An allosteric redox switch in domain V of ß2-glycoprotein I controls membrane binding and anti-domain I autoantibody recognition.

ß2-glycoprotein I (ß2GPI) is an abundant multidomain plasma protein that plays various roles in the clotting and complement cascades. It is also the main target of antiphospholipid antibodies (aPL) in the acquired coagulopathy known as antiphospholipid syndrome (APS). Previous studies have shown that ß2GPI adopts two interconvertible biochemical conformations, oxidized and reduced, depending on the integrity of the disulfide bonds. However, the precise contribution of the disulfide bonds to ß2GPI structure and function is unknown. Here, we substituted cysteine residues with serine to investigate how the disulfide bonds C32-C60 in domain I (DI) and C288-C326 in domain V (DV) regulate ß2GPI's structure and function. Results of our biophysical and biochemical studies support the hypothesis that the C32-C60 disulfide bond plays a structural role, whereas the disulfide bond C288-C326 is allosteric. We demonstrate that absence of the C288-C326 bond, unlike absence of the C32-C60 bond, diminishes membrane binding without affecting the thermodynamic stability and overall structure of the protein, which remains elongated in solution. We also document that, while absence of the C32-C60 bond directly impairs recognition of ß2GPI by pathogenic anti-DI antibodies, absence of the C288-C326 disulfide bond is sufficient to abolish complex formation in the presence of anionic phospholipids. We conclude that the disulfide bond C288-C326 operates as a molecular switch capable of regulating ß2GPI's physiological functions in a redox-dependent manner. We propose that in APS patients with anti-DI antibodies, selective rupture of the C288-C326 disulfide bond may be a valid strategy to lower the pathogenic potential of aPL.

Quantitation of Total and Free Thiol ß2-Glycoprotein I Levels for Diagnostic and Prognostic Purposes in the Antiphospholipid Syndrome.

ß2-Glycoprotein I is the major autoantigen in the antiphospholipid syndrome (APS), a prothrombotic disorder characterized by the occurrence of either venous or arterial thrombosis. In women it is also associated with an increased risk of obstetric complications such as recurrent miscarriages. We have identified that the plasma protein ß2-glycoprotein I in healthy individuals exists in an optimal ratio between two distinct forms, an oxidized and free thiol, reduced form. This ratio is disrupted in pathophysiological conditions associated with increased oxidative stress such as the APS, but also in the setting of age-related macular degeneration and gram-negative sepsis. We have developed assays that quantify plasma/serum levels of total and free thiol ß2-glycoprotein I which can potentially be used for risk stratification and prognostic purposes in the early stages of the aforementioned conditions.

Effect and mechanism of the aß2­GP I/rhß2­GP I complex on JEG­3 cell proliferation, migration and invasion.

Antiphospholipid antibody (aPL)­mediated antiphospholipid syndrome (APS) is an autoimmune disease. Upon binding to aPL, the primary antigen of aPL, ß2­glycoprotein I (ß2­GP I), induces abnormal immune function, which further activates downstream signaling pathways in the cell and eventually leads to APS. The present study aimed to determine whether ß2­GP I antigen and anti­ß2­glycoprotein I antibody (aß2­GP I), which belong to the aPL class of antibodies, may affect human chorionic epithelium cell (JEG­3) proliferation, migration and invasion. Recombinant human (rh)ß2­GP I protein was expressed using a prokaryotic expression system and aß2­GP I antibody was purified from the blood serum of 10 patients with recurrent pregnancy loss. JEG­3 cells were stimulated with rhß2­GP I and aß2­GP I separately or simultaneously, and serum immunoglobulin G of normal pregnant women was used as negative control. Using cell counting kit­8, cell cycle and transwell assays in addition to EdU staining, it was determined that aß2­GP I/rhß2­GP I complex markedly increased JEG­3 cell proliferation, migration and invasion. The results revealed that mRNA levels of inhibitor of nuclear factor (NF)­κB kinase subunit (IKKß), myeloid differentiation primary response protein MyD88 (MyD88), NF­κB and NF­κB inhibitor α (IκBα), as well as the protein levels of MyD88, IκBα and phospho(p)­IκBα in JEG­3 cells increased following incubation with the aß2­GP I/rhß2­GP I complex. The observed upregulation of p­IκBα protein suggested that IκBα­mediated inhibition of NF­κB was weakened. Furthermore, JEG­3 cells were transfected with PGMLV­NF­κB­Lu vector. Luciferase activity in JEG­3­NFκB­Luc1 and JEG­3­NFκB­Luc2 cells was enhanced following treatment with aß2­GP I/rhß2­GP I complex. The present study demonstrated that aß2­GP I/rhß2­GP I complex activates NF­κB through MyD88 signal transduction pathway, which further enhances JEG­3 cell proliferation, migration and invasion.

