Lupus Anticoagulant Activity Research Articles

Lupus anticoagulant (LA) testing: performance of clinical laboratories assessed by a national survey using lyophilized affinity-purified immunoglobulin with LA activity.

Lupus anticoagulant (LA) screens are frequently ordered in the workup of thrombophilic patients or women with fetal loss. The sensitivity and specificity of LA detection vary depending on the choice of tests, cutoff values, and results interpretation. This variation is detrimental to patient management because persistent LA positivity in patients with a history of thrombosis is a requisite for long-term anticoagulation therapy. Numerous surveys have been performed to assess the state of the art for LA diagnosis. The control plasmas used in these surveys were from LA-positive or -negative patients or were normal plasmas with monoclonal antibodies against human beta(2)-glycoprotein I (beta(2)-GPI) added. A large number of laboratories were asked to test a common set of lyophilized plasmas for LA, including three normal plasmas, to which increasing amounts of affinity-purified IgG from a patient positive for anti-beta(2)-GPI had been added, and three LA-negative plasmas: one normal, one with a coagulation deficiency, and one with heparin. Overall, 69, 68, and 59 of 70 participants were able to detect LA in plasmas with high, intermediate, and low potency (sensitivity, 99%, 97%, and 84%). Conversely, 69, 50, and 53 of 70 were able to rule out LA in the normal, heparinized, and deficient plasma (specificity, 99%, 71%, and 76%). Sensitivity for LA detection is satisfactory, whereas specificity could be improved. Surveys for LA detection should be carried out on a regular basis because they may help improve performance. Plasmas containing graded amounts of affinity-purified human anti-beta(2)-GPI may be used as a convenient source of well-characterized naturally occurring LA material.

Dimers of β2-Glycoprotein I Increase Platelet Deposition to Collagen via Interaction with Phospholipids and the Apolipoprotein E Receptor 2′

Patients with prolonged clotting times caused by lupus anticoagulant (LAC) are at risk for thrombosis. This paradoxal association is not understood. LAC is frequently caused by anti-beta2-glycoprotein I (beta 2GPI) antibodies. Antibody-induced dimerization of beta 2GPI increases the affinity of beta 2GPI for phospholipids, explaining the observed prolonged clotting times. We constructed dimers of beta 2GPI that mimic effects of beta 2GPI-anti-beta 2GPI antibody complexes, and we studied their effects on platelet adhesion and thrombus formation in a flow system. Dimeric beta 2GPI increased platelet adhesion to collagen by 150% and increased the number of large aggregates. We also observed increased platelet adhesion to collagen when whole blood was spiked with patient-derived polyclonal anti-beta 2GPI or some, but not all, monoclonal anti-beta 2GPI antibodies with LAC activity. These effects could be abrogated by inhibition of thromboxane synthesis. A LAC-positive monoclonal anti-beta 2GPI antibody, which did not affect platelet adhesion, prevented the induced increase in platelet adhesion by beta 2GPI dimers. Furthermore, increased platelet adhesion disappeared after preincubation with receptor-associated protein, a universal inhibitor of interaction of ligands with members of the low density lipoprotein receptor family. Using co-immunoprecipitation, it was shown that dimeric beta 2GPI can interact with apolipoprotein E receptor 2 (apoER2'), a member of the low density lipoprotein receptor family present on platelets. These results demonstrate that dimeric beta 2GPI induces increased platelet adhesion and thrombus formation, which depends on activation via apoER2'.

A simple method to discriminate between β2-glycoprotein I- and prothrombin-dependent lupus anticoagulants

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 beta2-glycoprotein I (beta2GPI) or prothrombin. In the present study, we describe a method to differentiate between LAC caused by antibodies directed against beta2GPI 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 beta2GPI- or prothrombin-dependent LAC activities. Addition of cardiolipin vesicles shortened the prolonged clotting time caused by anti-beta2GPI 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-beta2GPI or antiprothrombin antibodies with LAC activity. The effects of cardiolipin (CL) on beta2GPI-induced LAC activity were specific for contact activation mediated clotting assays. Possible explanations for these findings are the relatively high affinity of beta2GPI for cardiolipin, as determined by surface plasmon resonance analysis, and inhibition by anti-beta2GPI antibodies of the CL-induced prolongation of the PTT-LA.

