Free thiols introduced into β2GPI after reaction with TRX-1 and PDI; reduction of β2GPI on the platelet surface. Native or recombinant β2GPI was incubated with TRX-1 previously reduced by DTT (A) or TRX-R/NADPH (B) or with PDI reduced by DTT (C). Reduced TRX-1 was prepared by incubating 5μM TRX-1 with 25μM DTT or 10nM TRX-R/200μM NADPH for 1 hour at 37°C. Reduced PDI was prepared by incubating 500nM PDI with 50μM DTT. β2GPI (0.2μM) was incubated with the TRX-1 or PDI mixtures for 1 hour at 37°C. To label free thiols, N^a-(3-maleimidylpropionyl) biocytin (MPB; 100μM) was added to the β2GPI/TRX-1 or PDI solutions for 10 minutes at 37°C. The reaction was quenched with glutathione (200μM) for 10 minutes at 37°C. All reactions were performed in 20mM HEPES, 0.14M NaCl buffer, pH 7.4. The reactions were resolved under nonreducing conditions on SDS-PAGE (4%-12%) and then transferred to polyvinylidene fluoride membranes. MPB-labeled β2GPI was probed with streptavidin–horseradish peroxidase (1:1000). β2GPI was detected with the 4B2E7 monoclonal antibody 3.5 mg/mL or rabbit anti-β2GPI Ab 1.4 mg/mL. (A) Incorporation of MPB into β2GPI (at ∼ 70 kDa) after treatment with TRX-1 (lane 1). MPB cannot be introduced into β2GPI after treatment with the reductant DTT alone defining β2GPI as a substrate of TRX-1 (lane 2). Lanes 4 and 5 show detection of the reduced β2GPI forms on SDS-PAGE and the decreased immunoreactivity of anti-β2GPI monoclonal antibody to reduced β2GPI (± MPB) compared with nonreduced β2GPI (lane 6). (B) Coomassie staining of β2GPI incubated with TRX-1/TRX-R/NADPH/MPB and separated on SDS-PAGE shows one β2GPI band at ∼ 70 kDa in accordance with the MPB-labeled β2GPI band in the streptavidin–horseradish peroxidase blot (panel A lane 1) and the first β2GPI immunoreactive band on the anti-β2GPI blot (panel A lane 4). The remaining 3 β2GPI immunoreactive bands (between 50 and 70 kDa) apparent in the anti-b2GPI blot (panel A lane 4) are not detected on the Coomassie, showing that these β2GPI products have very low concentrations. (C) Incorporation of MPB into β2GPI after reaction with PDI. MPB-labeled β2GPI, after reduction with PDI, showed a minor shift in molecular size on the SDS PAGE (∼ 50 kDa, panel C lane 1) in comparison to TRX-1 treated β2GPI (panel A lane 1), showing that TRX-1 had a greater effect than PDI on the biotin labeling or denaturing of β2GPI. Numbers indicate the molecular weight markers. (D) Detection of reduced β2GPI on the platelet surface by a β2GPI-specific streptavidin-capture ELISA. Platelets (in buffer or pretreated with the TRX-R inhibitor 1-chloro-2, 4-dinitrobenzene [DNCB]) were incubated with or without β2GPI. MPB was added to label any free thiols formed. After acetone precipitation to remove nonincorporated MPB, the precipitated MPB-labeled proteins were applied to a streptavidin plate, which captured MPB-labeled proteins including (reduced) β2GPI. β2GPI in 20mM HEPES, 0.14M NaCl buffer, pH 7.4 buffer alone plus MPB served as a negative control (first column). As a positive control β2GPI reduced by TRX-1/TRX-/NADPH plus MPB was used. β2GPI incorporated MPB after incubation with platelets which was partially inhibited by DNCB. Platelets alone plus MPB gave a negligible signal on this ELISA. OD indicates optical density; β2, β2GPI; M, MPB; and TRN, TRX-1/TRX-R/NADPH. Data are expressed as mean ± SD, n = 3 in triplicate. *P < .02; **P < .01.