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Summary. Lupus anticoagulants (LAC) are a heterogeneous group of autoantibodies that prolong phospholipid-dependent clotting assays. The autoantibodies that cause LAC activity are predominantly directed against β2-glycoprotein I (β2GPI) or prothrombin. In the present study, we describe a method to differentiate between LAC caused by antibodies directed against β2GPI or prothrombin. Monoclonal antibodies, affinity purified patient antibodies, and selected patient samples were used to show that in an aPTT-based clotting assay (PTT-LA; Diagnostica Stago), the use of cardiolipin vesicles in the neutralization procedure discriminates between β2GPI- or prothrombin-dependent LAC activities. Addition of cardiolipin vesicles shortened the prolonged clotting time caused by anti-β2GPI antibodies with LAC activity, whereas this procedure further prolonged clotting times caused by antiprothrombin antibodies with LAC activity. In contrast, addition of phosphatidylcholine/phosphatidylserine vesicles corrected prolonged clotting times caused by either anti-β2GPI or antiprothrombin antibodies with LAC activity. The effects of cardiolipin (CL) on β2GPI-induced LAC activity were specific for contact activation mediated clotting assays. Possible explanations for these findings are the relatively high affinity of β2GPI for cardiolipin, as determined by surface plasmon resonance analysis, and inhibition by anti-β2GPI antibodies of the CL-induced prolongation of the PTT-LA.
Antiphospholipid antibodies (aPL) are traditionally classified as lupus anticoagulants (LAC) and anticardiolipin antibodies (aCL) according to their methods of detection with in vitro clotting tests or ELISA assays , respectively. LAC refers to immunoglobulins that prolong in vitro clotting assays [2–4], whereas aCL bind to immobilized cardiolipin (CL) [5,6], a negatively charged phospholipid. In contrast to infection-related aPL, autoimmune aPL are not directed to phospholipids alone, but to lipid-binding (plasma) proteins, notably β2-glycoprotein I (β2GPI) or prothrombin [7–11]. Although β2GPI itself has affinity for phospholipids, its affinity is strongly enhanced in the presence of anti-β2GPI antibodies, due to formation of bivalent complexes [12–15]. It is generally accepted that bivalent complexes compete more strongly with clotting factors for the catalytic phospholipid surfaces than monovalent β2GPI. We have recently described a similar mechanism of enhanced binding of divalent complexes for prothrombin-dependent LAC .
It has been shown that the presence of LAC correlates better with a history of thrombotic complications than in the presence of aCL [17,18]. It has been suggested that anti-β2GPI antibodies are more related to thrombotic complications than antiprothrombin-antibodies [19–22]. These observations were based on detection of these antibodies with an ELISA setup. The clinical relevance of anti-β2GPI and antiprothrombin antibodies with a functional activity (viz. induction of LAC activity) is unknown. A simple method to discriminate between LAC activity caused by anti-β2GPI and antiprothrombin antibodies therefore seems relevant for future clinical studies.
Guidelines for the detection of LAC have been published  and involve confirmation of the phospholipid-dependent nature of the inhibitory antibody. This confirmation step normally consists of addition of a high concentration of negatively charged phospholipids to abolish the inhibitory effects of the antibodies. The nature of the negatively charged phospholipids has never been defined and it is thought that all negatively charged phospholipids neutralize all LAC activity, irrespective of type of antibodies involved. Here we show that this basic assumption is incorrect as a neutralization procedure with cardiolipin vesicles in an aPTT-based assay only neutralizes LAC activity due to anti-β2GPI antibodies. This observation opens the possibility to discriminate between β2GPI- and prothrombin-dependent LAC activity.
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- Materials and methods
The results presented in this paper show that by using a PTT-LA test and two neutralization procedures with either CL or PS/PC vesicles, differentiation is possible between LAC activity caused by anti-β2GPI and antiprothrombin antibodies. Addition of increasing concentrations of CL to NPP spiked with mono- or polyclonal anti-β2GPI antibodies with LAC activity, or to patient plasma with LAC due to anti-β2GPI antibodies shortens the prolonged aPTTs (Fig. 2a,b). Addition of CL to NPP spiked with antiprothrombin antibodies with LAC activity or to patient plasmas with LAC due to antiprothrombin antibodies prolongs the aPTT clotting times (Fig. 2a,b). When the same experiments were done with PS/PC vesicles, we noted neutralization of LAC activity irrespective whether this was due to anti-β2GPI or antiprothrombin antibodies. In many patients, LAC activity is caused by a mixture of both types of antibody . We also performed experiments with NPP spiked with both anti-β2GPI antibodies and antiprothrombin antibodies. Here we found that addition of CL vesicles could reveal even the presence of weak anti-β2GPI antibodies (ratio 1.19) in a mixture of both types of antibodies (Fig. 3).
