Methods of Assessing Unbound PCSK9 or Effective PCSK9 Activity

Abstract

The invention provides methods of assessing unbound PCSK9 or effective PCSK9 activity in a subject based on the novel insights of the inventors that high levels of PCSK9 are bound to HDL in vivo and that this HDL can activate PCSK9 function. Specifically depleting HDL from a sample from a subject allows improved assessment of the level of unbound PCSK9. The invention provides such analytical methods, plus also associated methods of treatment and related kits.

Claims

1. A method of assessing unbound PCSK9 in a subject the method comprising: (a) providing a blood sample from the subject who is optionally diagnosed with, or believed to be at risk of, CVD; (b) specifically depleting at least HDL from the sample to remove HDL-bound PCSK9 from the sample; (c) assessing the level of unbound PCSK9 from the depleted sample.

2. A method as claimed in claim 1 further comprising assessing either total PCSK9 or PCSK9 bound to the HDL from the sample.

3. A method of assessing PCSK9 activity in a subject the method comprising: (a) providing a blood sample from the subject who is optionally diagnosed with, or believed to be at risk of, CVD; (b) assessing the amount of PCSK9 bound to the HDL from the sample, optionally by specifically depleting HDL from the sample to remove HDL-bound PCSK9 from the sample; (c) optionally assessing the amount of LDL-bound PCSK9 from the sample, optionally by specifically depleting ApoB and/or LDL from the sample; (d) correlating the amount of PCSK9 bound to HDL, or the ratio of PCSK9 bound to HDL compared to bound to LDL, with the PCSK9 activity.

4. A method as claimed in claim 3 wherein the ratio of PCSK9 bound to HDL compared to LDL bound is correlated with the PCSK9 activity

5. A method as claimed in any one of the preceding claims wherein the blood sample is a serum sample or plasma sample.

6. A method as claimed in any one of the preceding claims wherein the method comprises specifically depleting ApoB and/or LDL from the sample to remove LDL-bound PCSK9 from the sample.

7. A method as claimed in any one of the preceding claims wherein PCSK9 in the subject is assessed postprandially, optionally following a standard meal preceded by a period of fasting.

8. A method as claimed in any claim 7 wherein the level of unbound and bound PCSK9 or PCSK9 activity is assessed over a period of time postprandially, which is optionally up to 3, 4, 5, 6, 7 or 8 hours.

9. A method as claimed in any claim 8 wherein the unbound and bound PCSK9 or PCSK9 activity over the period of time are subject to area under the curve analysis for the subject.

10. A method as claimed in any one of the preceding claims wherein the subject is individually assessed.

11. A method as claimed in any one of the preceding claims wherein the subject is part of a subject group who are optionally diagnosed with, or believed to be at risk of, CVD, all of whom are assessed.

12. A method as claimed in any claim 11 wherein the group are stratified according to the result of the level of unbound PCSK9 from the depleted sample, and optionally the PCSK9 bound to the HDL from the sample and/or PCSK9 activity.

13. A method as claimed in any one of the preceding claims wherein the level of unbound PCSK9 from the depleted sample or PCSK9 activity is compared to a control, reference or threshold level.

14. A method as claimed in claim 13 wherein the reference level for unbound PCSK9 is the PCSK9 bound to the HDL from the sample or total PCSK9 in the sample, wherein optionally the ratio of unbound: HDL bound or unbound: total PCSK9 is calculated.

15. A method as claimed in claim 13 wherein the reference level is a measure of central tendency of unbound PCSK9 or PCSK9 activity, which is optionally a mean level, observed in one or more populations, wherein the one or more populations are optionally selected from a responsive group of subjects who have responded positively to treatment with a compound which is a statin or an inhibitor or putative inhibitor of PCSK9 or a non-responsive group of subjects who have not responded positively to treatment with a compound which is a statin or an inhibitor or putative inhibitor of PCSK9.

