Inhibition of APC anticoagulant activity on oxidized phospholipid by anti-{beta}2-glycoprotein I monoclonal antibodies

Abstract

Activated protein C (APC) anticoagulant activity and the ability to be inhibited by auto-antibodies associated with thrombosis are strongly augmented by the presence of phosphatidylethanolamine (PE) and phospholipid oxidation. beta(2)-glycoprotein I (beta(2)-GPI) is a major antigen for antiphospholipid antibodies present in patients with the antiphospholipid syndrome. We therefore investigated whether anti-beta(2)-GPI monoclonal antibodies (mAbs) could inhibit APC with similar membrane specificity. Five mouse mAbs that reacted with different epitopes on beta(2)-GPI were examined. Each inhibited the PE-, phospholipid oxidation-dependent enhancement of APC anticoagulant activity and required antibody divalency. A chimeric APC that retains anticoagulant activity but is relatively unaffected by protein S, PE, or oxidation was not inhibited by the antibodies. In purified systems, anti-beta(2)-GPI mAb inhibition of factor Va inactivation was greater in the presence of protein S and required beta(2)-GPI. Surprisingly, although the mAbs did increase beta(2)-GPI affinity for membranes, PE and oxidation had little influence on the affinity of the beta(2)-GPI antibody complex for the membrane vesicles. We conclude that antibodies to beta(2)-GPI inhibit APC function specifically and contribute to a hypercoaguable state by disrupting specific protein-protein interactions induced by oxidation of PE-containing membranes.

Figure 1.

Anti–β₂-GPI mAbs inhibit APC anticoagulant activity in a phospholipid oxidation–dependent manner. Plasma clotting was initiated with X-CP as described in “Materials and methods,” in the presence (□, ○) or absence (▪, •) of 0.2 μg/mL APC. Monoclonal IgG was incorporated in the clotting assays at the final concentrations indicated. Nonoxidized phospholipid (○, •); oxidized phospholipid (□, ▪). “Control” indicates addition of an irrelevant mAb IgG at the concentrations indicated. The normal range of the clotting time of pooled normal plasma using unoxidized PE-containing liposomes was 28.9±1.1 seconds SD (n = 15) in the absence of APC and 64.2±2.4 seconds (n = 9) in the presence of APC. The data represent 3 experiments run in duplicate.

Figure 2.

Both PE and oxidation are required to observe inhibition of APC activity by anti–β₂-GPI. PS/PC and PE/PS/PC vesicles were either not oxidized (▪) or oxidized () in the presence or absence of anti-β₂ mAb no. 1522 (120 nM) and APC (0.4 μg/mL) as indicated on the x-axis. Similar results were obtained with the other antibodies. Error bars indicate the SE of 2 or 3 determinations run in duplicate.

Figure 3.

Oxidation does not enhance PC-PtGla activity. PE/PS/PC phospholipid liposomes were oxidized with copper sulfate as described in “Materials and methods,” and plasma clotting was measured. Error bars indicate the SE of 3 determinations. □, 0.2 μg/mL APC-PtGla added; □, 0.2 μg/mL APC added; ○, no additions.

Figure 4.

Anti–β₂-GPI does not affect APC-Pt z-carboxyglutamic acid (Gla) activity but does inhibit APC/Pt1-22 activity. PE/PS/PC vesicles were either not oxidized (▪) or oxidized () and used in clotting assays in the presence or absence of 250 nM anti–β₂-GPI mAb no. 1522 and anticoagulant enzyme as indicated on the x-axis. Similar results were obtained with APC and APC-PtGla with the other antibodies. Error bars indicate the SE of 2 determinations run in duplicate.

Figure 5.

Domain mapping of the anti–β₂-GPI mAbs. Monoclonal antibody (5 nM) was mixed with varying concentrations of inhibitor as indicated and tested for binding to intact β₂-GPI in ELISA as described in “Materials and methods.” The names over the panels indicate the domains of β₂-GPI present in the inhibitor being tested. The percent binding remaining is indicated on the y-axis. □, mAb no. 1529; •, mAb no. 1519; ○, mAb no. 1522; ▴, mAb no. 1527.

Figure 6.

Inhibition of APC inactivation of factor Va by anti–β₂-GPI requires oxidized phospholipid in purified systems. Factor Va (50 nM) was reacted with APC (2.5 pM) on nonoxidized (left panels) or oxidized (right panels) phospholipid vesicles in the absence (top panels) or presence (bottom panels) of anti–β₂-GPI no. 1522 (1 μM) for the times indicated. Factor Va remaining (y-axis) was determined by clotting assay in factor V–deficient plasma as described in “Materials and methods.”× indicates no other additions; ▴, + protein S; ○, +β₂-GPI; □, + protein S +β₂-GPI. Similar results were obtained with mAbs 1519 and 1529.

Figure 7.

Binding of β₂-GPI-mAb complex to liposomes does not necessarily require PE or oxidation. Nonoxidized (▪, •, ▴) or oxidized (□, ○, ▵) liposomes (10 μg/mL) were used to study the binding of β₂-GPI–anti-β₂-GPI complexes in the presence of 2 mM calcium. Complexes were formed by mixing the mAb no. 1522 with 2.25 times its concentration of β₂-GPI to ensure saturation of the mAb before addition to the liposomes. Concentrations indicated on the x-axis refer to those of the mAb. (A) PS/PC vesicles; (B) PE/PS/PC vesicles; (C) vesicles contain 40% PE plus 2% PS (○, •), 5% PS (▵, ▴), or 20% PS (□, ▪), with the remainder of vesicles comprised of PC. Binding isotherms of panel C were performed with a different set of liposomes than panels A and B, resulting in slightly different maximum binding values for the 20%PS/PE/PC vesicles (□, ▪) than observed in panel B. Similar results were obtained with the other mAbs. Is/lo is the ratio of signal intensity after reagent addition (Is) to the baseline value (lo).