The purification of reduced ß2-glycoprotein I showed its native activity in vitro.

BACKGROUND: New evidence has shown that reduced ß2-glycoprotein I (ß2GPI) has anti-oxidative stress and anti-inflammatory activity. However, the details are still poorly understood. This study aims to prepare stable reduced ß2GPI with its native bioactivity in vitro. METHODS: Human ß2GPI was purified from plasma first with perchloric acid precipitation and then purified with a series of chromatography methods including Sephadex G-25 desalting, SP HP, AF-heparin HC-650 M, and Sephacryl S-200. The purified human ß2GPI was reduced with thioredoxin-1 (TRX-1) activated by DL-dithiothreitol (DTT). Glutathione (GSH) was selected to block the free thiols in reduced ß2GPI. LC/MS was used to verify the location of free thiols. Western blot analysis was used to detect ß2GPI immunoreactivity. MTS and flow cytometry were conducted to investigate its biological effect on oxidative-stress-induced death of human umbilical vein endothelial cells (HUVECs). The levels of tumour necrosis factor-alpha (TNF-α),interleukin-6 (IL-6) interleukin-10 (IL-10),interleukin-12P70 (IL-12P70),interferon-gamma (IFN-γ) and monocyte chemoattractant protein -1(MCP-1) in mouse serum were quantified to assess its anti-inflammatory activity in lipopolysaccharide (LPS)-mediated systemic inflammation. RESULTS: We obtained approximately 10 mg ß2GPI (purity 98.7%) from 200 ml plasma. The protein yield was 0.05 mg/ml plasma. ß2GPI was then reduced by TRX-1/DTT in vitro; the free thiols were detected on Cys288 and Cys326 in domain V of ß2GPI. The GSH blockage stabilized the reduced ß2GPI in vitro. This reduced ß2GPI can be recognized by the anti-ß2GPI antibody, can significantly reduce the death of HUVECs after H2O2 treatment and can significantly decrease the levels of TNF-α, IL-6,IFN-γ and MCP-1 in mice upon LPS stimulation. CONCLUSION: Stable reduced ß2GPI can be obtained in vitro by TRX-1 deoxidation followed by the blockage of thiols with GSH. This reduced ß2GPI maintains the same immunological activity as oxidized ß2GPI and has the ability to counter the oxidative stress induced by H2O2 in HUVECs and inflammation in LPS-mediated inflammation in mice.

Measuring IgA Anti-ß2-Glycoprotein I and IgG/IgA Anti-Domain I Antibodies Adds Value to Current Serological Assays for the Antiphospholipid Syndrome.

INTRODUCTION: Currently available clinical assays to detect antiphospholipid antibodies (aPL) test for IgG and IgM antibodies to cardiolipin (aCL) and ß2-glycoprotein I (aß2GPI). It has been suggested that testing for IgA aPL and for antibodies to Domain I (DI), which carries the key antigenic epitopes of ß2GPI, could add value to these current tests. We performed an observational, multicenter cohort study to evaluate the utility of IgG, IgM and IgA assays to each of CL, ß2GPI and DI in APS. METHODS: Serum from 230 patients with APS (n = 111), SLE but not APS (n = 119), and 200 healthy controls were tested for IgG, IgM and IgA aCL, aß2GPI and aDI activity. Patients with APS were further classified into thrombotic or obstetric APS. Logistic regression and receiver operator characteristic analyses were employed to compare results from the nine different assays. RESULTS: All assays displayed good specificity for APS; IgG aCL and IgG aß2GPI assays however, had the highest sensitivity. Testing positive for IgA aß2GPI resulted in a higher hazard ratio for APS compared to IgM aß2GPI. Positive IgG, IgM or IgA aDI were all associated with APS, and in subjects positive for aCL and/or aß2GPI, the presence of aDI raised the hazard ratio for APS by 3-5 fold. IgG aCL, aß2GPI, aDI and IgA aDI were associated with thrombotic but not obstetric complications in patients with APS. CONCLUSION: Measuring IgG aDI and IgA aß2GPI and aDI may be useful in the management of patients with APS, particularly thrombotic APS.

