INTRODUCTION

The antiphospholipid syndrome manifests as either venous or arterial thrombosis, and in females recurrent fetal loss. Antiphospholipid antibodies are an essential diagnostic feature of the syndrome. Antiphospholipid antibodies represent a heterogenous group of antibodies, targeting protein antigens that bind to anionic phospholipids and anionic phospholipids per se.¹

In this chapter we review the methodology used to detect antiphospholipid antibodies, which can be detected by two methods: using the enzyme linked immunosorbent assay (ELISA) for the detection of a heterogenous group of antibodies that fall under the umbrella term anticardiolipin antibodies (aCL) and clotting assays for determination of the lupus anticoagulant (LA).¹ We also examine the dominant antibodies in APS, which are those targeting β₂GPI^2–4 and prothrombin.⁵ The nature of the dominant antigenic targets is also examined within this context. Pathogenic mechanisms proposed for thrombosis and fetal loss, based on experimental evidence, are reviewed along with mechanisms responsible for the generation of autoantibodies.

DEFINITION OF THE ANTIPHOSPHOLIPID SYNDROME

Diagnosis of the antiphospholipid syndrome requires at least one laboratory criterion and one clinical criterion.⁶ The laboratory criterion involves the detection of moderate to high titers of IgM or IgG anticardiolipin antibodies 12 weeks apart. Alternatively, the detection of the lupus anticoagulant on two separate occasions 12 weeks apart is required. The presence of anti-β₂GPI Abs (either IgG or IgM) 12 weeks apart also constitutes positive serology for the syndrome, and represents a modification of the Sapporo criteria undertaken in Sydney in 2004 during the Eleventh International Congress on antiphospholipid antibodies.⁶

The clinical criteria include the occurrence of venous, arterial, or small-vessel thrombosis anywhere in the circulation. The obstetric criteria include three recurrent miscarriages, in the absence of an alternate explanation, before 10 weeks gestation, or a miscarriage after or including the 10th week of gestation or delivery of a child before the 34th week of gestation due to preeclampsia, eclampsia, or intrauterine growth retardation.⁶ A number of clinical features have been noted to be associated with the syndrome, although they do not constitute a clinical criterion on their own. These include valvular heart disease, livedo reticularis, thrombocytopaenia, transverse myelopathy, and renal small artery vasculopathy.⁶ The catastrophic antiphospholipid syndrome is characterized by widespread intravascular thrombosis.⁷

ANTICARDIOLIPIN ELISA

The aCL ELISA involves microtitre polystyrene plates coated with cardiolipin in ethanol and then dried by evaporation by leaving the ELISA plate open to air.⁸ The plate is subsequently washed with phosphate buffered saline (PBS), and blocked with either fetal or bovine serum or bovine albumin.⁸ The plate is then washed with PBS, and the patient’s samples are applied at a dilution of 1/50 to 1/100.⁸ After incubation and washing of the plates, the labeled secondary antibody (directed against either IgG or IgM) is applied. The aCL levels are then calculated from a standard curve created from a sufficient number of standards.⁸

It was initially believed that the antibodies from patients with APS were binding directly to cardiolipin in this test, and hence the name. It was subsequently found that the antibodies were binding to anionic phospholipid binding proteins (which bound and became immobilized on the cardiolipin-coated plate). These proteins were contained within the patient’s sample and within the blocking buffer.⁹ The dominant antigenic target in patients with APS was subsequently found to be β₂GPI, an abundant plasma protein.^2–4 Other protein antigenic targets have been described, including annexin V¹⁰ and protein S¹¹. The detection of these latter antibodies in APS has been noted in case reports in the literature, and their utility in the clinical diagnosis of APS or as mediators of pathogenesis remains unknown. Anti-prothrombin antibodies are not detected using this method.⁸

TESTS FOR THE LUPUS ANTICOAGULANT

Four sequential steps have been determined to be important in the performance of lupus anticoagulant. They are prolongation of a phospholipid dependent clotting time, absence of correction of the prolonged clotting time on mixing the sample obtained from the patient with normal plasma (demonstrating that the prolongation is not due to a coagulation factor deficiency), evidence of phospholipid dependence (demonstrated by reversal of the prolonged clotting time by the addition of excess phospholipid), and exclusion of specific inhibition of any one coagulation factor.⁸ Two tests are usually performed to detect lupus anticoagulant, as no one test can detect them all. The tests are usually the dilute Russell Viper Venom time (DRVVT) and either an activated partial thromboplastin time (aPTT) or a Kaolin Clotting Time (KCT).⁹