The basic criteria for the detection of LAC are (i) prolongation of a phospholipid-dependent coagulation test; (ii) evidence of an inhibitor demonstrated by mixing studies with normal plasma; and (iii) confirmation of the phospholipid-dependent nature of the inhibitory antibody by adding extra phospholipids. Commercial tests for the detection of LAC differ with respect to composition and concentration of phospholipids used for detection and conformation assays. These differences probably affect the sensitivity and specificity of the tests. Indeed, a large number of publications have shown that the correlation between different tests for the detection of LAC is unacceptable low [31,32]. Thus far, no guidelines have been proposed on the nature and composition of the phospholipids to be used because of the assumption that all negatively charged phospholipids will neutralize all different types of LAC activity irrespective of the test system used. Here we show for the first time that this assumption is incorrect. We observed major differences for cardiolipin in its ability to neutralize LAC activity. Cardiolipin neutralized anti-β2GPI antibody-dependent LAC in an APTT-based assay while cardiolipin did not neutralize anti-β2GPI antibody-dependent LAC activity in a dRVVT or a dPT. These differences were not found with antiprothrombin antibody dependent LAC activity.
It is noteworthy that when cardiolipin vesicles were added to NPP, we observed a prolongation of the clotting time with PTT-LA reagents (Fig. 1a). This effect was specific for aPTT based clotting tests, and it was not observed for a dPT or dRVVT. We suppose that interference of CL with the contact activation pathway is the cause of the observed increase in clotting time, probably due to the adsorption of high molecular weight kininogen (HMWK) and/or prekallikrein by cardiolipin, thereby depleting the plasma from these essential cofactors of the contact activation (V. Pengo, personal communication). To our knowledge, this interference of cardiolipin with aPTT-based assays has not been described before. Why addition of CL prolongs the aPTT is unclear at the moment, however, this phenomenon enhances the differences in neutralization patterns for β2GPI- and prothrombin-dependent LAC activity.
The effects of the monoclonal antibodies directed against β2GPI on the prolongation of the PTT-LA by cardiolipin are much stronger than the effects of the autoantibodies against β2GPI isolated from patients, especially at higher concentrations of cardiolipin. These differences are due to the higher affinity of the monoclonal antibodies for β2GPI compared to the human antibodies.
The interaction of β2GPI with negatively charged phospholipids was first shown by Shousboe . Later on, by using different techniques, the interaction of β2GPI with negatively charged phospholipids was demonstrated in several other studies [13,14,34–39], but quantitative data on the binding to CL are scarce. Also data on the interaction of prothrombin with negatively charged PS/PC surfaces have been described extensively [30,40–47], but studies on the interaction of prothrombin with CL are lacking. We show here with SPR analysis using purified β2GPI and prothrombin that in the absence or presence of calcium, the affinity of β2GPI for CL is, respectively, 40 and 10 times higher compared to that of prothrombin (Table 4). This relatively high affinity of β2GPI for CL might provide an explanation for the contrasting effects that addition of CL vesicles has on prolonged clotting times caused by either anti-β2GPI or antiprothrombin antibodies with LAC activity. However, as the differences were not seen with clotting assays initiated with tissue factor or Russell's viper venom, competition for catalytic surfaces could not be the only explanation. Addition of CL vesicles to normal plasma by itself caused a prolongation of aPTT-based clotting assays. We presume that besides competition between β2GPI–anti-ß2GPI antibody complexes and clotting factors for the available catalytic surface, the complexes also interfere with the CL-induced prolongation of the PTT-LA.
In conclusion, with a modified PTT-LA test, in which cardiolipin and PS/PC vesicles are used as confirmation reagent, one can discriminate between β2GPI and prothrombin-dependent LAC activities in patient plasmas. This modified PTT-LA test is suitable for use in clinical studies that evaluate the importance of β2GPI- and prothrombin-mediated LAC. Further studies of the specificity of phospholipids for the detection and neutralization of specific LAC inducing antibodies are of major importance to improve our assays used for the laboratory diagnosis of LAC.