Development of a high yield expression and purification system for Domain I of Beta-2-glycoprotein I for the treatment of APS.

BACKGROUND: In this paper we describe a novel method to achieve high yield bacterial expression of a small protein domain with considerable therapeutic potential; Domain I of Beta-2-glycoprotein I (ß2GPI). ß2GPI is intrinsic to the pathological progression of the Antiphospholipid Syndrome (APS). Patients develop autoantibodies targeting an epitope located on the N-terminal Domain I of ß2GPI rendering this domain of interest as a possible therapeutic. RESULTS: This new method of production of Domain I of ß2GPI has increased the production yield by ~20 fold compared to previous methods in E.coli. This largely scalable, partially automated method produces 50-75 mg of pure, folded, active Domain I of ß2GPI per litre of expression media. CONCLUSION: The application of this method may enable production of Domain I on sufficient scale to allow its use as a therapeutic.

Optimization of unnicked ß2-glycoprotein I and high avidity anti-ß2-glycoprotein I antibodies isolation.

Patient biological material for isolation of ß2-glycoprotein I (ß2GPI) and high avidity IgG anti-ß2-glycoprotein I antibodies (HAv anti-ß2GPI) dictates its full utilization. The aim of our study was to evaluate/improve procedures for isolation of unnicked ß2GPI and HAv aß2GPI to gain unmodified proteins in higher yields/purity. Isolation of ß2GPI from plasma was a stepwise procedure combining nonspecific and specific methods. For isolation of polyclonal HAv aß2GPI affinity chromatographies with immobilized protein G and human ß2GPI were used. The unknown protein found during isolation was identified by liquid chromatography electrospray ionization mass spectrometry and the nonredundant National Center for Biotechnology Information database. The average mass of the isolated unnicked purified ß2GPI increased from 6.56 mg to 9.94 mg. In the optimized isolation procedure the high molecular weight protein (proteoglycan 4) was successfully separated from ß2GPI in the 1st peaks with size exclusion chromatography. The average efficiency of the isolation procedure for polyclonal HAv anti-ß2GPI from different matrixes was 13.8%, as determined by our in-house anti-ß2GPI ELISA. We modified the in-house isolation and purification procedures of unnicked ß2GPI and HAv anti-ß2GPI, improving the purity of antigen and antibodies as well as increasing the number of tests routinely performed with the in-house ELISA by ~50%.

Identification of the binding site for fondaparinux on Beta2-glycoprotein I.

Antiphospholipid syndrome (APS) is an autoimmune disease with clinical manifestations of thrombosis and pregnancy complications. Beta2-glycoprotein I (ß2GPI) is the major antigen for the APS-related antibodies. Heparin, low-molecular weight heparin and the synthetic pentasaccharide fondaparinux are commonly used for prophylaxis and treatment of thrombosis in patients with antiphospholipid syndrome. These antithrombotic drugs bind and activate antithrombin III to inactivate blood clotting proteases. Heparin and heparin derivatives might have a direct beneficial effect in APS via binding to ß2GPI and interfering with prothrombotic properties of ß2GPI/antibody complexes. We compared fondaparinux to heparin regarding its ability to bind ß2GPI and inhibit the binding of ß2GPI/antibody complexes to negatively charged phospholipids and endothelial cells. Although heparin and fondaparinux bind ß2GPI at therapeutically relevant doses, neither fondaparinux nor heparin was efficient in inhibition of the binding of ß2GPI/antibody complexes to negatively charged phospholipids and endothelial cells. Our studies suggest that these drugs do not act on pathological properties of ß2GPI/antibody complexes, emphasizing the need for a new treatment specific for ß2GPI-related thrombosis in APS. We observed that the binding interface of fondaparinux on ß2GPI does not include the lysine residues known to be critical for binding of heparin. The docking model of the ß2GPI complex with fondaparinux is in agreement with multiple experimental observations.

Anti-ß2GP1 antibodies have variable effects on platelet aggregation.