Thrombogenicity of β2-glycoprotein I–dependent antiphospholipid antibodies in a photochemically induced thrombosis model in the hamster

AbstractWe previously showed that β2-glycoprotein I (β2GPI)–dependent lupus anticoagulants (LAs) form bivalent antigen-antibody complexes with high affinity for phospholipids; these complexes are responsible for their in vitro anticoagulant effect. We now studied the role of these bivalent complexes in arterial thrombosis in the hamster. Three monoclonal antibodies (mAbs) raised against human β2GPI were selected on the basis of their cross-reactivity with hamster β2GPI. Two of these, one with LA activity, 5H2, and one with only anticardiolipin properties, 11E8, were infused at 0 to 10 mg/kg prior to photochemically induced vessel damage. 5H2 promoted thrombus formation dose dependently, raising the thrombus size from 6.0 arbitrary units (AU) in controls (n = 9) to 65.0 AU in the high-dose group (10 mg/kg, n = 6,P= .007). The LA−mAb 11E8 and mAb 27A8, reactive with human β2GPI exclusively, did not significantly promote thrombus formation. In a second set of experiments, intact mAb 5H2 was compared to its fragments. Intact mAb 5H2 at 3.3 mg/kg and the equimolar dose of F(ab′)2fragments (2.2 mg/kg) promoted thrombus formation equally well (55.8 AU, n = 8 and 62.5 AU, n = 7, respectively); mAb 5H2-derived Fab′ fragments were inactive. Immunohistochemical analysis showed platelet-rich thrombi, with 5H2 or its F(ab′)2fragments mainly bound to individual platelets. Our results indicate that bivalent immune complex formation plays an important role in the genesis of arterial thrombosis by certain antiphospholipid antibodies. Cellular activation via the Fc portion of these immune complexes, however, is not essential, because F(ab′)2fragments of 5H2 still promote thrombus formation.

Effects of beta2-glycoprotein I and monoclonal anticardiolipin antibodies in platelet interaction with subendothelium under flow conditions.

To evaluate whether the effect of human monoclonal anticardiolipin antibodies (aCL) on platelet interaction with the subendothelium under flow conditions is dependent on beta(2)-glycoprotein I (beta(2)GPI). Three monoclonal IgM aCL with anti-beta(2)GPI activity (TM1B3, GR1D5, and EY2C9) obtained from patients with antiphospholipid syndrome, a monoclonal aCL with lupus anticoagulant activity but without anti-beta(2)GPI activity (FRO) obtained from a patient with a splenic lymphoma, and a control monoclonal IgM without aCL activity were used. TM1B3, GR1D5, EY2C9, FRO, and control IgM (30 microg/ml) were added to reconstituted blood containing gel-filtered platelets (200 x 10(9)/liter), factor VIII (100 units/dl), and fibrinogen (1.5 gm/liter). Samples were perfused (wall-shear rate 800 seconds(-1)), with and without the addition of purified beta(2)GPI (20 microg/ml), through annular chambers containing collagen-rich denuded vascular segments, and the percentages of surface covered by platelets and by thrombi were evaluated. No differences in the percentages of surface covered by platelets and by thrombi were observed among samples with TM1B3, GR1D5, EY2C9, FRO, and control IgM added when reconstituted blood samples without beta(2)GPI were used. However, a significant increase in the percentage of surface covered by platelets was observed in the presence of TM1B3, GR1D5, and EY2C9 but not in the presence of FRO when samples containing beta(2)GPI were used. Increased thrombi formation was induced by TM1B3 and GR1D5 but not by EY2C9 or FRO in samples with added beta(2)GPI. Monoclonal aCL require anti-beta(2)GPI activity to promote platelet interaction with the subendothelium under flow conditions.

Treatment and monitoring of patients with antiphospholipid antibodies and thrombotic history (Hughes syndrome).