16. A method as claimed in claim 13 wherein the reference level is based on past measurements of unbound PCSK9 or PCSK9 activity in the same subject.

17. A method as claimed in any one of claims 1 to 16 wherein the depletion in step (b) or (c) is performed by one or more of (i) column chromatography, which is optionally immunodepletion (ii) centrifugation, (iii) electrophoresis, or (iv) precipitation, which is optionally immunoprecipitation.

18. A method as claimed in claim 17 wherein the column chromatography is selected from affinity chromatography, size exclusion chromatography, which is optionally HPLC.

19. A method as claimed in any one of claims 1 to 18 wherein the assessing, optionally in in step (c), is performed using an immunoassay; an aptamer-based method; or mass spectrometry.

20. A method as claimed in any one of claims 1 to 19 wherein the level of unbound PCSK9 from the depleted sample is calculated by subtracting PCSK9 bound to the HDL from the sample from total PCSK9 in the sample.

21. A method of: selecting a subject for treatment with a compound which is a statin or an inhibitor or putative inhibitor of PCSK9; or classifying a subject according to their likelihood of responding to treatment with a compound which is a statin or an inhibitor or putative inhibitor of PCSK9; or predicting the response of a subject to treatment with a compound which is a statin or an inhibitor or putative inhibitor of PCSK9; or determining whether an anti-CVD effect is likely to be produced in a subject by treatment with a compound which is a statin or an inhibitor of PCSK9; or estimating the level of in vivo binding of an antibody directed against PCSK9 in the subject assessing the response of a subject who has previously been treated with a statin or an inhibitor or putative inhibitor of PCSK9; the method comprising: (i) performing a method of assessing unbound PCSK9 or PCSK9 activity in the subject according to any one of claims 1 to 20 (ii) using the result of the level of unbound PCSK9 from the depleted sample or PCSK9 activity, and optionally the PCSK9 bound to the HDL from the sample to respectively: select the subject; classify the subject; predict the response; determine whether an anti-CVD effect is likely to be produced; estimate the level of in vivo binding of an antibody directed against PCSK9 in the subject; assess the response.

22. A method as claimed in any one of claims 1 to 21 further comprising treating or further treating a subject selected in accordance with the level of unbound PCSK9 or PCSK9 activity from the depleted sample, and optionally the PCSK9 bound to the HDL from the sample, with a compound which is a statin or inhibitor or putative inhibitor of PCSK9.

23. A method for assessing the efficacy of a compound which is a statin or an inhibitor or putative inhibitor of PCSK9 which is putatively therapeutic for CVD, the method comprising the steps of: (a) selecting a treatment group who have been diagnosed with, or believed to be at risk of, CVD and who have been classified as being likely to be responsive to treatment with such a compound according to a method of claim 21; (b) treating members of the treatment group with the compound for a treatment timeframe; (c) deriving physiological outcome measures for the treatment group; (d) comparing the outcomes at (d) with a comparator arm of which is optionally a placebo or minimal efficacy comparator arm; (e) using the comparison in (d) to derive an efficacy measure for the compound.

24. A method of treating CVD comprising administering a compound which is a statin or an inhibitor of PCSK9 to a subject that has been determined to be responsive to the compound based on the level of serum PCKS9 in the subject not bound to HDL or the proportion of the non-bound PCSK9 to the HDL-bound PCSK9 or to the total PCSK9 or the PCSK9 activity.

25. A method of treating CVD comprising administering a compound which is a statin or an inhibitor of PCSK9 to a subject, wherein the subject has previously been selected for such treatment according to the method of claim 21.

26. A method of treating CVD comprising administering a compound which is a statin or an inhibitor of PCSK9 to a subject, wherein the method comprises selecting the subject for such treatment according to a method of claim 21.

27. A compound which is a statin or an inhibitor or putative inhibitor of PCSK9 for use in a method of treating a subject diagnosed with, or believed to be at risk of, CVD, wherein the subject that has been determined to be responsive to the compound based on the level of expression of PCKS9 in the subject not bound to HDL or the proportion of the non-bound PCSK9 to the HDL-bound PCSK9 or to the total PCSK9 or the PCSK9 activity.