AIM: To investigate the effects of affinity-purified rabbit anti-ß2GP1, and anti-ß2GP1 purified from patients with systemic lupus erythematosus (SLE), on adenosine diphosphate (ADP)-induced aggregation. METHODS: Whole blood was collected and processed to obtain platelet poor plasma (PPP) from normal controls (n = 15) and SLE patients (n = 15). Using PPP, anti-ß2GP1 titres were determined using an ELISA and IgG fractions isolated using a HiTrap protein G column. Anti-ß2GP1 was purified from two SLE patients using purified ß2GP1 coupled to a HiTrap NHS-activated HP column. RESULTS: The effect of rabbit and human derived anti-ß2GP1 (0-100 µg/mL), on ADP (2.5, 5 µM) induced platelet aggregation were investigated using light transmission aggregometry. Rabbit anti-ß2GP1 significantly inhibited all parameters of 5 µM ADP-induced platelet aggregation; %Max (p = 0.028), %AUC (p = 0.014) and slope (p < 0.001). In contrast, anti-ß2GP1 purified from SLE patients significantly enhanced the %Max (p = 0.031) and %AUC (p = 0.007) in a concentration dependent manner, but inhibited the slope (p < 0.05) of 5 µM ADP-induced platelet aggregation. CONCLUSION: Our data suggest anti-ß2GP1 purified from different species have variable effects on in vitro platelet aggregation. The disparity between rabbit and human anti-ß2GP1 may be due to the heterogeneous nature of anti-ß2GP1, varying avidity or different antibody binding specificities between species.

In vitro effect of anti-ß2 glycoprotein I antibodies on P-selectin expression, a marker of platelet activation.

OBJECTIVE: Antiphospholipid antibodies (aPL) associated with thrombembolic events and/or pregnancy morbidity characterize the so-called antiphospholipid syndrome (APS). Beta2glycoprotein I (ß2GPI) represents the major target antigen for aPL, but the pathogenic role of anti-ß2GPI antibodies (aß2GPI) is still unclear. Some authors assume they play a role in activating platelets. The effects of aß2GPI antibodies on platelet P-selectin expression were evaluated in this study. METHODS: Aß2GPI antibodies in the plasma of a pregnant APS patient were isolated by affinity chromatography during two different stages (catastrophic and quiescent) of the disease. Gel filtered platelets (100,000/µl) from healthy volunteers were incubated with ß2-GPI (20 µg/ml) and with different concentrations (5, 25 e 50 µg/ml) of aß2GPI antibodies. P-selectin surface expression on platelets was assessed by flow cytometry using a specific fluorescent antibody directed against P-selectin. RESULTS: Aß2GPI antibodies induced platelet activation only in the presence of thrombin receptor activator for peptide 6 (TRAP-6), a platelet agonist, at a subthreshold concentration. Aß2GPI antibody enhancement on platelet surface P-selectin expression was stronger in the catastrophic than in the quiescent phase of the disease (47% versus 15%). CONCLUSIONS: TRAP-6 dependent platelet activation by aß2GPI antibodies is consistent with the "two hit" pathogenetic hypothesis for thrombosis. Aß2GPI antibodies induce higher platelet P-selectin expression during the active rather than in the acute phases.

Chemical synthesis and characterization of wild-type and biotinylated N-terminal domain 1-64 of beta2-glycoprotein I.

The antiphospholipid syndrome (APS) is a severe autoimmune disease associated with recurrent thrombosis and fetal loss and characterized by the presence of circulating autoantibodies (aAbs) mainly recognizing the N-terminal domain (DmI) of beta2-glycoprotein I (beta2GpI). To possibly block anti-beta2GpI Abs activity, we synthesized the entire DmI comprising residues 1-64 of beta2GpI by chemical methods. Oxidative disulfide renaturation of DmI was achieved in the presence of reduced and oxidized glutathione. The folded DmI (N-DmI) was purified by RP-HPLC, and its chemical identity and correct disulfide pairing (Cys4-Cys47 and Cys32-Cys60) were established by enzymatic peptide mass fingerprint analysis. The results of the conformational characterization, conducted by far- and near-UV CD and fluorescence spectroscopy, provided strong evidence for the native-like structure of DmI, which is also quite resistant to both Gdn-HCl and thermal denaturation. However, the thermodynamic stability of N-DmI at 37 degrees C was remarkably low, in agreement with the unfolding energetics of small proteins. Of note, aAbs failed to bind to plates coated with N-DmI in direct binding experiments. From ELISA competition experiments with plate-immobilized beta2GpI, a mean IC(50) value of 8.8 microM could be estimated for N-DmI, similar to that of the full-length protein, IC(50)(beta2GpI) = 6.4 microM, whereas the cysteine-reduced and carboxamidomethylated DmI, RC-DmI, failed to bind to anti-beta2GpI Abs. The versatility of chemical synthesis was also exploited to produce an N-terminally biotin-(PEG)(2)-derivative of N-DmI (Biotin-N-DmI) to be possibly used as a new tool in APS diagnosis. Strikingly, Biotin-N-DmI loaded onto a streptavidin-coated plate selectively recognized aAbs from APS patients.

