1 Supplemental Figure Legends Supplemental Figure 1 ELISA scheme to measure plasma total, mature and furin-cleaved PCSK9 concentrations. 4 Plasma mature and furin-cleaved PCSK9s were measured by a sandwich ELISA using specific 5 combinations of Mabs. A combination of Mab 1FB and HRP-labeled Mab B1E was used for 6 total PCSK9 quantification. A combination of Mab B1E and HRP-labeled Mab G1D was 7 used for the mature PCSK9 quantification, while Mab B1G and HRP-labeled Mab B1E were 8 used for the furin-cleaved PCSK9. 9 10 Supplemental Figure Titration curves of the PCSK9 ELISA. 11 The ELISA was performed as described in the Supplemental Materials and Methods. The 1 purified rhpcsk9 was used as the primary calibrator for the ELISAs of total and mature PCSK9, 1 and the cell lysate containing rhδ18pcsk9 was used to calibrate the ELISA of furin-cleaved 14 PCSK9. The titration curves were made using serial dilutions (1:1,80 to 1:0) of purified 15 rhpcsk9 (closed circles), rhpcsk9 culture medium (open circles), or human plasma (open 16 triangles) for the total (A) and mature PCSK9 (B), and using serial dilutions (1:56 to 1:4) of 1
1 cell lysate of rh 18PCSK9 (closed circles), purified rhpcsk9 digested with furin (open circles), or human plasma (open triangles) for furin-cleaved PCSK9 (C). Each point represents the mean of triplicate determinations. 4 5 Supplemental Figure Standard curves in the ELISA for total, mature and furin-cleaved 6 PCSK9s. 7 The standard curve was made using serial dilutions (1:100 to 1:6,400) of 000 ng/ml purified 8 rhpcsk9 for total (A) and mature PCSK9 (B), and using serial dilutions (1:10 to 1:640) of cell 9 lysates rh 18PCSK9 for furin-cleaved PCSK9 (C). Each point represents the mean of triplicate 10 determinations. 11 1 Supplemental Figure 4 Correlation between plasma HDL-C reduction and mature PCSK9 1 reduction in FH homozygotes and FH heterozygotes. 14 (A) Correlation between plasma HDL-C reduction (Y-axis) and mature PCSK9 reduction 15 (X-axis) in FH homozygotes after a single LDL-A treatment with DS columns (N=7). 16 (B) Correlation between plasma HDL-C reduction (Y-axis) and mature PCSK9 reduction
1 (X-axis) in FH heterozygotes after a single LDL-A treatment with DS columns (N=11).
1 Supplemental Materials and Methods Construction, expression, and purification of recombinant PCSK9 proteins Human PCSK9 cdna was obtained by RT-PCR from mrna of HepG cells and a C-terminal 4 His 6 tag was added as described [1]. Briefly, PCR was carried out using the following primers: 5 5 -gacgaattccagcgacgtcgaggcgctcatggttg- and 6 5 -gacgaattctcagtgatggtgatggtgatgctggagctcctgggaggcctgcgcc- for mature PCSK9 (1-69 aa), 7 and 5 -agtcaagcttcaggccagcaagtgtgacagtcat- and 5 -agtagtcgacctggagctcctgggaggcctg- for 8 18PCSK9 (19-69 aa), which corresponds to the furin-cleaved PCSK9. The respective 9 cdnas were subcloned into the pef1 mammalian expression vector to yield pef1/pcsk9 10 or pef1/ 18PCSK9 vector. CHO-K1 cells stably transfected with the pef1/pcsk9 or 11 pef1/ 18PCSK9 vector were cultured. The mature form of recombinant human PCSK9 1 (rhpcsk9) from the culture medium was partially purified by affinity column chromatography 1 using Talon metal affinity resin (Clontech, Mountain View, CA), followed by anion exchange 14 chromatography using DEAE Sepharose CL-6B (GE Healthcare, Uppsala, Sweden). The purity 15 of rhpcsk9s and rhpcsk9 (1 µg) digested with or without recombinant human furin (0.5 µg; 16 R&D Systems) was confirmed by SDS-PAGE followed by silver staining or immunoblotting. 17 For rhδ18pcsk9, transfectant cells were collected by trypsinization, and suspended in TBS 18 containing 1% NP-40. After centrifugation of the cell suspension at 15,000 X g for 0 min at 1
1 4 C, the supernatant was collected and used as a calibrator for furin-cleaved PCSK9 ELISA. For immunoblotting, purified rhpcsk9 and rh 18PCSK9 in the cell lysate were detected with anti-tetra-his antibody (Qiagen, Valencia, CA). The bands were detected with an enhanced 4 chemiluminescence kit (Perkin-Elmer, Norwalk, CT). 5 6 Production of monoclonal antibodies against PCSK9 7 Balb/c mice were immunized using a DNA-based or standard immunization method with 5 µg 8 purified rhpcsk9 [1], and spleen cells from mice were fused with Sp/0 myeloma cells. The 9 supernatants of hybridoma cells were screened by ELISA using plates coated with purified 10 rhpcsk9 (100 ng/well) and by immunoblotting. Positive hybridoma cells were cloned at least 11 four times by limiting dilution and injected intraperitoneally into pristane-primed balb/c mice. 1 The IgG fraction was isolated from ascitic fluid using protein A-Sepharose FF (GE Healthcare, 1 Piscataway, NJ), dialyzed against PBS at 4 C, and stored at -80 C. The specificities of each 14 monoclonal antibody (Mab) obtained by standard immunization (1FB) and by DNA-based 15 immunization (B1G, B1E and G1D), respectively, were confirmed by ELISA and 16 immunoblotting against purified rhpcsk9. The Mab isotype was characterized using an Isostrip 17 mouse Mab isotyping kit (Roche Diagnostics, Basel, Switzerland), and was IgG1 for all Mabs. 18
