospholipids phosphatidylcholine and phosphatidylethanolamine,20~48~50~67 although a recent case report describes one patient with LAC and an antibody reactive with phosphatidylethanolamine who had thrombosis.68 Early work52 suggested differences in phospholipid specificities of SLE and syphilis aPL, but it now appears that phospholipid specificities are similar.4g*50 SLE aPLs are best inhibited by CL in a pure form, whereas syphilis aPL are best inhibited by CL as part of the VDRL antigen (CL, cholesterol, and 50,52*6gIt seems most likely phosphatidylcholine. that these aPLs recognize different structural epitopes. The reactive epitope on the phospholipid is not yet defined, although some important components of the epitope have been clarified. Both the phosphodiester group and a negatively charged polar head appear necessary. The length and degree of saturation of the fatty acid chains also play an important role. SLE aPLs bind best to Cl 8:l (18 carbon atoms and 1 double bond per chain) phosphatidylglycerol (PG), and binding is greater to Cl8 than to C14:O or C16:O PGs. SLE aPLs do not bind to C 12:O PG.” Phase of the phospholipid also may be important in determining antibody binding.‘l Although this work has not been widely reproduced, it raises further issues about structural specificity of the phospholipid epitope. Animal models for aPL syndrome recently have been described.72 MRL/lpr and New Zealand black/New Zealand white (NZB/ NZW) (Fl ) are common “autoimmune” strains of mice. While MRL/lpr mice produce aPL, mice NZB/NZW (Fl) mice do not. MRL/lpr have lower platelet counts and fewer offspring per litter than do NZB/NZW (Fl) or Balb/C

SAMMARITANO,

GHARAVI, LOCKSHIN

mice. Like aPL in patients with SLE, aCLs in MRL/lpr mice bind to anionic rather than neutral phospholipids, encompass all four IgG subclasses, and are not inhibited by DNA. Although clear differences in antibody characteristics between SLE and syphilis aPL have been described, it is still unclear which, if any, antibody characteristics are important in predicting clinical complications, Qamar et a173 evaluated high titer IgG aPL sera from SLE patients with and without fetal loss for IgG subclass, avidity, light chain type, and phospholipid specificity. None of the specified antibody characteristics distinguished between the patients with and without fetal loss. Complications due to aPL may depend on both the presence of the antibody and another, as yet unidentified, inciting factor or conditions. Mechanisms of Pathogenicity Any or all of the major components of the clotting system may be involved in aPL pathogenicity, including the coagulation cascade (many of these steps are phospholipid dependent), platelet activation and aggregation, and endothelial cell function. Interference with each of these has been postulated as a possible mechanism. Endothelial cells are crucial contributors to the normal homeostasis maintained between the procoagulant and fibrinolytic pathways. Anticoagulant substances produced by the endothelial cell include heparinlike molecules that accelerate serine protease inactivation by antithrombin III; thrombomodulin, which binds thrombin and alters its specificity, thus activating protein C and blocking thrombin-catalyzed procoagulant processes (protein C inactivates factors V, and VIII,); prostacyclin, a prostaglandin with vasodilatory effects; and tissue plasminogen activator (tPA) that activates plasminogen. Procoagulant substances synthesized by the endothelial cell include tissue factor, von Willebrand factor, platelet activating factor, and plasminogen activator inhibitor (PAI).74,75 Antiendothelial cell antibodies have been described in SLE sera by several groups,76-81 and have been postulated to cross-react with or represent a part of the spectrum of aPLs. Direct binding of these antibodies to endothelial cells could result in damage and immune-complex deposition. Cines et al” showed binding of SLE

ANTlPHOSPHOLlPlD

ANTIBODY

SYNDROME

(but not normai) IgG and heat-aggregated IgG to cultured human endothelial cells, with resulting complement activation and disruption of the monolayer. Others79*80 have evaluated both antiendothelial cell antibodies and aPLs in SLE sera; significantly higher levels of antiendothelial cell antibodies were found in LAC SLE sera (as compared with SLE sera without LAC),” and partial inhibition of antiendothelial cell antibody binding was found after preincubation with CL liposomes. 79 In contrast to this, Rosenbaum et al found no relation between antiendothelial cell antibodies and aPL in patients with SLE, scleroderma, and rheumatoid arthritis, and found no correlation of antiendothelial cell antibodies with thrombosis. 78 It has been sugg ested that these antibodies may be related to the presence of vasculitis in SLE76 and may stimulate tissue factor production by endothelial cells,” but their significance and their relation to aPL have not been proven. A second hypothesis, based on interference by aPL with normal endothelial cell function, is aPL-impaired release of arachidonic acid from cell membrane phospholipids with decreased prostacyclin production. Several groups have documented decrease in prostacyclin production in in vitro systems with addition of SLE sera82-84; however, this work has not been reproduced by all investigators.85s6 Carreras et al’* initially described inhibition of prostacyclin prcriuction in rat aorta rings and pregnant human myometrium with the addition of IgG isolated from a LAC-positive patient with pregnancy loss and arterial thrombosis. Arachidonic acid abolished this inhibitory effect. This group later reported on 14 LAC-positive sera, 8 of which inhibited prostacyclin production including 6 which were from patients with thrombosis.83 Although some investigators have found increased thromboxane synthesis in addition to decreased prostacyclin,84 others have not reached similar conclusionss5~86 and the role of prostacyclin inhibition remains unclear. By binding to endothelial cells, aPL may also interfere with the function of endothelial cellassociated thrombomodulin.87-90 Comp et al described inhibition of protein C activation by IgG from two of seven patients with nonspecific inhibitors.” Cariou et al investigated effcits of IgG on endothelial cell function in 16 patients