Plasmin-clipped beta(2)-glycoprotein-I inhibits endothelial cell growth by down-regulating cyclin A, B and D1 and up-regulating p21 and p27.

beta(2)-Glycoprotein-I (beta(2)gpI), an abundant plasma glycoprotein, functions as a regulator of thrombosis. Previously, we demonstrated that plasmin-clipped beta(2)gpI (cbeta(2)gpI) exerts an anti-angiogenic effect on human umbilical vein endothelial cells (HUVEC). The present study was focused on the molecular background responsible for this phenomenon. cbeta(2)gpI strongly reduced HUVEC growth and proliferation as evidenced by the MTT and BrdU assay and delayed cell cycle progression arresting HUVEC in the S-and G2/M-phase. Western blot analysis indicated that cbeta(2)gpI inhibited cyclin A, B and D1, and enhanced p21 and p27 expression. Activity of p38 was down-regulated independently from the cbeta(2)gpI incubation time. Phosphorylation of ERK1/2 was not changed early (30 and 60 min) but became enhanced later (90 min, 4h). JNK activity was reduced rapidly after cbeta(2)gpI treatment but compared to controls, increased thereafter. Annexin II blockade prevented growth inhibition and cell cycle delay evoked by cbeta(2)gpI. We assume that cbeta(2)gpI's effects on HUVEC growth is mediated via cyclin A, B and D1 suppression, up-regulation of p21 and p27 and coupled to modifications of the mitogen-activated protein (MAP) kinase signalling pathway. cbeta(2)gpI may represent a potential endogenous angiogenesis-targeted compound, opening the possibility of a novel tool to treat cancer.

{beta}2 Glycoprotein I ({beta}2GPI) binds platelet factor 4 (PF4): implications for the pathogenesis of antiphospholipid syndrome.

Antiphospholipid syndrome (APS) is an autoimmune thrombophilia characterized by arterial/venous thrombosis and/or pregnancy morbidity in the presence of antiphospholipid antibodies that mainly recognize beta2 glycoprotein I (beta2GPI). To investigate potential platelet ligands of beta2GPI, platelet membrane proteins from healthy persons and patients with APS were passed through a beta2GPI-affinity column. By using mass spectrometry, platelet factor 4 (PF4) appeared as the dominant beta2GPI binding protein. PF4 could bind in vitro, with high-affinity, recombinant beta2GPI, and the binding was abrogated by soluble beta2GPI. Coprecipitation experiments further confirmed this interaction. In silico molecular docking showed that PF4 tetramers can bind 2 beta2GPI molecules simultaneously. Size exclusion chromatography confirmed that anti-beta2GPI antibodies selectively interact with complexes composed of (beta2GPI)(2)-(PF4)(4). In addition, as shown by the beta2GPI antigenicity evaluation, the reactivity of APS sera was higher against PF4-beta2GPI complex than against beta2GPI alone. On complex formation, anti-beta2GPI-beta2GPI-PF4 significantly induced platelet p38MAPK phosphorylation and TXB2 production, mainly through F(ab')(2) fragments of antibodies. In summary, this study makes evident that beta2GPI forms stable complexes with PF4, leading to the stabilization of beta2GPI dimeric structure that facilitates the antibody recognition. This interaction can probably be involved in the procoagulant tendency of APS.

Detection of serum beta(2)-GPI-Lp(a) complexes in patients with systemic lupus erythematosus.

