24 : Mechanistic and Diagnostic Considerations Jef M. M. C. Arnout and Jos Vermylen

Introduction

Antiphospholipid syndrome (APS) is defined as the association of antiphospholipid (aPL) with arterial or venous , recurrent foetal loss, thrombo- cytopenia, or neurological disorders [1–3]. The gradual development of the notion APS started in the 1950ss with the recognition of two laboratory curiosities in a subset of patients with systemic (SLE). In these patients, rheumatologists frequently found a chronic biological false positive test for syphilis, whereas hematologists described a non-specific inhibitor manifested by prolongation of the whole blood clotting time and the , without reduction of any specific clotting factor then measurable [1–3]. The non-specific coagulation inhibitor which appeared not to be associated with a bleeding tendency was named the “lupus ” by Feinstein and Rapaport [4] and was regarded as a laboratory curiosity until Bowie et al [5] drew the attention to the high prevalence of thrombotic complications in SLE patients with this “anticoagu- lant.” The LA was later also found to be associated with obstetric complications and thrombocytopenia [6]. Only in the 1980s did it became clear that antibodies interacting with anionic phospholipids are responsible for the in vitro LA effect and the chronic biological false positive syphilis serology [7]. This led to the development of better-defined LA tests and the so-called anticardiolipin test in which antibodies binding to solid phase cardiolipin (aCL) are measured [8, 9]. With these improved assays, the majority of SLE patients with a LA also had elevated aCL levels and a statistically significant relation between these 2 types of aPL was observed. It is now well established that persistently present aCL and LA in patients with SLE are associ- ated with thrombosis and pregnancy morbidity [10]. This association is now termed APS [11]. Some patients with similar clinical symptoms and laboratory findings but not suffering from SLE or a closely related autoimmune disease are diagnosed as having a “primary APS” [12]. The availability of a sensitive assay for aCL has been crucial for the further characterization of aPL. Affinity purification of aCL led to the discovery that, in contrast to what the term aPL suggests, aCL do β not bind to cardiolipin per se but to 2-glycoprotein I bound to anionic phospho- lipid surfaces [13–15].

291 292 Hughes Syndrome Antigenic Targets of aPL β Soon after the discovery that 2-glycoprotein I was involved in the binding of aCL to cardiolipin, it was reported that a subpopulation of aCL possesses LA activity and that certain LAs are directed against prothrombin. It was also reported that aCL β bind to 2-glycoprotein I even in the absence of PL [16]. The affinity of the interac- β tion of these antibodies with fluid phase 2-glycoprotein I is, however, low. It is now generally accepted that autoimmune aPL have in common that they are directed against with affinity for PL or negatively charged surfaces. The main anti-

Figure 24.1. Structure of human β2-glycoprotein I based on amino acid sequence, disulphide mapping, and crys- tallographic data. The five repeating sushi domains are indicated with roman numbers. CHO denotes N-linked glycosylation sites, 1 denotes amino terminal end, and 326 denotes carboxyterminal end. The hydrophobic flex- ible loop Ser311–Lys317 is indicated by arrow A; the positively charged amino acids interacting with the anionic phospholipid headgroups are marked in grey. Arrow B indicates the plasmin sensitive cleavage site at position Lys317–Thr318. Lupus Anticoagulants: Mechanistic and Diagnostic Considerations 293

β gens are 2-glycoprotein I and prothrombin, although a number of recognizing other PL binding proteins, like C, protein S, annexin V, comple- ment factor H, high- and low-molecular-weight kininogen, prekallikrein, factor XI, tissue factor pathway inhibitor (TFPI), factor VII/VIIa, etc., have been found in sera of patients with APS [17–19].

