The concentration of Plg in the plasma of WT and KO mice as determined by ELISA was 15.8 ± 1.2 and 14.4 ± 1.3 mg/dL, respectively (mean ± SD of four determinations in duplicate from four mice). From each plasma sample, Plg was isolated by lysine-Sepharose affinity chromatography. In both cases, the product contained a single band migrating in the same position as standard Plg when analyzed on 4–12% SDS-PAGE gradient gels stained with Coomassie Blue (Figure 1A). In addition, Plg samples from WT and KO mice reacted against anti-mouse Plg (Figure 1B).

To evaluate whether mouse Plg contained OxPtdPCs we subjected Plg isolated from WT and KO mice to immunoblot analyses using T15. We found that both sets of samples reacted strongly with the OxPtdPC-specific monoclonal antibody (Figure 1B). Pre-treatment with denaturing agents, extraction with organic solvents and 1–2 cycles of freezing/thawing failed to affect T15 reactivity (not shown). These results indicated that OxPtdPCs are covalently linked to Plg.

To investigate whether Plg-bound oxPtdPCs are metabolized by Lp-PLA₂ in vitro we incubated Plg isolated from WT and KO mouse plasma with a pure, enzymatically active, preparation of human Lp-PLA₂. Both sets of samples exhibited the same T15 reactivity as incubated (24 h at 37 °C), but untreated, controls (Figure 2). These in vitro studies strongly suggested that Lp-PLA₂ does not metabolize oxPtPC linked to Plg.

To further investigate this issue, we quantified the oxPtdPC molecules bound to Plg isolated from WT and KO mice by ELISA (see “Methods”) (Figure 3A). The assay was highly reproducible over a wide range of concentrations (1.56 to 100 nmol/L, Figure 3B). From the values obtained we calculated an oxPtdPC:Plg ratio of one for both Plg samples, establishing that under basal conditions, deletion of Lp-PLA₂ does not affect the extent of Plg conjugation with oxPtdPCs.

The studies in mice were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the committee on the Ethics of Animal Experiments of the University of UTAH (animal welfare assurance number A3031-01).

BSA, Tween-20, SDS, ε-amino caproic acid, 4-(2-Aminoethyl)-benzene sulfonylfluoride, and N-α-tosyl-L-lysine chloromethylketone hydrochloride were from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Immobilon-P membranes were from Millipore Corp. (Billerica, MA, USA) and Superblock blocking buffer and the Supersignal^® West Dura Extended Duration Substrate were purchased from Thermo Scientific (Rockford, IL). Coomassie staining solution (Page Blue) was from Fermentas Inc. (Glen Burnie, MD, USA). Precast acrylamide gels were from Invitrogen Corp. (Carlsbad, CA, USA). All chemicals were of reagent grade.

Human recombinant Lp-PLA₂ (Pafase^®) was a generous gift from ICOS Corporation (Bothell, WA, USA).

The murine T15 antibody-secreting cell line, BH8 (IgM) that reacts with oxPtdPC antigens was a gift from Dr. John F. Kearney (University of Alabama) and is heretofore referred to as T15. This cell line was maintained in the Frank W. Fitch Antibody Facility of the University of Chicago. The antibodies in the culture medium were isotyped and contained a high titer of IgM antibodies (greater than 90%) as verified by the major Coomassie stained band on SDS-PAGE.

Affinity purified rabbit anti-mouse Plg polyclonal antibodies were obtained from Cell Sciences (Canton, MA, USA). Horseradish peroxidase (HRP)-labeled secondary antibodies [goat anti-mouse IgM (μ chain specific) and donkey anti-rabbit IgG] were from Sigma-Aldrich.

Plasma from healthy mice was utilized to purify Plg using the procedure of Deutsch and Mertz [9], followed by purification on G25 Sepharose columns. We also employed commercial preparations of mouse Plg purchased from Enzyme Research Laboratories (South Bend, IN, USA). Quantitative determination of Plg in mouse plasma and in purified samples was conducted using an ELISA kit (Kamiya Biomedical Co., Seattle, WA, USA). The Plg standard concentrations ranged from 6.25 to 200 ng/mL.