Patients with Hughes (antiphospholipid) syndrome who develop an initial thrombosis have an increased risk of subsequent thrombotic events. Current therapy to prevent recurrent thrombosis is controversial. While it seems clear that anticoagulant treatment is a better option than anti-aggregants alone, there is no consensus regarding the duration and intensity of oral anticoagulation. The risk of bleeding, the main complication of anticoagulant treatment, and the need for frequent monitoring of the International Normalized Ratio to measure the anticoagulant effect of warfarin concern patients and physicians. In addition, there is some debate about the validity of the International Normalized Ratio in patients with lupus anticoagulant activity. The development of new therapies that target more specific pathogenic mechanisms is highly warranted.

Acquired activated protein C resistance is associated with the co-existence of anti-prothrombin antibodies and lupus anticoagulant activity in patients with systemic lupus erythematosus.

Venous thromboembolism (VTE) is one of the common manifestations in the anti-phospholipid (aPL) syndrome. We examined the levels of IgG antibodies (Abs) to beta2-glycoprotein I (beta2-GP I) and prothrombin, lupus anticoagulant (LA) activity, activated protein C resistance (APC-R), and factor V Leiden in 96 patients with systemic lupus erythematosus (SLE); 19 with VTE and 77 without VTE. Acquired APC-R, which was not found in any patient with the factor V Leiden mutation, was present in 33 (34.4%) out of the 96 patients with SLE. The presence of acquired APC-R was a strong risk factor for VTE. The SLE patients were divided into four groups according to the results of enzyme-linked immunosorbent assay (ELISA) and LA activity for each aPL Abs: ELISA+, LA+; ELISA+, LA-; ELISA-, LA+; and ELISA-, LA-. A significant association was observed between APC-R and the co-existence of anti-beta2-GP I Abs and LA activity or of anti-prothrombin Abs and LA activity. There was no association between APC-R and the presence of anti-beta2-GP I Abs, anti-prothrombin Abs, or LA activity alone. However, when multivariate logistical regression analysis was performed, it was clear that only the co-existence of anti-prothrombin and LA activity was a significant risk factor for APC-R. These findings indicate that the co-existence of anti-prothrombin Abs and LA activity may be an important factor in the pathogenesis of acquired APC-R in patients with SLE.

Antiphospholipid antibodies induced in mice by immunization with a cytomegalovirus-derived peptide cause thrombosis and activation of endothelial cells in vivo.

To characterize the binding and functional properties of antiphospholipid antibodies (aPL) induced by immunization with a viral peptide and to determine whether aPL are pathogenic in vivo. Ten murine monoclonal aPL were generated from spleen cells of PL/J mice immunized with TIFI, a phospholipid-binding peptide spanning Thr(101)-Thr(120) of ULB0-HCMVA from human cytomegalovirus (CMV), which shares structural similarity with the phospholipid-binding site of beta(2)-glycoprotein I (beta(2)GPI). The antibodies generated had aPL activity that was inhibited by cardiolipin liposomes, and this inhibition was enhanced in the presence of beta(2)GPI. Some of the antibodies exhibited binding to cultured endothelial cells in vitro, and some had lupus anticoagulant activity. Injection with 2 of the monoclonal aPL in mice resulted in a significant increase in the number of leukocytes adhering to endothelial cells and enhanced thrombus formation in vivo. These results indicate that aPL induced by immunization with a phospholipid-binding CMV peptide are pathogenic in vivo. The results also suggest a mechanism (molecular mimicry) by which pathogenic aPL may be generated in patients with antiphospholipid syndrome.

Anti-mitochondrial M5 type antibody represents one of the serological markers for anti-phospholipid syndrome distinct from anti-cardiolipin and anti-beta2-glycoprotein I antibodies.