Relevant Structures of β2-Glycoprotein I and Prothrombin β 2-glycoprotein I (apolipoprotein H) is mainly synthesized by hepatocytes and to a lesser extent by endothelial cells and placental cells [20]; its plasma concentration is β approximately 3 µmol/L. 2-glycoprotein I consists of a single polypeptide chain of 326 amino acids and is composed of 5 homologous domains of approximately 60 amino acids, designated short consensus repeats/complement control protein repeats, or “sushi” domains [21, 22]. These are designated as domain I to domain V, from the N terminus to the C terminus (Fig. 24.1). Domains III and IV are heavily glycosylated. Domain V contains a 6-residue insertion and a 19-residue C-terminal tail resulting in a C-terminal loop consisting of 20 amino acids cross-linked by an β additional disulfide bond [22]. The crystal structure of 2-glycoprotein I has been defined [23, 24]. The overall shape is that of an elongated fishhook, domain V being at right angle to the aligned first 4 domains, and making contact with the phospho- lipid surface. A hydrophobic core consisting of 6 amino acids (Ser311–Lys317) would penetrate deep within the phospholipid bilayer; it is surrounded by 14 posi- tively charged residues, which stabilize the binding to phospholipids via electrosta- β tic interactions with the anionic phospholipid head groups. 2-glycoprotein I is sensitive to cleavage by plasmin between Lys 317 and Thr318 [25]. The cleaved form β binds much less avidly to negatively charged phospholipids. Binding of 2-glyco- protein I to phospholipid does not involve calcium ions. Prothrombin is a 579 amino acid long single chain glycoprotein, whose plasma concentration is approximately 1.5 µmol/L. Prothrombin is built up by an amino terminal GLA domain, in which the 10 γ-carboxyglutamic acid residues are concen- trated and through which prothrombin binds to negatively charged phospholipid in the presence of Ca2+ ions [26]. Two kringle domains, K1 and K2, and a serine pro- tease domain follow the GLA domain [see Fig. 24.2(A)]. The two kringle domains contain a highly conserved pentapeptide CRNPD, shared by all kringle proteases. Prothrombin contains 2 cleavage sites for factor Xa and 2 cleavage sites for throm- bin, respectively, located at positions 271 and 320 and positions 155 and 284. Prothrombinase-catalyzed activation of prothrombin occurs by factor Xa mediated consecutive cleavages at positions 320 and 271, resulting in the generation of meizothrombin, prothrombin fragment 1+2, and α . This is followed by thrombin cleavage at position 155 giving rise to prothrombin fragment 1 and pro- thrombin fragment 2. Depending on the enzyme and the conditions used, pro- thrombin can be degraded into various fragments [see Fig. 24.2(B)] that may be used for epitope mapping. β The plasma concentrations of 2-glycoprotein I and prothrombin are remarkably similar. As will be discussed later, the LA phenomenon depends on surface occu- pancy. Presumably, micro-molar concentrations of the phospholipid binding pro- teins are required to allow adequate surface coverage. 294 Hughes Syndrome

Figure 24.2. Panel (A) shows a simplified structure of prothrombin. The amino terminal GLA domain is followed by two kringle domains and a catalytic domain, stabilized by an internal disulphide bridge. The cleavage sites for thrombin (IIa) and activated factor X (Xa) are indicated. Panel (B) gives the prothrombin fragments that may be formed after digestion by thrombin or activated factor X. Lupus Anticoagulants: Mechanistic and Diagnostic Considerations 295 Anticoagulant Mechanism of LA in vitro

The in vitro anticoagulant effect of LA was originally explained by the assumption that these antibodies compete with clotting factors for anionic phospholipids acting β as catalytic surface for coagulation reactions. With the discovery of 2-glycoprotein I and prothrombin as cofactors for aPL, the question rose how antibodies may enhance the binding of these proteins to phospholipids. This question has been β addressed first for so-called 2-glycoprotein I–dependent lupus anticoagulants. A β few characteristics of the 2-glycoprotein I–phospholipid interaction β should be noted. 2-glycoprotein I, although binding with high affinity to pure neg- atively charged phospholipids such as cardiolipin and phosphatidylserine, has only a weak affinity for physiological procoagulant phospholipids [27], explaining why this protein is at most a poor anticoagulant. To become anticoagulant, the complex β β of 2-glycoprotein I and patient anti– 2-glycoprotein I antibody should have a β higher affinity for phospholipid than that of 2-glycoprotein I alone. However, β patient anti– 2-glycoprotein I antibodies by themselves only have a relatively weak β affinity for 2-glycoprotein I. This makes a scenario unlikely, where antibody β binding causes a conformational change in 2-glycoprotein I that favors phospho- lipid binding. A second possible scenario is much more plausible (see Fig. 24.3).