28. A compound which is a statin or inhibitor or putative inhibitor of PCSK9 for use in a method of treating a subject diagnosed with, or believed to be at risk of, CVD, wherein the subject has previously been selected for such treatment according to the method of claim 21.

29. A compound which is a statin or an inhibitor or putative inhibitor of PCSK9 for use in a method of treating a subject diagnosed with, or believed to be at risk of, CVD, wherein the method comprises selecting the subject for such treatment according to a method of claim 21.

30. Use of a compound which is a statin or an inhibitor of PCSK9 in the preparation of a medicament for use in a method of treating a subject diagnosed with, or believed to be at risk of, CVD, wherein the subject that has been determined to be responsive to the compound based on the level of expression of PCKS9 in the subject not bound to HDL or the proportion of the non-bound PCSK9 to the HDL-bound PCSK9 or to the total PCSK9 or the PCSK9 activity.

31. Use of a compound which is a statin or an inhibitor of PCSK9 in the preparation of a medicament for use in a method of treating a subject diagnosed with, or believed to be at risk of, CVD, wherein the subject has previously been selected for such treatment according to the method of claim 21.

32. Use of a compound which is a statin or an inhibitor of PCSK9 in the preparation of a medicament for use in a method of treating a subject diagnosed with, or believed to be at risk of, CVD, wherein the method comprises selecting the subject for such treatment according to a method of claim 21.

33. A method, compound for use, or use according to claim 15 or any one of claims 21 to 32 wherein the compound is a statin.

34. A method, compound for use, or use according to claim 15 or any one of claims 21 to 32 wherein the compound is an inhibitor of PCSK9.

35. A method, compound for use, or use according to claim 15 or any one of claims 21 to 32 wherein the compound binds directly to PCSK9, inhibiting its interaction with LDLR and/or intemalisation of LDLR and/or targeting of LDLR for lysosomal degradation.

36. A method, compound for use, or use according to claim 15 or any one of claims 21 to 32 wherein the compound is an antibody molecule.

37. A method, compound for use, or use according to claim 15 or any one of claims 21 to 32 wherein the compound is selected from Table T.

38. A method, compound for use, or use according to any one of claims 1 to 37 wherein said CVD comprises at least one of coronary atherosclerosis, dyslipidemia, type II dyslipidemia, hypercholesterolemia and myocardial infarction.

39. A kit for use in a method of any one of claims 1 to 26 which comprises: (a) means for collecting serum, plasma or full blood from the subject; and/or (b) means for specifically depleting at least HDL from the sample to remove bound PCSK9 from the sample; and/or (c) means assessing the level of PCSK9 from the depleted plasma sample; and (d) instructions for use in the method.

40. A kit as claimed in claim 39 which comprises means for specifically depleting ApoB and/or LDL from the sample.

Description

FIGURES

[0151] FIG. 1. NMR lipoprotein profiling reveals PCSK9-HDL association. NMR lipoprotein analysis was conducted in the community based prospective Bruneck cohort (year 2000 evaluation, n=668). ELISA-based measurements of circulating PCSK9 were correlated against several lipoprotein attributes including; particle number (P), lipid contents (L), phospholipids (PL), total cholesterol (C), cholesterol esters (CE), free cholesterol (FC), triglycerides (TG) and lastly, each lipid class is also represented as a percentage of total lipids (perc).

