**2.6. Inhibition of the LMW activator activity by anti-HATKTAK rabbit IgG antibody**

Antibody against HATKTAK was raised in a rabbit by intracutaneous injection of keyholelimpet-conjugated CHATKTAK (6 injections at 2-week intervals) by Sigma Aldrich Japan (Ishikari city, Japan). The IgG antibody was refined using a protein A column. The control IgG antibody was obtained from the same rabbit at day 0 of immunization.

By indirect enzyme-linked immunosorbent assay (ELISA) [33], specificity of the anti-HATKTAK rabbit antibody was examined. 100 l of antigens involving Apo CIII and Apo CIII-related peptides (1 g/ml in PBS) was immobilized to each well of a polystyrene microplate by incubation at 4 °C overnight. 1000-fold-diluted anti-HATKTAK rabbit antibody or l000-fold-diluted anti-Apo CIII gout IgG (Gene Tex, Inc., San Antonio, TX) was used as the primary antibody. Peroxidase-conjugated anti-rabbit IgG (Fab') or anti-goat IgG (Fab') (Histofine simple stain MAX-PO(R) or -PO(G), respectively, Nichirei Bioscience, Tokyo, Japan) was used as the secondary antibody. Substrate solution consisted of 10 ml of 0.5 M citrate buffer, 10 ml of 0.3% hydrogen peroxide and 10 mg of orthophenylene diamine (Wako, Osaka, Japan). 2 M sulfuric acid was used as a stopping solution. Absorbance was determined at 450 nm in a model 550 microplate reader (Bio-Rad, Tokyo, Japan).

To examine the effect of anti-HATKTAK antibody on an LMW activator, a x102-diluted lowmolecular-weight fraction of the platelet release products prepared from fresh platelet- rich plasma and a x106-diluted one from platelet-rich plasma stored for 120 hours were prepared. Then, they were incubated with a one-hundredth volume of anti-HATKTAK IgG or control IgG at room temperature for 30 min, applied to a protein A column to remove the immune complexes and the residual antibodies, and used as the source of the LMW activator.

#### **2.7. Ion exchange chromatography**

724 Lipoproteins – Role in Health and Diseases

**2.4. Preparation of platelet lysate** 

platelet lysate.

Germany).

**antibody** 

**2.5. Peptide synthesis** 

to obtain the platelet release products in the supernatant.

These platelets were washed twice in PBS supplemented with 6.7 mM EDTA and twice in PBS, and adjusted to a concentration of 4x105/l. The platelet suspension was stimulated with 0.1 unit/ml thrombin at 37 °C for one minute in the presence or absence of 4 mEq/l Ca++, then cooled immediately on an ice-water bath and centrifuged at 960 g for 15 minutes at 4 °C

Platelet suspensions obtained from platelet-rich plasma as described above were frozen at - 15 °C, thawed and centrifuged at 1,500 g for 30 minutes. The supernatant was used as the

Peptides such as S1-K21, H18-R40, H18-K24, H18-K21 and T22-K24 of Apo CIII were synthesized by Sawady Technology (Tokyo, Japan). Other peptides that contain part or all of H18-K24 (HATKTAK) of Apo CIII were produced by Thermo Fisher Scientific GmbH (Ulm,

**2.6. Inhibition of the LMW activator activity by anti-HATKTAK rabbit IgG** 

IgG antibody was obtained from the same rabbit at day 0 of immunization.

determined at 450 nm in a model 550 microplate reader (Bio-Rad, Tokyo, Japan).

complexes and the residual antibodies, and used as the source of the LMW activator.

To examine the effect of anti-HATKTAK antibody on an LMW activator, a x102-diluted lowmolecular-weight fraction of the platelet release products prepared from fresh platelet- rich plasma and a x106-diluted one from platelet-rich plasma stored for 120 hours were prepared. Then, they were incubated with a one-hundredth volume of anti-HATKTAK IgG or control IgG at room temperature for 30 min, applied to a protein A column to remove the immune

Antibody against HATKTAK was raised in a rabbit by intracutaneous injection of keyholelimpet-conjugated CHATKTAK (6 injections at 2-week intervals) by Sigma Aldrich Japan (Ishikari city, Japan). The IgG antibody was refined using a protein A column. The control

By indirect enzyme-linked immunosorbent assay (ELISA) [33], specificity of the anti-HATKTAK rabbit antibody was examined. 100 l of antigens involving Apo CIII and Apo CIII-related peptides (1 g/ml in PBS) was immobilized to each well of a polystyrene microplate by incubation at 4 °C overnight. 1000-fold-diluted anti-HATKTAK rabbit antibody or l000-fold-diluted anti-Apo CIII gout IgG (Gene Tex, Inc., San Antonio, TX) was used as the primary antibody. Peroxidase-conjugated anti-rabbit IgG (Fab') or anti-goat IgG (Fab') (Histofine simple stain MAX-PO(R) or -PO(G), respectively, Nichirei Bioscience, Tokyo, Japan) was used as the secondary antibody. Substrate solution consisted of 10 ml of 0.5 M citrate buffer, 10 ml of 0.3% hydrogen peroxide and 10 mg of orthophenylene diamine (Wako, Osaka, Japan). 2 M sulfuric acid was used as a stopping solution. Absorbance was Cation exchange chromatography was performed using a MONO S HR5/5 column (GE Healthcare UK Ltd.). Samples were dissolved in 20 mM Tris-HCl, pH 8.0 (buffer A). The adsorbed materials were recovered by elution with a linear increase in buffer B (buffer A + 1 M NaCl).