Figure 24.3. LA-positive aPL form stable bivalent immune complexes on PL surfaces. The affinity of the bivalent immune complex for coagulation active PL surfaces is higher than that of the monomeric PL-binding protein such as β2-glycoprotein I (β2-GPI) or prothrombin (FII) alone. 296 Hughes Syndrome

Indeed, it has been shown that in the presence of a physiologic procoagulant phos- β pholipid surface, some anti– 2-glycoprotein I IgGs cross-link 2 phospholipid-bound β 2-glycoprotein I molecules and thereby attach these with high affinity to the surface [28–30]. The surface occupancy by bivalent complexes impedes the clotting reaction and accounts for the LA phenomenon. β Studying a series of murine monoclonal antibodies against human 2-glycopro- tein I, it became apparent that several antibodies, although detected in the aCL assay, did not affect phospholipid dependent clotting tests [30]. Apparently, only β β LA-positive anti– 2-glycoprotein I antibodies cross-link 2 2-glycoprotein I mole- β cules in such a way that both 2-glycoprotein I molecules of the bivalent complex can interact efficiently with the phospholipid surface. Because the affinity increases with the number of available binding sites, these particular bivalent complexes have β a significantly higher affinity for coagulation active phospholipids than 2-glyco- protein I alone and remain attached on the surface. This then explains the observa- tion that a significant proportion of patients have antibody binding to immobilized β 2-glycoprotein I (a positive anti-cardiolipin assay), yet lack LA activity in their β plasma [31]. Autoimmune anti– 2-glycoprotein I antibodies are indeed a heteroge- neous group of antibodies. Patients further often have polyclonal antibodies with different specificities. It was therefore not surprising that studies on the domain- specific location of the epitopes recognized by these antibodies showed binding to β various domains of 2-glycoprotein I , mainly V, IV, and I [32–34]. However, certain groups claimed that a dominant epitope was located on domain IV [33] while others provided evidence for a dominant epitope on domain I [34,35]. It recently became β clear that this discrepancy is due to the orientation of 2-glycoprotein I on the dif- β ferent micro-titer plates used for binding studies and that the majority of anti– 2- glycoprotein I antibodies bind to domain I [36]. Clear discrimination at the epitope β level between LA-positive and -negative anti– 2-glycoprotein I antibodies has not yet been reported. In the same manner, only a proportion of prothrombin dependent aPL show LA activity [37]. Some autoimmune antibodies to prothrombin can markedly enhance binding of prothrombin to negatively charged phospholipid [38, 39], again likely via the formation of bivalent prothrombin antibody complexes [40]. The epitopes recognized by autoimmune antiprothrombin antibodies have not yet been fully characterized. Binding to prethrombin 1, fragment 1+2, and fragment 1 has been reported [38, 41, 42]. Antibodies recognizing prethrombin 1 are likely to be directed against a conformational epitope on kringle 2 because binding usually disappears under reducing conditions and because binding to thrombin appears to be very exceptional. Puurunen et al [42] have described a more precise epitope for anti-prothrombin antibodies located on kringle 2, between amino acid 210 and 229. This amino acid sequence contains a conserved pentapeptide CRNPD shared by all kringle proteins, such as plasminogen and factor XII. Puurunen showed that a high proportion of autoimmune anti-prothrombin antibodies cross-react with plasmino- gen. Whether this conserved sequence is indeed a dominant epitope on prothrom- bin is questionable because antiprothrombin antibodies do not cross-react with factor XII, another kringle protein sharing the same conserved pentapeptide [43]. Patient antibodies to prothrombin seem to be polyclonal or oligoclonal in nature as antibodies recognizing various prothrombin fragments may occur simultaneously [38]. Lupus Anticoagulants: Mechanistic and Diagnostic Considerations 297 aPL and Risk for Thrombosis