1 Measurement of plasma mature and furin-cleaved PCSK9 concentrations Plasma mature and furin-cleaved PCSK9s were measured by an ELISA using a specific combination of Mabs as previously described (Supplemental Fig. 1) [1]. The absorbance was 4 measured at 450 nm with a microplate reader. The intra- and inter-assay coefficients of variation 5 of the ELISAs in serum samples (the total and mature PCSK9 ranged from.9 to 560.5 6 ng/ml and furin-cleaved PCSK9 ranged from 9.4 to 9.1 ng/ml) were 1.9-.8% (n=10) and 7.9-7.8% (n=10), respectively. No interference with either ELISA was observed with 8 hemoglobin (5.0 g/l), bilirubin (0. g/l), or triacylglycerol (4.5 g/l). 9 10 Standardization of ELISA for the mature and furin-cleaved PCSK9s in plasma 11 The purified rhpcsk9 was used as the primary calibrator for the ELISAs of total and mature 1 PCSK9, and the cell lysate containing rhδ18pcsk9 was used to calibrate the ELISA of 1 furin-cleaved PCSK9. To obtain a calibration curve in the ELISA for total and mature PCSK9, 14 dilutions of the primary calibrator were made in PBS containing 0.1% Tween 0 and 0.% BSA 15 (sample diluent) to provide 0.0- ng of rhpcsk9 protein per well (1.-000 ng/ml). When 16 the rhpcsk9 culture medium, as a secondary calibrator, was diluted in sample diluent to cover 17 the same range of PCSK9 concentration, the curve was identical to that obtained with the 18 primary calibrator (Supplemental Fig. ). Similarly, the calibration curve was made using
1 dilutions of the cell lysate of rh 18PCSK9 for furin-cleaved PCSK9. The linearity of each ELISA was up to 000 ng/ml for total and mature PCSK9, and up to 400 ng/ml for furin-cleaved PCSK9, and was suitable for quantifying each form of PCSK9 concentration to as 4 low as 9.1 ng/ml for the total and mature PCSK9, and 6.8 ng/ml for furin-cleaved PCSK9 to 5 provide 0.006-4.0 ng of rh 18PCSK9 protein per well (6.-400 ng/ml). The linearity in each 6 system was also confirmed with serially diluted rhpcsk9 culture medium and plasma samples 7 of several concentrations (6.4 to 544. ng/ml for total and mature PCSK9, and 10.8 to 94.1 8 ng/ml for furin-cleaved PCSK9). The plasma was diluted 150-fold, which gave an absorbance 9 between 0.5 and 1.. 10 11 References 1 [1] Ishihara M, Kujiraoka T, Iwasaki T, Nagano M, Takano M, Ishii J, Tsuji M, Ide H, 1 Miller IP, Miller NE, Hattori H 005 A sandwich enzyme-linked immunosorbent assay for 14 human plasma apolipoprotein A-V concentration. J Lipid Res 46:015-0 15 16 4
Supplemental Table 1. Patient characteristics of the 18 FH subjects in this study Duration of Mutations in LDLR, Medication- Patient Medication Medication Treated plasma LDL-A Age Sex Type of FH PCSK9, or CAD anticoagulants or no. -statin -Ezetimibe volume (ml) treatment LDLRAP1 gene antiplatelets (year) 1 59 Male Homo LDLR + Pitavastatin - Aspirin 5000 6 6 Female Homo LDLR + Atorvastatin + Aspirin 6000 56 Male Homo LDLRAP1 + Atorvastatin + Aspirin 6000 7 4 8 Female Homo None + Atorvastatin - Aspirin 4000 6 5 56 Female Homo N/A + Atorvastatin + Warfarin, Ticlopidine 5000 4 6 47 Female Homo N/A - Rosuvastatin + - 000 0 7 Female Homo N/A - - - - 6000 15 8 65 Female Hetero LDLR, PCSK9 + Rosuvastatin + Aspirin 000 0 9 58 Male Hetero LDLR + Rosuvastatin + Aspirin, 5000 0 Clopidogrel sulfate 10 66 Male Hetero LDLR + Simvastatin - Aspirin, Ticlopidine 4000 0 11 76 Female Hetero LDLR + Pitavastatin + Aspirin 4000 0 1 75 Male Hetero None + Atorvastatin + Aspirin 4000 6 1 60 Female Hetero None + Rosuvastatin + Aspirin 4000 9 14 45 Female Hetero None + - - Aspirin, 1500 0 Clopidogrel sulfate 15 76 Male Hetero None + Pitavastatin + Aspirin 900 0 16 50 Female Hetero N/A + Pitavastatin + 17 81 Male Hetero N/A + Pitavastatin + Asprin, Clopidogrel sulfate 5000 Asprin, Sarpogrelate hydrochloride 5000 11 18 6 Male Hetero N/A + Atorvastatin - Aspirin, Ticlopidine 5000 10 Abbreviations: CAD, coronary artery disease; FH, familial hypercholesterolemia; N/A, genetic testing was not performed.
