24 Lupus Anticoagulants: Mechanistic and Diagnostic Considerations Jef M

24 Lupus Anticoagulants: Mechanistic and Diagnostic Considerations Jef M. M. C. Arnout and Jos Vermylen Introduction Antiphospholipid syndrome (APS) is defined as the association of antiphospholipid antibodies (aPL) with arterial or venous thrombosis, recurrent foetal loss, thrombocytopenia, 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 lupus erythematosus (SLE). In these patients, rheumatologists frequently found a chronic biological false positive test for syphilis, whereas hematologists described a non-specific coagulation inhibitor manifested by prolongation of the whole blood clotting time and the prothrombin time, 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 anticoagulant” 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 “anticoagulant.” 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 associated 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 phospholipid 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 proteins 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 autoantibodies recognizing other PL binding proteins, like protein C, protein S, annexin V, complement 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 phospholipid surface. A hydrophobic core consisting of 6 amino acids (Ser311–Lys317) would penetrate deep within the phospholipid bilayer; it is surrounded by 14 positively 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-glycoprotein 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 thrombin, 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 thrombin. This is followed by thrombin cleavage at position 155 giving rise to prothrombin fragment 1 and prothrombin fragment 2. Depending on the enzyme and the conditions used, prothrombin 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 proteins 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 antibody 2-glycoprotein I–phospholipid interaction β should be noted. 2-glycoprotein I, although binding with high affinity to pure negatively 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 phospholipid 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–

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