Although aPL are mainly measured to fulfill the classification criteria for an APS, they are more and more used to estimate the risk for (recurrent) thrombosis or pregnancy morbidity in a given patient. There is growing consensus that LA are stronger risk factors for thrombosis than aCL. It was firstly shown that the presence of a LA correlates better with a history of thrombotic complications than the pres- ence of aCL. The results from a systematic review of the literature formally estab- lished that, in patients with previous thrombosis and/or SLE, the presence of a LA is a stronger risk factor for future thrombotic complications than the presence of aCL [44].

Pathogenic Mechanisms of aPL: Surface Mediated Thrombogenic Characteristics

In view of the strong association between the presence of (certain) aPL and throm- bosis, it is tempting to believe that aPL are involved in the thrombogenesis. Several prothrombotic mechanisms have been proposed which have in common that they interfere with surface mediated phenomena. The formation of bivalent immune complex described above is also a surface- β dependent phenomenon because the binding between anti– 2-glycoprotein I anti- bodies and its antigenic target in solution is hampered by the intrinsically low affinity nature of this interaction. The observation that LAs are more closely associ- ated with thrombotic events than aCL also provides an argument in favor of the hypothesis that the prothrombotic activity of aPL may be a surface mediated phe- nomenon. Anionic enriched PL surfaces, needed for the formation of stable bivalent immune complexes, are hardly available within a healthy vessel because negatively charged PLs are normally sequestered within the inner leaflet of the PL bilayer of the cell membrane. Such surfaces, however, become available during normal haemostatic processes [45–47]. aPL do probably not cause thrombosis by themselves but rather influence the thrombotic process once negatively charged PL becomes exposed. aPL would there- fore function as a “second hit.” There is indirect clinical support for the concept of a double hit phenomenon. First, not all patients with aPL develop thrombosis but those who do develop thrombosis have a high rate of recurrence. Secondly, arterial events are almost always followed by arterial events and is most likely followed by another venous thrombosis [48]. There are several “anticoagulant” surface mediated processes with which aPL are thought to interfere. One major anticoagulant pathway involves protein C and protein S [49]. Protein C, activated by thrombin on endothelial thrombomodulin, and protein S bind to negatively charged PL through their GLA domains; activated protein C in association with protein S cleaves factor Va and factor VIIIa on the PL surface and thereby inactivates the intrinsic tenase and prothrombinase reactions. Occupancy of the surface by immune complexes could impede these interactions and thereby promote further thrombin generation and thrombus growth. Indeed, 298 Hughes Syndrome

