Free thiol formation within β2GPI is enhanced within minutes on incubation with EAhy926 cells. (A-B) TRX-1/DTT–treated nβ2GPI incubated with EAhy926 cells results in an increase in MBP labeling compared with TRX-1/DTT–treated nβ2GPI incubated with empty wells (P ≤ .001; n = 4). (C-D) rβ2GPI (1μM) was pretreated with DTT-activated human TRX-1 (1.75μM) and then incubated in wells coated with and without EAhy926 cells for 0, 5, and 15 minutes. The rβ2GPI/TRX-1 mixture was then labeled with MPB after each respective incubation time. EAhy926 cells enhanced MPB labeling of TRX-1/DTT–treated rβ2GPI within 5 minutes (**P ≤ .007; n = 4) and was maintained at 15 minutes (*P ≤ .04; n = 4). (E) The rβ2GPI/TRX-1/DTT MPB-labeled mixture was then subjected to nickel chromatography, and the degree of relative MPB labeling of equal amounts of purified rβ2GPI from cell-coated and empty wells (750 ng of protein/lane) was determined with streptavidin–horseradish peroxidase (HRP). The loss of MPB-labeled TRX-1 after nickel purification confirms the efficiency of the rβ2GPI purification process. (F-G) The increase in MPB labeling of TRX-1/DTT–treated his-tagged rβ2GPI after cell incubation before nickel purification (**P ≤ .004; n = 3) is also observed after nickel purification (*P ≤ .04; n = 3). Membranes were stripped and probed with anti–TRX-1 to ensure equal protein loading between wells. rβ2GPI versus native β2GPI is approximately 7 kDa smaller because of nonmammalian glycosylation by insect cells.