Lipids were extracted from purified Plg by a modified Bligh and Dyer method [10] in that chloroform was replaced by anhydrous diethyl ether in a ratio of 1:2 v/v (ether:methanol). Plg in 0.8 mL 50 mM Tris-HCl containing 0.1 M NaCl, pH 7.4 was added to the ethyl ether-methanol mixturedropwise and rotated at 4 °C for 6 h. The mixture was then centrifuged and the precipitated protein washed three times with diethyl ether. The washed precipitate was dried with argon gas and dissolved in the Tris buffer. The protein recovery was close to 85% of the original starting material. An alternative delipidation technique was also carried out using a 3:2 v/v mixture of ethanol:diethyl ether as already published for apo(a) [1]. In both delipidation techniques the Plg was soluble in aqueous buffers.

SDS-PAGE (gradients of 4–12% polyacrylamide) was performed on a Novex system (Novex, San Diego, CA) for 1.5 h at constant voltage (120 V) at 22 °C, as described [1,2]. The samples were prepared by heating at 95 °C for 5 min in sample buffer [94 mM phosphate buffer (pH 7.0), 1% SDS and 2 M urea, with or without 3% mercaptoethanol]. Following electrophoresis, the gels were placed on Immobilon-P sheets (Millipore Corp., Bedford, MA) previously wetted in 48 mM Tris, 39 mM glycine (pH 8.9). Blotting was performed on a horizontal semi-dry electroblot apparatus (Amersham Biosciences) at 0.8–1 mA/cm² for 45 min at 23 °C.

After electroblotting, the Immobilon-P sheets were blocked in Superblock (Thermo Scientific, Inc. Rockford, IL. USA) for 1 h at 23 °C followed by incubation with rabbit anti-mouse Plg IgG or, in the case of T15, by incubation in 10% Superblock for 18 h at 4 °C. Bound antibodies were visualized using secondary antibodies conjugated to HRP; donkey anti-rabbit IgG for Plg, and goat anti-mouse IgM (μ-chain specific) for oxPtdPC. The blots were developed with Supersignal^® West Dura Extended Duration Substrate from Thermo Scientific, Inc. (Rockford, IL).

We utilized a novel sandwich ELISA by which we measured T15 immunoreactivity in Plg isolated from mouse plasma. Polystyrene microtiter plates (flat-bottom 96-well EIA plates) were coated with 100 μL of T15 [400 ng/well, dissolved in TBS buffer [50 mmol/L Tris-HCl, 0.15 mol/L NaCl (pH 7.5)] and incubated overnight at room temperature, in 10 mmol/L Tris, 0.15 mol/L NaCl (pH 7.6). Unbound antibodies were removed by washing with TBS supplemented with 0.1% BSA and 0.02% Tween-20. Non-specific binding sites were blocked with 1% BSA in TBS for 1.5 h. After three washes with TBS supplemented with 0.02% Tween-20 (TBST), we added 100 μL of each dilution of the mouse Plg standard and samples (diluted in TBST) and incubated the plates for 2 h at room temperature. After three washes with TBST, bound Plg was detected by incubation with a specific, HRP-conjugated anti-mouse Plg polyclonal antibody for 1 h in TBST. The wells were washed three times with TBST, the chromogenic substrate 3,3′,5,5′-tetramethylbenzidine was added and, after an appropriate incubation period at room temperature, the reactions were stopped with 3 mol/L H₂SO₄. T15 immunoreactivity was quantitated by assessing the absorbance at 450 nm using a Versamax microplate reader (Molecular Devices, Sunnyvale, CA). The concentrations of the Plg standard ranged from 1.56 to 100 nmol/L.

Based on the standard curve, the absorbance of each sample reacting with T15 was converted to nmol/L of Plg. This number was divided by the concentration (nmol/L) of Plg in each mouse sample. This ratio was defined as the “T15 equivalent”.

The generation of Pla2g7^−/− mice took place at the University of Utah and was recently described in detail [11] Briefly, a targeting vector lacking exons 3 and 4 of the mouse Pla2g7 gene was introduced into embryonic stem cells and a resulting chimeric male was bred to C57BL/6J females. The progeny was genotyped as previously described [11].

Mouse Plg (1 to 3 μg) was incubated with recombinant Lp-PLA₂ (4 μg) in PBS for up to 24 h at 37 °C. The reactions were stopped with Pefabloc and the samples were stored frozen at −20 °C until subsequent analyses.