LAs can induce “activated protein C resistance” [50]. A second anticoagulant mech- anism involves tissue factor pathway inhibitor. This protein binds to negatively charged PL and to factor Xa on PL. This protein complex then links to the tissue factor–factor VIIa complex, and shuts off further tissue factor mediated clotting. Again, occupancy of the PL surface by immune complexes may impede this interac- tion, leading to prolonged thrombin generation [51]. While these interferences of the immune complexes with anticoagulant pathways have been elegantly demonstrated in vitro, it is difficult to unequivocally extrapo- late these observations in vivo because the immune complexes could impede assem- bly of the procoagulant complexes to the same extent and the overall result would then be neutral. However, under certain pathological conditions, this may not be the case. Oxidation is believed to play an important role in inflammatory diseases such as atherosclerosis. In addition it was reported that oxidation of phos- phatidylethanolamine containing liposomes potentiates the anticogulant effect of the protein C pathway without affecting the procoagulant pathway. Total IgG from patients with aPL and thrombosis appears to selectively inhibit the anticoagulant effect of phospholipid oxidation [52]. The prothrombotic action of aPL may not be limited to inhibition of surface mediated anticoagulant pathways. Chesterman’s group provided evidence that enhanced antibody-mediated deposition of prothrombin on PL may lead to increased thrombin generation in conditions of flow [53]. We suggest that the deposition of immune complexes on slightly activated cells may induce further cell activation, leading to generation of micro-vesicles that para- doxically provide a much larger PL surface resulting in enhanced thrombin genera- tion [54, 55] (see Fig. 24.4). This pathogenetic model was confirmed in an in vivo model of thrombosis in the hamster in whom slight radical induced endothelial injury of a carotid artery had β been provoked after intravenous injection of monoclonal antibodies to human 2- glycoprotein I [56]. One of the tested antibodies had LA activity in hamster plasma, β a second bound immobilized hamster 2-glycoprotein I but was without LA activity, β a third had no affinity for hamster 2-glycoprotein I. The antibody with LA activity markedly enhanced arterial thrombosis, the second antibody without LA activity hardly enhanced thrombosis, the third antibody being inactive. F(ab)2 fragments of the LA antibody also enhanced thrombosis, whereas Fab fragments were inactive. These findings confirm that bivalency is required for thrombosis enhancement but also that the Fc component of the antibody is not compulsory for this enhancement. Immunohistological examination revealed that the antibody was mainly localized focally within the platelet thrombus, suggesting thrombus growth spreading from the foci of immune complex deposition on activated platelets. These experiments are in agreement with a double hit scenario: following mild endothelial damage, a small platelet thrombus develops (first hit); the slightly activated platelets expose β negatively charged PL; this leads to patchy deposition of bivalent 2-glycoprotein I–antibody complexes; these complexes cause further platelet activation and throm- bus growth (second hit). The fact that Fc receptor activation is not compulsory for the prothrombotic action is intriguing and may suggest that another type of receptor-triggered process is involved. Direct evidence for this was obtained from in vitro experiments under β β flow conditions showing that recombinant 2-glycoprotein I–dimer, like 2-glyco- protein I–antibody dimers, enhances platelet activation and deposition on collagen Lupus Anticoagulants: Mechanistic and Diagnostic Considerations 299

Figure 24.4. Putative pathogenic mechanism in APS: as a consequence of an initial damage, anionic PLs are exposed on cells of the blood, on endothelium, or on trophoblasts. In the presence of aPL and their antigenic targets such as β2-glycoprotein I bivalent immune complexes with increased affinity for these PL surfaces are formed. The deposition of immune complexes causes further cellular activation probably via cross-linking apolipoprotein E receptor 2 (apoER2) and a signal transducing mechanism involving p38MAP kinase and this in addition to the potential role of the platelet FcγRIIa receptor; for platelets this would lead to release of granule contents and formation of micro-vesicles, which would paradoxically provide a much larger negatively charged PL surface, and therefore enhance rather than inhibit thrombin generation. surfaces [57]. This platelet deposition appeared to be dependent on cross-linking and activation of the apolipoprotein E receptor 2.

Laboratory Diagnosis of LA SSC Criteria for LA Testing

According to the recommendations of the SSC, the laboratory diagnosis of a LA should be based on the following criteria: prolongation of a phospholipid depen- dent clotting test, in particular when the phospholipid content of the test system is low; lack of correction of the prolonged clotting time by addition of a small amount of normal plasma (thereby excluding clotting factor deficiency); correction by the presence of a higher concentration of phospholipid or by use of a reagent that is poorly responsive to the LA effect [58]. Detection of LA is delicate and sometimes impossible when the patient is already treated with heparin or oral anticoagulants. 300 Hughes Syndrome

Because autoimmune aPL are chronic, the presence of a LA should be confirmed on a second sample taken several weeks later. It should be emphasized that a 6-week interval may not be sufficient to distinguish persistent LA from those seen in rela- tion with infectious disease because the latter may persist for several months [59]. It is very hard to give precise recommendations on which assays to use for the detection of LA. Selection of assay systems with optimal sensitivity and specificity is almost impossible due to the lack of a gold standard (a well-defined LA-positive patient population). Given the fact that various assays may detect different types of LA [60, 61] and that not all LA are associated with thrombosis, the best way to select optimal screen assays should be based on clinical trials. According to a recent sys- tematic review of the literature however, the risk of thrombosis at present appears to be independent of the laboratory tests used to identify LA [44].