The predominant antibodies responsible for the LA effect are anti-β₂GPI Abs¹² and anti-prothrombin antibodies.⁵ Recently it has been demonstrated that anti-β₂GPI Abs with the lupus anticoagulant effect strongly correlate with thrombosis.¹³ Furthermore, anti-β₂GPI Abs with LA activity appear to recognize the epitope Gly-40/Arg-43 in domain I of the β₂GPI molecule.¹⁴ Anti-prothrombin antibodies with LA activity appear to be less specific for the diagnosis of APS.¹³

The LA is an in vitro phenomenon that does not correlate with in vivo activity. Otherwise, it would be associated with a bleeding diathesis rather than with the prothrombotic tendency associated with APS. The LA phenomenon can be explained mechanistically as follows. Clotting in vitro requires that an anionic phospholipid surface be present to allow for the prothrombinase complex to form, which leads to thrombin generation, which then leads to the generation of fibrin and hence clotting. Anti-β₂GPI Abs and anti-prothrombin Abs in complex with their respective antigens (β₂GPI and prothrombin) bind with very high affinity to anionic phospholipids, thereby competitively inhibiting the binding and formation of the prothrombinase complex and leading to prolongation of clotting.⁹ The addition of excess phospholipids ensures that there are adequate binding sites for the prothrombinase complex to form, thereby removing the competitive inhibition.⁹

ANTI-β₂GPI ELISA

The finding that patients with APS require β₂GPI for binding in the aCL ELISA led to the testing of the β₂GPI ELISA system without cardiolipin.¹⁵ It has been found that for the β₂GPI ELISA to work, β₂GPI has to be coated onto a negatively charged surface. This surface may be a preirradiated polystyrene microtitre plate.¹⁵ The reasons for this requirement have been closely studied, and the different explanations suggested are not necessarily mutually exclusive. one explanation is that the negatively charged surface allows β₂GPI to bind via domain V.¹⁶ This may lead to a conformational change of the molecule that leads to exposure of a cryptic epitope on domain I, thus allowing antibodies to bind.¹⁶ Another explanation is that the negatively charged surface allows the β₂GPI molecules to cluster closely together, and this has been shown to enhance the avidity of the antibodies for the antigenic target.¹⁷ Anti-β₂GPI Abs tend to be low affinity and do not tend to form antibody antigen complexes in plasma.¹⁸

The β₂GPI molecule is composed of five domains.⁹ Studies have been performed to determine the epitope specificity of the antibodies associated with the syndrome. It has been found that in APS the dominant antigenic target is in domain I.¹⁹ The fine epitope specificity is at Gly-40/Arg-43.^20,21 In the majority of infections, anti-β₂GPI Abs are not detected.²² In leprosy infection and atopic dermatitis associated with childhood, anti-β₂GPI Abs have been detected. However, in these clinical situations the dominant antigenic target lies in domain V.^23,24 It has been proposed that developing a diagnostic test that can detect anti-β₂GPI Abs directed against the epitopes in domain I may further improve the specificity of testing for the antiphospholipid syndrome.¹⁴

β₂GPI

β₂GPI is found in human plasma at a mean concentration of 4 μM.²⁵ It is conserved among mammals, there being 60 to 80% amino acid sequence identity among the human, bovine, canine, and murine proteins.⁹ It is composed of 326 amino acids.²⁶ In crystallographic analysis, its structure is J shaped.²⁶ It is divided into five domains, each domain being typical of the complement control protein module.²⁶ The first four domains are composed of 60 amino acids.²⁶ Each domain contains cysteine moieties, which allow the formation of disulfide bridges by linking the first with the third and the second with the fourth cysteine residues.²⁶ The third and fourth domains are heavily glycosylated.²⁶ The fifth domain contains an extra 20 amino acids at the C-terminus.²⁶ The C-terminus is unusual in that it terminates with a cysteine moiety, which forms a disulfide bridge with a cysteine found between the standard second and third cysteine residue positions in the fifth domain.²⁶ The fifth domain, between amino acids 281 and 288, contains a region that contains multiple positively charged lysine residues and is critical for phospholipid binding.²⁶