The aim of this study was to characterize the antigen specificity and to evaluate the diagnostic and prognostic value of anti-mitochondrial M5 type antibodies (AMA M5). Fifty-eight patients selected on the basis of their AMA M5 positivity were investigated in relationship to their clinical and serological profile. Cross-absorption studies, Western blotting and immunoprecipitation analysis were carried out for AMA M5 antigen specificity characterization. Most patients had a diagnosis of systemic lupus erythematosus (SLE) (65.5%) or of primary anti-phospholipid syndrome (PAPS) (24%); all the patients were positive for IgG or IgM anti-cardiolipin (anti-CL) antibodies and 49% of them also displayed lupus anticoagulant (LA) activity. Anti-beta2-glycoprotein I (beta2-GPI) IgG were detectable in 30/38 sera (78.9%) and IgM in 34/38 (89.4%). While anti-CL and anti-beta2-GPI IgG antibodies were significantly associated with history of thrombosis and fetal loss, AMA M5 displayed a statistical association only for thrombocytopenia and recurrent fetal loss. Absorption with human beta2-GPI both in free solution or in solid phase as well as with CL liposomes or CL/beta2-GPI liposome complexes did not affect AMA M5 fluorescence. While AMA M5 activity is absorbed by whole mitochondrial preparations, no specific reactivities against several human, bovine and rat mitochondrial proteins could be detected in Western blotting and immunoprecipitation studies. AMA M5 appear to be detectable in both primary and secondary APS, displaying a strong association with the presence of thrombocytopenia and fetal loss. Although strictly related to anti-phospholipid antibodies, AMA M5, anti-CL and anti-beta2-GPI antibodies represent distinct serological markers of the APS.

Anti-prothrombin antibodies combined with lupus anti-coagulant activity is an essential risk factor for venous thromboembolism in patients with systemic lupus erythematosus.

Anti-prothrombin antibodies (anti-prothrombin) and anti-beta2-glycoprotein I antibodies (anti-beta2-GP I) are the most common and characterized anti-phospholipid antibodies (aPL) detected using specific enzyme-linked immunosorbent assay (ELISA) systems. Recently, lupus anti-coagulant (LA) activity detected by a phospholipid-dependent coagulation assay was reported to be associated with anti-prothrombin and/or anti-beta2-GP I. Here we show that the co-existence of IgG anti-prothrombin and LA activity might be an essential risk factor for venous thromboembolism (VTE) in patients with systemic lupus erythematosus (SLE). We examined not only the levels of antibodies to prothrombin and anti-beta2-GP I (both IgG and IgM isotypes) using an ELISA system, but also LA activity detected using both diluted Russell's viper venom time (dRVVT) and STACLOT LA test in 124 patients with SLE. The SLE patients were divided into four groups according to the results of ELISA and LA assay results for each aPL: group A, ELISA+ and LA+ group B, ELISA+ and LA-; group C, ELISA- and LA+ group D, ELISA- and LA-. Regarding IgG anti-prothrombin, the prevalence of VTE was significantly higher in group A (16/35 cases, 45.7%, P < 0.001, Fisher's exact probability test) than in the other groups (B, 2/30, 6.7%; C, 1/22, 4.5%; D, 1/37, 2.7%). With respect to IgM anti-prothrombin and IgG or IgM anti-beta2-GP I, the prevalence of VTE was higher in both groups A and C than in group D, but no statistical difference in prevalence was found between groups A and C. Multivariate logistic regression analysis of risk factors for VTE confirmed that the co-existence of IgG anti-prothrombin and LA activity was the only significant risk factor for VTE (odds ratio, 19.13; 95% confidence intervals, 4.74-77.18).

Antiphospholipid antibody levels in intravenous immunoglobulin (IVIg) preparations.

The antiphospholipid syndrome (APS) is characterized by various clinical manifestations and by elevated levels of antiphospholipid antibodies. Passive induction of APS by infusion of these antibodies has been demonstrated in animal models. Intravenous immunoglobulin (IVIg) is one of the therapeutic options in APS. In this study, five commercially used preparations of IVIg were tested for the presence of elevated levels of cardiolipin, beta-glycoprotein-I, phosphatidylserine, antinuclear, and double-stranded DNA autoantibodies, as well as for lupus anticoagulant activity. The absence of abnormal elevated levels of any of these autoantibodies in five different IVIg preparations provides additional evidence for the safety of IVIg use in APS.

Complexes of anti-prothrombin antibodies and prothrombin cause lupus anticoagulant activity by competing with the binding of clotting factors for catalytic phospholipid surfaces.