Discrimination Between LA and Factor VIII Inhibitors

Differentiation between LA and specific factor inhibitors, although extremely impor- tant in view of their different clinical consequences, often remains difficult. In a survey of the European Concerted Action on (ECAT), a sample with a slow acting auto-immune factor VIII inhibitor at 10 Bethesda Units was included [61]. As much as 20% of the participating laboratories interpreted their results as compatible with the presence of a LA. In several cases, this interpretation was based on the combination of a prolonged partial thromboplastin time (aPTT) which did not entirely correct upon mixing with normal plasma and a prolonged confirm aPTT with reduced phospholipids. As clinical laboratories receive less and less information on the clinical condition of the patients from whom samples for LA diagnosis are obtained, the use of factor assays should be encouraged whenever a LA is diagnosed in an aPTT based system. Laboratories should be aware that potent LA and specific factor VIII inhibitors may behave very similarly in vitro (an example is given in Table 24.1). In the presence of a potent LA, the apparent activity of various clotting factors in a one-stage assay may be reduced. However, a high titer factor VIII inhibitor may also cause a false reduction of factors IX, XI, and XII and therefore mimic a LA. Determining factor levels in diluted plasma may be of help to differenti- ate between a LA and a factor VIII inhibitor. In the first case, the apparent clotting

Table 24.1. An example of the effect of a strong lupus anticoagulant and a high titer FVIII inhibitor on factor assays.

Plasma predilution in buffer 1/10 1/20 1/40

Lupus anticoagulant FVIII (%) 1.6 8.5 39.9 FIX (%) 8.3 28.9 57.2 FXI (%) 5.9 19.7 51.8 FXII (%) 21.3 44.1 66.4

Factor VIII inhibitor FVIII (%) 0.5 0.5 0.5 FIX (%) 12.6 18.2 27.3 FXI (%) 15.4 21.6 31.3 FXII (%) 22.5 29.7 35.7

Note that not all LA or factor VIII inhibitors behave similarly. Lupus Anticoagulants: Mechanistic and Diagnostic Considerations 301 factor activity increases as higher plasma dilutions are being tested. In the latter case, the factor VIII level remains low in diluted plasma.

Importance of LA Potency or Titer

For aCL, the thrombotic risk seems to increase with the antibody titer. Only few attempts have been made to study the relation between the LA potency or titer and the clinical manifestations of the APS. A first feasibility study to quantify LA was performed by the Groupe d’Etudes sur l’Hémostase et la Thrombose (GEHT) [60]. The LA potency was determined in samples from 40 patients with APS and 29 LA-positive asymptomatic individuals using LA screening assays calibrated with a normal plasma pool spiked with β equimolar combinations of a LA positive anti- 2-glycoprotein I monoclonal anti- body (moab) and an LA-positive anti-prothrombin moab at concentrations ranging from 0 to 60 µg/mL. Eight reagent–instrument combinations were used in this study. Patients with APS had coagulation profiles distinct from those with asympto- matic LA, the former having significantly higher LA titers measured using a calibrated dilute Russell Viper Venom Time (dRVVT). The largest difference was β found for the 2 dRVVT assays, which were more responsive to 2-glycoprotein I–dependent LA. This study not only showed that quantification of LA activity in clinical samples using calibrator plasmas spiked with moabs against protein targets of aPL is feasible, but also suggested that there might be a relation between the titer (or potency) and the risk for thrombosis. That such relation indeed exists was subsequently confirmed in a prospective study of 678 patients with SLE [62]. In this study, the immediate risk of deep venous thrombosis increased 34% with each 5 seconds prolongation of the dRVVT.