Certain proteases [such as plasmin and activated factor XI (FXIa)] have been found to be able to cleave β₂GPI between Lys 317 and Thr 318, abolishing its ability to bind to anionic phospholipids.^27,28 β₂GPI is predominantly generated in liver.⁹ The physiologic function of β₂GPI is not clearly delineated as yet, although progress has been made in this field. β₂GPI knockout mice have been generated. The hemostatic system has been studied in these mice, as has the reproductive system. In the hemostatic system it has been demonstrated that in vitro plasma from β₂GPI knockout mice displayed an impairement of thrombin generation compared to plasma from wild-type mice.²⁹ With regard to reproduction, β₂GPI knockout mice produce litters that are smaller than wild-type mice. Furthermore, they display mild placental abnormalities.³⁰

In the past, a number of functions were attributed to β₂GPI based on its ability to bind the anionic phospholipid phosphatidylserine. Phosphatidylserine is expressed on apoptotic cells.³¹ It is also expressed by activated platelets and activated endothelial cells.¹ It was suggested that β₂GPI may bind to apoptotic cells, thereby allowing for their clearance.³¹ Its binding to activated platelets has been suggested to enable it to play an anticoagulant role by inhibiting ADP-induced aggregation of washed platelets³² and inhibition of the initiation of the contact pathway by competitively inhibiting FXII binding to the negatively charged surface.³³ The ability of β₂GPI to bind to phosphatidylserine on cell surfaces in conditions that mimic the in vivo milieu has recently been called into question by the finding that monomeric β₂GPI in fluid phase displays negligible binding, suggesting that any physiologic function is unlikely to be mediated by binding to phosphatidylserine in the absence of cofactors yet to be defined.³⁴

β₂GPI has recently been found to directly bind factor XI.³⁵ Factor XI plays an important role in the amplification of thrombin generation.³⁶ It can be activated either by FXIIa or thrombin.³⁶ In vivo evidence is accumulating that FXI activation may play an important role in thrombus propagation.³⁷ FXI-deficient mice display poorly formed and friable thrombus formation on ferric chloride-induced carotid artery injury, in contrast to complete blockage in the wild type.³⁸ In primates, FXI plays an important role in thrombus propagation.³⁹ It is interesting to note that β₂GPI binding to FXI inhibits its activation by FXIIa and thrombin.²⁸ When β₂GPI is clipped by plasmin or FXIa, even though it retains its ability to bind FXI it loses its ability to inhibit its activation.³⁵ This suggests that in vivo β₂GPI may regulate FXI activation.³⁵

It has been demonstrated that clipped β₂GPI, which can be generated either by plasmin²⁷ or activated FXI,²⁸ can bind to plasminogen and inhibit its conversion to plasmin by tissue plasminogen activator.⁴⁰ This set of results suggests that β₂GPI may provide a regulatory link between the FXI activation pathway and fibrinolysis.

PROTHROMBIN

Prothrombin is generated and secreted by the liver. It contains 579 amino acids. The mean concentration in the plasma is approximately 1 to 2 μM.⁴¹ It is a vitamin-K-dependent glycoprotein. It is the zymogen precursor of thrombin.⁴² Unlike β₂GPI, its physiologic function is well characterized. It represents a key point in the coagulation cascade. Thrombin is responsible for local fibrin formation, and upon binding to thrombomodulin for activating protein C (which serves to limit clot propagation in the region of intact and healthy endothelium).⁴³ Thrombin also binds to the platelet surface via the GPIb-IX-V receptor, and by cleaving the protease-activated receptors 1 and 4 (PAR1 and PAR4).⁴³

During its biosynthesis in the liver, prothrombin undergoes γ-carboxylation.⁴² These γ-carboxyglutamic residues (known as the Gla domain, and located on fragment 1 of the prothrombin molecule) are essential for the calcium-dependent binding of prothrombin to phosphatidylserine.⁹ The prothrombinase complex (Xa/Va/Ca²⁺/phospholipid) activates prothrombin to thrombin by cleavage at several sites. The Gla domain is removed during this process, and hence thrombin does not have a phospholipid binding site.⁹

AUTOANTIBODY PRODUCTION MECHANISMS