85

with LAC.87,88 They found no inhibition of prostacyclin production, normal release of tPA and PAI, and significantly lower rates of protein C activation on endothelial cells in the presence of LAC IgG (43% inhibition). Thrombomodulininduced changes in thrombin specificity occur as a phospholipid-dependent process, and neutralization of the IgG inhibitory effect was seen with incubation with excess phospholipid. Inhibition of endothelial cell protein release has also been postulated as a mechanism for aPL action. In 1979, Angeles-Can0 et al studied 28 patients with SLE for platelet function, coagulation, and fibrinolytic parameters.” They found two correlations with thromboembolic disease: increased levels of factor VIII-von Willebrand factor and absence of plasminogen activator (tPA) release after venous occlusion. Others92,93 similarly find lower levels of tPA activity, but not tPA antigen, after venous occlusion in patients with SLE, suggesting a possible stimulatory effect on endothelial cell release of PAI. Higher levels of PA1 in SLE-LAC patients have been demonstrated.94 Khamashta et a195 recently described binding of aPL to frozen-thawed platelets, but not intact platelets, suggesting that a “perturbation” step must first occur before aPL can bind platelet phospholipid. Similarly, Rauch et al found little binding of human hybridoma LAC antibodies to intact platelets in vitro,96 and Misra et al found aCL reacted only with activated lymphocytes.97 Because phosphatidylserine and other anionic phospholipids to which aPL are known to bind are predominantly on the interior of the platelet membrane, platelet activation seems likely, and may be related to aPL-associated thrombocytopenia. Whether aPL-platelet interaction alone could serve as the mechanism for thrombosis is unclear, as is the issue of what serves as the inciting event for this process. Cortelazzo et al, in evaluating LAC effects on platelets in 10 patients, report one IgM LAC from a patient with WaldenStrom’s macroglobulinemia that stimulated platelet activation. 98 No other groups report aPL binding to intact platelets. Other mechanisms for aPL action have been postulated. Several groups have reported a functional deficiency of protein S in LAC sera,99-102 including a recent case report describing two aPL-positive patients with functional protein S

86

SAMMARITANO,

deficiency, cerebral venous thrombosis, and coumarin skin necrosis.“’ Similarly, a functional deficiency of antithrombin III has been described in patients with SLE, some with LAC.103,104 Finally, Sanfelippo et al evaluated three patients with LAC with evidence of inhibitory activity directed against prekallikrein as well as phospholipid. lo5Prekallikrein, among other functions, serves as a cofactor participating in plasminogen activation by factor XII. Of interest is a placental anticoagulant protein, also found in other vascular tissue, which functions in vitro to inhibit the phospholipiddependent activation of prothrombin by the prothrombinase complex (prothrombin, factor X,, factor V,, and phospholipid.)10”‘08 Because aPL binds to anionic phospholipid, it is possible that fetal loss secondary to placental thrombosis and insufficiency may result from interference with this natural anticoagulant. Very recently a normal plasma cofactor was reported to be required for aPL to bind to PL in the ELISA assay.108a The importance of this cofactor is not yet known.

CLINICAL

ASPECTS

Although thromboses have long been associated with the LAC, the sensitivity of the anticardiolipin immunoassay and its potential for standardization has focused new attention on clinical complications. As the number of prospective, retrospective, and especially case report studies grow, it may seems as though there is little left that has not yet been associated with the aPL. This is misleading. The literature supports a well-defined clinical syndrome. Many groups have identified the following clinical associations: venous and arterial thromboses~17*19~1W-116 recurrent fetal loss 109,110,112,117-119 thrombocytopenia,s-',l'O-l22 Coombs’ positivity,6.121hemolytic anemia,122livedo reticularis,110~123-126 valvular lesions,127-131 and other Clinical complications neurological diseases.132-136 seem largely limited to high-titer aPL in SLE and the aPL syndrome4*6’31;infection137-142and drugassociated143’144 aPL are not generally believed to be associated with clinical complications, although Triplett et a12shas described thrombotic complications in association with procainamide-induced aPL.

GHARAVI, LOCKSHIN

Prevalence of aPL

The prevalence of aPL in SLE varies with type of assay and population studied. Our estimates of the frequencies in various clinical settings are shown in Table 4. LAC has been identified in from 6% to 71% of SLE patients35*46,111;as described above, this varies widely with the clotting assay chosen. In a review of the literature, Derksen found only 49% of reported cases to be associated with SLE. Twelve percent were drug induced (most commonly phenothiazine, procainamide, hydralazine, and dilantin), 6% were associated with lupus-like disease, and 33% were ascribed to miscellaneous etiologies including hematologic malignancy, cancer, hepatitis, vasculitis, infection, and unexplained thrombosis or fetal loss in apparently healthy people.“’ aCLS have been identified in from 18% to 6 1% of SLE patients.4,'09,"0"45"51 Many other populations have been evaluated for the presence of aCL. In the general population the prevalence seems to be from 0% to 14%.30~109~148*149~152-154 The lower figure is most likely to be true. Syphilis patients may have elevated aCL levels by ELISA, but usually of low titer and IgM isotype.4 Acute infection was long recognized as a common cause of a BFP-STS,” and may be a significant cause of transiently elevated aCL. Vaarala et a113’ evaluated 149 paired sera with antibody-proven recent infections including common viral infections such as adenovirus, rubella, chicken pox, mumps, and also mycoplasma. Thirty-two percent sera had significant levels of aCL, with 10% of these IgG. On repeat samples, aCL levels were often declin-

Table 4: Estimated

Prevalence

of aPL and LAC

in Various Patient Populations aPL

LAC

%+

%+

Normal

c2

tl

Normal pregnancy