Supplemental Table. Change of plasma mature and furin-cleaved PCSK9 and lipids before and after a LDL-A treatment with DS column Patient no. Mature PCSK9 (ng/ml) Furin-cleaved PCSK9 (ng/ml) TC LDL-C HDL-C TG ApoA-I ApoA-II ApoB ApoC-II ApoC-III ApoE Lp(a) before after before after before after before after before after before after before after before after before after before after before after before after before after 1 90 07 6 6 45 11 7 75 8 18 70 84 7 5 1 65 6.4.5 15 5.8 8.6.9 0 5 57 188 46 18 95 90 55 65 4 1 80 19 6 50 18 14 198 49 1.6 <0.5 6.4.1 7.1 1.6 4 11 414 166 106 1 55 94 95 59 5 9 14 1 89 81 6 1 1 5.9.0 14 5.0 9.4 1. N/A N/A 4 51 0 7 187 46 159 6 1 18 8 6 58 19 18 105 18 1.8 0.9 4.6..8 0.5 66 1 5 46 7 75 8 19 98 184 75 18 16 85 6 5 48 16 14 169 66 <0.5 <0.5.9 1.4 4.4 1.7 6 16 6 86 406 10 47 91 105 6 47 40 5 11 111 101 6 158 41.4 1.8 8.0 4. 6.7.0 1.6 7 41 94 57 17 60 57 1 46 6 100 15 104 84 1 17 18 1 1.5 1.0 9..8 5. 0.7 1 8 617 81 111 69 160 80 14 5 6 4 50 15 76 67 19 96 7.9 1.8 7.8 4..7 1. 86 7 9 694 76 77 8 68 85 18 0 48 4 184 64 16 1 45 41 14 6.5.4 1 4.7 4.4 1.1 8 10 14 176 1 17 147 48 11 5 17 14 90 4 57 55 17 16 88 0.5 1.1 5.6.5.1 0.9 4 11 11 404 181 45 1 198 66 16 16 54 47 9 16 19 17 6 9 10 4.. 9.1 5.0.5 0.6 7 8.7 1 49 194 9 0 19 70 140 8 7 104 4 97 88 19 10 7. 1.1 7.6 4..8 1.1 9.7 1 56 17 79 46 400 10 67 104 16 1 84 16 45 41 15 1 45 69 4.1 1.4 8.8.5 6.0 1. 51 17 14 7 60 5 1 8 19 17 109 77 69 16 7 17 156 8 4 15 77 6.7 5.1 1 11 4.4.5 10 59 15 5 46 57 54 44 11 19 80 1 0 104 17 9 91 10 5 4.9. 1 6.6 5.1 1. 70 6 16 51 00 59 8 189 58 1 0 44 6 65 1 111 91 1 5 110 1. 1.0 6.7.8 4.0 0.6 6 8 17 5 154 9 1 14 54 8 7 5 4 1 91 71 17 8 1.1 <0.5 4.8.6. 0.5 1 18 45 141 54 11 91 170 6 1 91 4 66 58 0 18 146 50 1.8 <0.5 6..1 4. 1.4 49 19 Abbreviations: HDL-C, high-density lipoprotein-cholesterol; LDL-C, low-density lipoprotein-cholesterol; Lp(a), lipoprotein (a); TC, total cholesterol; TG, triglyceride
Supplemental Table. Laboratory data in FH heterozygotes before and after a single LDL-A treatment with DM column Before After Reduction (%) TC 187 ± 6 8 ± 4 a 57 LDL-C 17 ± 1 44 ± 4 a 66 HDL-C 9 ± 9 ± 17 b 6 TG 96 ± 44 9 ± 4 b 59 Apo A-I 109 ± 45 85 ± 6 a Apo A-II ±.7 18 ±. a Apo B 100 ± 11 5 ± 18 a 66 Apo C-II.1 ± 1.7 1.6 ± 1. b 50 Apo C-III 8.1 ±.7 4.4 ±.0 a 49 Apo E.4 ± 0.6 1.5 ± 0.6 a 56 Lp(a) 51 ± 8 18 ± 16 a 68 Abbreviations: HDL-C, high-density lipoprotein-cholesterol; LDL-C, low-density lipoprotein-cholesterol; Lp(a), lipoprotein (a); TC, total cholesterol; TG, triglyceride All values are shown as mean ± SD. N=5 a P<0.01, b P<0.05 vs. the respective values before LDL-A