We investigated the mechanism by which anti-prothrombin antibodies cause lupus anticoagulant (LAC) activity. Addition of affinity-purified anti-prothrombin antibodies from LAC-positive plasma samples (alpha-FII-LAC+) to normal plasma induced LAC activity. Upon increasing the phospholipid concentration, LAC activity was neutralized. Addition of purified alpha-FII-LAC+ to normal plasma strongly inhibited factor Xa formation. No inhibition was measured when alpha-FII-LAC+ were added to prothrombin-deficient plasma or when purified anti-prothrombin antibodies from LAC-negative plasma samples (alpha-FII-LAC-) were added. When a combination of prothrombin and alpha-FII-LAC+ was added to the purified clotting complex, a strong inhibition of factor Xa and IIa formation was seen. The alpha-FII-LAC+ alone or a combination of prothrombin and alpha-FII-LAC- did not show inhibition. Ellipsometry studies showed that, in the presence of alpha-FII-LAC+, the affinity of prothrombin for a phospholipid surface increased dramatically, whereas a much lower increase was observed with alpha-FII-LAC-. Our results show that complexes of prothrombin and anti-prothrombin antibodies with LAC activity inhibit both prothrombinase and tenase. The antibodies increase the affinity of prothrombin for the phospholipid surface, thereby competing with clotting factors for the available catalytic phospholipid surface, a mechanism similar to that of anti-beta2-glycoprotein I antibodies.

Prevalence of Different Anti-Phospholipid Antibodies in Systemic Lupus Erythematosus and Their Relationship with the Antiphospholipid Syndrome

The antiphospholipid syndrome is a complex disorder (1) that is related to a set of unusual and fascinating antibodies. Recent research is defining these antibodies and their relationship to features of the syndrome. The antiphospholipid syndrome is defined by the association of arterial and/or venous thrombosis, recurrent miscarriages, and anti-phospholipid (aPL) antibodies (1). The syndrome may be either idiopathic (primary) or may occur in the setting of an associated disease (secondary), most frequently systemic lupus erythematosus (SLE) or other autoimmune diseases (2)(3). Lupus anticoagulants and anti-cardiolipin antibodies at medium to high titers are laboratory indicators of the condition (4). Lupus anticoagulants behave as acquired inhibitors of coagulation and prolong phospholipid-dependent in vitro coagulation tests (5), whereas anti-cardiolipin antibodies bind cardiolipin and other anionic phospholipids and are detected in solid-phase immunoassays (6). Despite their name, aPL antibodies recognize plasma proteins bound to suitable anionic (not necessarily phospholipid) surfaces rather than anionic phospholipids. Among them, β2-glycoprotein I (β2-GPI) (7)(8) and prothrombin (9) are the most common and investigated antigenic targets. β2-GPI is required by the majority of autoimmune anti-cardiolipin antibodies to react with cardiolipin in immunoassays (7)(8). Specific subgroups of anti-β2-GPI (10) and anti-prothrombin (11) antibodies are responsible for the lupus anticoagulant activity in phospholipid-dependent coagulation tests. In addition to β2-GPI and prothrombin, (activated) protein C (12), protein S (12), annexin V (13), high- and low-molecular weight kininogens (14), factor XII (15), thrombomodulin (16), and tissue-type plasminogen activator (17) have been reported to be antigenic targets of aPL antibodies. Because all of these proteins are involved in the initiation and control of blood coagulation, it …

Dimers of β2-Glycoprotein I Mimic thein Vitro Effects of β2-Glycoprotein I-Anti-β2-glycoprotein I Antibody Complexes

Anti-beta(2)-glycoprotein I antibodies are thought to cause lupus anticoagulant activity by forming bivalent complexes with beta(2)-glycoprotein I (beta(2)GPI). To test this hypothesis, chimeric fusion proteins were constructed of the dimerization domain (apple 4) of factor XI and beta(2)GPI. Both a covalent (apple 4-beta(2)GPI) and a noncovalent (apple 4-C321S-beta(2)GPI) chimer were constructed. As controls, apple 2-beta(2)GPI and apple 4-C321S-beta(2)GPI-W316S, in which beta(2)GPI-W316S is not able to bind to phospholipids, were made. In a phospholipid binding assay, apple 4-beta(2)GPI and apple 4-C321S-beta(2)GPI were able to bind to phospholipids with an affinity 35 times higher than that of plasma-derived beta(2)GPI and apple 2-beta(2)GPI. Apple 4-C321S-beta(2)GPI-W316S did not bind at all. Only apple 4-beta(2)GPI and apple 4-C321S-beta(2)GPI were able to bind to adhered platelets as shown by immunofluorescence. Using the prothrombin time, which was the most responsive coagulation assay, the clotting time was approximately doubled when 200 microg/ml apple 4-beta(2)GPI or apple 4-C321S-beta(2)GPI was added. Addition of 200 microg/ml plasma-derived beta(2)GPI, apple 2-beta(2)GPI, or apple 4-C321S-beta(2)GPI-W316S did not affect clotting time. Clotting time could be corrected with the addition of extra phospholipids, which is indicative for lupus anticoagulant activity. An additional increase in clotting times for apple 4-beta(2)GPI or apple 4-C321S-beta(2)GPI was achieved by the addition of monoclonal antibodies against beta(2)GPI. In conclusion, dimerization of beta(2)GPI explains the in vitro observed effects of beta(2)GPI-anti-beta(2)GPI antibody complexes.