BACKGROUND: Circulating beta(2)-glycoprotein-I-oxidized low-density lipoprotein (beta(2)-GPI-ox-LDL) complexes have been found in patients with systemic lupus erythematosus (SLE) and other autoimmune diseases as a contributor to the development of autoimmune-mediated atherosclerosis. In vitro study showed that beta(2)-GPI also bound with high affinity to atherogenic lipoprotein (a) [Lp(a)] which shares structural similarity to LDL. We examined the existence and clinical significance of serum complexes of beta(2)-GPI with Lp(a) in SLE patients. METHODS: A "sandwich" ELISA was developed for measuring serum concentrations of beta(2)-GPI-Lp(a) complexes, using rabbit anti-human beta(2)-GPI antibody as capturing antibody, and quantitating with antibody against apo(a). Forty-seven SLE patients and 42 healthy controls were studied. RESULTS: Both Lp(a) (400+/-213 mg/l vs. 181+/-70 mg/l) and ox-Lp(a) (27.07+/-22.30 mg/l vs. 8.20+/-4.55 mg/l) concentrations were higher in SLE patients than in controls (P<0.0001). beta(2)-GPI-Lp(a) complexes were detectable in both controls and SLE. The complexes levels in SLE were higher than in controls (0.96+/-0.41 U/ml vs. 0.59+/-0.20 U/ml, P<0.0001) and was positively correlated with ox-Lp(a) (P<0.001). CONCLUSIONS: We report the existence of beta(2)-GPI-Lp(a) complexes in both controls and SLE patients. The complexes levels increase in SLE.

Intracellular trafficking of beta2-glycoprotein I complexes with lipid vesicles in macrophages: implications on the development of antiphospholipid syndrome.

Beta(2)-glycoprotein I (beta(2)GPI) is known as a major autoantigen for antiphospholipid antibodies. Our recent data show that binding of beta(2)GPI to oxidized low-density lipoprotein (oxLDL) or to liposomes containing anionic phospholipid(s) may facilitate the presentation of beta(2)GPI's epitope by macrophages/dendritic cells to autoreactive T cells. In the present study, we investigated intracellular trafficking of beta(2)GPI and its complexes with oxLDL or liposomes containing phosphatidylserine (PS-liposomes) in mouse macrophage-like J774 cells. A relatively small amount of non-complexed beta(2)GPI was taken up and stagnated in the late endosome after incubating for 16h. In contrast, beta(2)GPI complexes with oxLDL or PS-liposomes were transported into the lysosome. In the presence of the IgG anti-beta(2)GPI autoantibody, WB-CAL-1, beta(2)GPI/oxLDL complexes were rapidly incorporated into intracellular space and were finally localized in the lysosome. Interestingly, in vitro pulses by beta(2)GPI/oxLDL complexes together with WB-CAL-1 led to the expression of membranous CD36 as well as Fcgamma type I receptors (FcgammaRI). These observations suggest that IgG immune complexes of beta(2)GPI/oxLDL provide not only FcgammaRI- but also scavenger receptor-mediated uptake of beta(2)GPI/oxLDL complexes by macrophages. Thus, beta(2)GPI/oxLDL complexes as a major atherogenic autoantigen and IgG anti-beta(2)GPI autoantibodies may facilitate antigen presentation and foam cell formation in antiphospholipid syndrome.

Binding of antiphospholipid antibodies to discontinuous epitopes on domain I of human beta(2)-glycoprotein I: mutation studies including residues R39 to R43.

OBJECTIVE: Pathogenic antiphospholipid antibodies (aPL) bind the self antigen N-terminal domain (domain I) of beta(2)-glycoprotein I (beta(2)GPI), with residues G40-R43 being important. However, peptides homologous to other regions of domain I have also been shown to bind aPL. Furthermore, there are no published reports of the effects of altering R39, which has greater surface exposure than the G40-R43 residues. METHODS: We used a novel, efficient method of production and purification of human domain I by Escherichia coli to create multiple mutants of domain I. These domain I mutants were then screened for binding to a range of polyclonal IgG purified from patients with antiphospholipid syndrome, using both solid-phase and fluid-phase assays. RESULTS: E coli-expressed purified domain I selectively bound IgG derived from patients with antiphospholipid syndrome. In region R39-R43, the R39S mutation had the greatest effect in terms of reducing binding to a panel of aPL in the fluid phase (mean +/- SD inhibition 14 +/- 18.5% versus 44.1 +/- 31.7% for G40E and 62.9 +/- 25.7% for wild-type domain I). Conversely, altering both D8 and D9 to S8 and G9, respectively, had the effect of enhancing binding to aPL in the fluid phase. Adding the remainder of the domain I-II interlinker resulted in enhanced binding over wild-type in the solid phase but not the fluid phase. CONCLUSION: The binding of aPL to beta(2)GPI domain I is complex and likely to involve discontinuous epitopes that include R39 in addition to G40-R43, the domain I-II interlinker, and possibly D8 and D9. Domain I variants with enhanced binding to aPL compared with wild-type domain I may aid in the development of novel therapies.