Annexin A5 resistance studies. (A) Effect of aPL mAb on inhibition of prothrombinase reaction by annexin A5. Factor Xa, factor Va, and prothrombin added to the PL bilayer yielded a baseline thrombin generation rate (TGR) of 2402 ± 210 pM/min (mean ± SD). Addition of annexin A5 significantly reduced the TGR to 358 ± 18 pM/min (P < .0001). Addition of the aPL mAb to the system containing the annexin A5 reversed the anticoagulant effect of annexin A5 and increased the TGR (713 ± 87 pM/min compared with 347 ± 28 pM/min for the control mAb, P < .01), a reversal that was further enhanced by the addition of β2GPI (859 ± 77 pM/min compared with 421 ± 64 pM/min for the control mAb, P < .01). (B) Effects of spiking plasma with aPL IgG on annexin A5 anticoagulant activity. aPL or control IgG was added to a normal plasma. The annexin A5 anticoagulant ratio for the phospholipid preincubated with aPL IgG and β2GPI was significantly less (mean ± SD, 143 ± 11%) than the phospholipid preincubated with control IgG and β2GPI (180 ± 8%, P = .01) (n = 3 pairs of different IgG). (C) Effects of depleting aPL IgG from aPL plasmas on annexin A5 anticoagulant activity. aPL plasmas that were pretreated with protein G-Sepharose significantly increased annexin A5 anticoagulant ratios (mean ± SD, 235 ± 28%) compared with plasmas pretreated with Sepharose 2B (193 ± 48%, P < .05). In control plasmas, no significant difference on annexin A5 anticoagulant activity was observed between plasmas pretreated with protein G-Sepharose (239 ± 15%) and plasmas pretreated with Sepharose 2B (250 ± 12%) (n = 10 pairs of aPL and control plasmas). (D) Effects of varying aPL plasma concentration on annexin A5 anticoagulant activity. Varying dilutions of aPL plasma in control plasma were incubated with TF-aPTT reagent-phospholipid. The annexin A5 anticoagulant ratio progressively decreased as the concentration of aPL plasma was increased in control plasma. (Each point is the mean of 2 aPL patient plasmas.)