Immunologic and hematologic properties of antibodies to prothrombin and plasminogen in a mouse model.

Antibodies to prothrombin have been associated with venous and arterial thrombosis, and they cross-react with a structurally closely related protein plasminogen. We immunised 16 mice with human prothrombin and 15 mice with human plasminogen. Mice immunised with prothrombin developed cross-reactive antibodies to plasminogen (12/16), beta2-glycoprotein I (4/16), tissue-type plasminogen activator (6/16) and cardiolipin (11/16). Mice immunised with plasminogen developed cross-reactive antibodies to prothrombin (8/15), tissue-type plasminogen activator (2/12) and cardiolipin (5/12). Functional effects of antibodies were examined. Immunisation with prothrombin induced lupus anticoagulant activity in 9/14 mice. In mice immunised with plasminogen, radial fibrinolysis was inhibited in 8/10 and plasminogen activation in the chromogenic assay was inhibited in 9/11. No cross-functionality was observed. In conclusion, antibodies to prothrombin and plasminogen cross-react in vivo. Antibodies to prothrombin and plasminogen have different functional profiles, immunisation with prothrombin leads to prolonged blood clotting time, and immunisation with plasminogen induces antibodies interfering with fibrinolysis.

Quantification of Lupus Anticoagulants in Clinical Samples Using Anti- β2GP1 and Anti-Prothrombin Monoclonal Antibodies

Quantification of lupus anticoagulant (LA) in clinical samples is hampered by the lack of a suitable standard of activity. We evaluated the use of mAbs displaying LA activity for this purpose. As most patient samples contain both beta2Glycoprotein I (beta2GP1) and prothrombin dependent LA, a combination of two mAbs, one of each specificity, was added to normal plasma in a concentration from 0 to 60 microg/ml. Eight assay systems using different reagents and instruments were used. The calibration curves were linear for all but one, with marked differences between the responsiveness to each mAb. A panel of plasmas from 69 patients with persistent LA diagnosed using the SSC-ISTH criteria was tested. An antiphospholipid syndrome (APS) was present in 40, whereas 29 were asymptomatic. LA activities of individual plasmas varied between assays (p < 10(-4)), but homogeneous subgroups were identified. In a majority of samples, LA activity displayed a prothrombin-dependent profile, with a variable contribution of beta2GP1-dependent activity. The latter was associated to beta2GP1 antibodies detected by solid-phase immunoassay. By using 3 dilute Russell viper venom time assays, higher LA titers were found in APS, compared to asymptomatic patients (p <0.05).

Improved detection of lupus anticoagulants by the dilute Russell's Viper venom time.

Accurate and timely detection of lupus anticoagulants (LAs) is of diagnostic and prognostic importance due to the association of persistent LAs with thrombotic disease. A review of LA screening results by kaolin clotting time and dilute Russell's Viper venom time (dRVVT) on 2843 patient samples demonstrated that only 40.7% (417 of 1024) of elevated dRVVT ratios could be interpreted as consistent with the presence of an LA by confirmatory procedures. Apart from those due to the effects of anticoagulant therapy, the remainder generated inconclusive interpretations, necessitating significant numbers of costly repeat investigations. Manipulation of dRVVT assay conditions by increasing confirmatory reagent concentration, and altering venom concentration to maintain analytical parity with the standard assay, revealed LAs not fully neutralized by confirmatory tests at standard concentrations. Further experiments were performed using Russell's Viper venom reagents from five different manufacturers to demonstrate that the findings were not a reagent-specific phenomenon. Higher detection rates were achieved using multiple conventional assays but samples remained that required a modified confirmatory test to demonstrate LA activity. A previously unreported group of LAs was identified with raised dRVVT ratios that failed to correct with any of the dRVVT assays but demonstrated significant correction with all reagents in the modified confirmatory test. Use of modified confirmatory tests enhances sensitivity and specificity, and doubles LA detection rates by dRVVT. Adoption of the technique will significantly increase cost-effectiveness of LA detection in clinical practice.