Importance of the Antigenic Target of LA β There is growing evidence that 2-glycoprotein I–dependent aPL may be more strongly associated with thrombosis than prothrombin-dependent aPL. The results of a 4-year prospective study based on the Italian registry show that the risk for thrombosis is highest in APS patients who in addition to a LA also have medium or β high titer aCL [63]. Because autoimmune aCL are 2-glycoprotein I–dependent aPL, this observation may suggest that the LA associated with thrombosis may be pre- β β dominantly directed against 2-glycoprotein I. Galli and coworkers noted that 2- glycoprotein I–dependent LA more strongly prolong the dRVVT than prothrombin-dependent LA, whereas the effects of these two types of LA on the kaolin clotting time (KCT) are opposite [64]. She also found the dRVVT to be a better predictor of thrombosis than the KCT in a historical cohort of 100 consecu- tive LA-positive patients [65]. However, it should be noted that there also is evi- dence for a prothrombotic role of anti-prothrombin antibodies [66, 67]. β Two recent reports describe a method to discriminate 2-glycoprotein I–depen- dent LA from prothrombin dependent ones. The first method, by Simmelink et al β [68], is based on the high affinity interaction between 2-glycoprotein I and pure β cardiolipin. 2-glycoprotein I and LA dependent on this protein are adsorbed on cardiolipin vesicles which are added to patient plasma. Shortening of a LA sensitive aPTT in the presence of cardiolipin vesicles is an indication for the presence of a 302 Hughes Syndrome

β 2-glycoprotein I–dependent LA. The second method, by Pengo et al [69], is even β more elegant and is based on an increased affinity of 2-glycoprotein I for coagula- tion active phospholipids when the calcium concentration or the ionic strength in the reaction mixture is lowered [70]. A further prolongation of a LA sensitive screening assay such as the dRVVT or the dilute prothrombin time after lowering β the calcium concentration from 10 to 5 mM is an indication for the presence of a 2- glycoprotein I–dependent LA in a patient sample. On the contrary, the clotting time is slightly shortened with lower calcium concentrations in case of prothrombin con- taining immune complexes, because calcium is involved in the binding of pro- thrombin to phospholipids. The availability of relatively simple clotting based β assays to differentiate 2-glycoprotein I from prothrombin dependent LA now makes it possible to more properly address the question whether the risk for throm- bosis is to some extent linked to the target antigen of the LA.

Quality Control

Despite internationally accepted guidelines [58] and many efforts to better stan- dardize LA assays, several national and international inter-laboratory surveys and workshops have shown that the accuracy of LA testing still is far from optimal. In the first French interlaboratory survey on LA, only 41% of 4500 laboratories made a correct diagnosis of LA [71]. In the U. K. national external quality assessment scheme (NEQAS), a weak LA was not found by more than 50% of the laboratories and a factor IX deficient plasma was diagnosed as LA positive by 26% of the labora- tories [72]. A major shortcoming in this regard is the lack of internationally accepted reference and control materials. For internal quality assessment, laborato- ries and reagent manufacturers still have to rely on LA-positive patient samples. A set of LA-positive reference plasmas with a range of potencies has been pre- pared by the LA working party of the British Society for Haematology [73]. These plasmas were obtained by pooling several LA-positive plasmas and diluting them in LA-negative plasma. However, the supply of such materials is very limited and the fact that each new pool will have different characteristics probably makes this approach less suited to prepare real reference materials. β LA positive anti– 2-glycoprotein I and anti-prothrombin moabs are now avail- able [74, 75]. These antibodies recognize the same target proteins as human LA and share the same mechanism of action. In addition, they may be combined and can be produced in large quantities with a constant behavior over time. Therefore, normal plasma pools spiked with certain amounts of these moabs have the potential to serve as reference and control materials for LA testing. The Scientific and Standardization subcommittee on LA and phospholipid-dependent antibodies of the International Society on Thrombosis and Haemostasis is currently preparing such international reference materials.

Acknowledgments

The work of J. Arnout is supported by a grant from the Flemish Fund for medical scientific research Levenslijn 7.0032.98 and FWO G.0226.01. Lupus Anticoagulants: Mechanistic and Diagnostic Considerations 303 References

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