Antiphospholipid Antibodies and the Protein C Pathway

Among the mechanisms suggested for the prothrombotic activity of lupus anticoagulant and antiphospholipid antibodies is the direct inhibition of the anticoagulant activated protein C (APC) pathway. Although some pathological antibodies may be directed towards the proteins involved, we hypothesize that populations exist which selectively inhibit the APC complex as a result of differences in the phospholipid requirements of this complex as compared to those of the procoagulant complexes. The most prominent feature is the requirement for the presence of phosphatidylethanolamine in the membrane for APC anticoagulant function. This mimics the requirements for inhibitory activity of at least a subset of autoantibodies associated with thrombosis. The role of oxidation of the phospholipid in APC function and antibody reactivity is also discussed.

Phospholipid-Bound β2-Glycoprotein I Induces the Production of Anti-Phospholipid Antibodies

Apoptotic-cell-bound β2-glycoprotein I (β2GPI), but not apoptotic cells or β2GPI alone, can induce the production of anti-phospholipid (anti-PL) antibodies (Ab) in normal mice. Although it is presumed that β2GPI binds to anionic phospholipid (PL) exposed on the apoptotic cell membrane, the precise nature of this complex and its immunogenicity is unclear. To address these issues, we investigated the structure and immunogenicity of human β2GPI in the presence of different PL that may be expressed on the surface of apoptotic cells. BALB/c mice were immunized intravenously (iv) with β2GPI in the presence of cardiolipin (CL), phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylcholine (PC), or PS/PC (25%/75%) vesicles. Cardiolipin+β2GPI induced the highest levels of anti-β2GPI and anti-CL IgG Ab and lupus anticoagulant (LA) activity, while β2GPI with PC or PS/PC vesicles produced no significant anti-PL Ab. PS+β2GPI was somewhat immunogenic, but less so than PG+β2GPI. β2GPI was immunogenic in the presence of native (CLN), but not hydrogenated (CLH), CL. Circular dichroism analysis demonstrated that the structure of β2GPI was altered specifically by interaction with CLN, but not other anionic PL, including CLH. Similarly, the structure of CLNwas affected by interaction with β2GPI, as detected by31P nuclear magnetic resonance. These findings demonstrate that β2GPI complexed with CLNis structurally altered, highly immunogenic, and induces the production of IgG anti-PL Ab. Furthermore, the structural modification and the generation of immunogenic epitopes on β2GPI upon interaction with CLNrequire the presence of unsaturated fatty acid chains, suggesting a role for oxidation in this process.

Haemostatic abnormalities and lupus anticoagulant activity in patients with Gaucher disease type I.

Gaucher disease (GD) type I (McKusick 230800), the most prevalent lysosomal storage disorder, is due to β-glucocerebrosidase (EC 3.2.1.45) deficiency. It is characterized by the storage of uncleaved β-glucocerebroside in the cells of the reticuloendothelial system, leading to bone marrow infiltration, hepatosplenomegaly and skeletal lesions. Other clinical features include haematological changes with anaemia and thrombocytopenia (Beutler and Grabowski 1995). Enzyme replacement therapy has become available and is effective in reversing hepatosplenomegaly and haematological abnormalities (Charrow et al 1998). Patients with GD type I have a bleeding tendency partially due to thrombocytopenia; a combined deficiency of clotting factors and inhibitors has also been reported (Billet et al 1996; Hollak et al 1997). We undertook this study to gain further insight into the pathophysiology of haemostatic abnormalities associated with Gaucher disease. The effect of enzyme replacement therapy on haemostasis was also monitored in comparison with changes of other laboratory parameters reflecting the status of the disease.