[0152] FIG. 2. Confirmation of PCSK9 association with HDL. ApoB containing lipoproteins were immunoprecipitated from plasma (n=14) and the resulting apolipoprotein profile was quantified by MRM-MS using heavy labelled peptide standards (a), alongside the quantification of PCSK9 by ELISA (b). HDL was immunodepleted from plasma using anti-human HDL specific IgY antibody columns (n=8). The apolipoprotein profile after HDL depletion was quantified by MRM-MS and compared to matched non-depleted plasma (c). The plasma concentration of PCSK9 after HDL depletion was assessed by ELISA (b). 15 ug of non-depleted plasma, HDL-depleted plasma and HDL-fraction were run by SDS-PAGE and total protein was stained to confirm efficient HDL depletion and isolation (n=4 paired representative samples) (e). The presence and enrichment of PCSK9 and APOA1 in the HDL fraction was confirmed by western blot (n=4 paired representative samples) (f).

[0153] FIG. 3. The HDL proteome as defined by high resolution mass spectrometry. HDL was isolated from 172 patients with varying CVD-related phenotypes and was analysed by both labelfree and multiplexed TMT proteomic methods. Proteins that were quantifiable in all HDL samples across the CVD cohort by label-free discovery-based MS were considered as the core HDL proteome (n=191, 66 proteins). Proteins are grouped based on Reactome pathway analysis and functionality. Relative contribution to the proteome is estimated through the use of total PSM/mW, taking into account the bias of MS-signal due to protein size. Orange=Apolipoproteins, Pink=Lipid Metabolism, other colours represent related protein clusters. Number represents total Peptide Spectrum Matches/Molecular Weight (PSM/m). Colours are obtained by conditional formatting.

[0154] FIG. 4. PCSK9 is a stable member of the HDL proteome. The coefficients of variation in HDL protein abundances, as measured by label-free mass spectrometry, were calculated across the whole cohort (a). Correlations between the MS and ELISA measurements of PCSK9 in HDL (b) and circulating versus HDL-bound PCSK9 levels (c) are represented. Linear regression analysis was used to determine strength of relationship.

[0155] FIG. 5. HDL-bound PCSK9 alteration in CVD. ELISA-based quantification of PCSK9 in plasma n=202 and HDL n=167 was conducted and the variation in PCSK9 across the multiple clinical and CVD phenotypes are shown. The variation in plasma and HDL-bound PCSK9 as a result of sex (a, b), statin use (c, d) and as a result of time over a 6-month period post-PCI (e, f) are represented. p-values reported were obtained through the non-parametric Mann-Whitney test, * p<0.05, ** p<0.001. The variation in plasma and HDL-bound PCSK9 across CVD phenotypes is also represented (g, h). The non-parametric Kruskal-Wallis test with Benjamini-Hochberg FDR correction was used; * p<0.05, * p<0.01, ** p<0.001.

[0156] FIG. 6. Characterisation of the HDL proteome and lipidome by mass spectrometry.

[0157] Proteins correlating with PCSK9 within the core HDL proteome are shown (a), protein correlations are ranked 1 to 65, representing the strength of correlation. The quantitative analysis of the HDL lipidome was conducted using the Biocrates AbsoluteIDQ p400 kits, on a high-resolution Thermo Scientific Q-Exactive HF mass spectrometer. 365 lipid species were quantified in the HDL samples across the cohort (n=149). The sum of each lipid species in a respective class was taken and a Pearson correlation matrix was generated against the HDL apolipoprotein profile, as well as PCSK9 and PLTP. A hierarchical cluster analysis was conducted upon the resulting matrix, being represented in heat map form (b).

[0158] FIG. 7. Post-prandial PCSK9 kinetics. Postprandial plasma samples were obtained from 20 individuals at 8, hourly time points. HDL was immuno-precipitated from postprandial plasma samples and label-free quantitative proteomic analysis was conducted on the isolated HDL samples and significant protein changes over the 8 hr time period are represented in a heat map (a). Two distinct clusters of protein changes emerged at at baseline and at 4 hours, and these clusters are graphically represented (b). Significance was determined using the repeated-measure one-way ANOVA test, with Benjamini Hochberg FDR correction.

[0159] FIG. 8. PCSK9 is undetectable in human LDL isolated by ultracentrifugation. The PCSK9 content of LDL and HDL from the same individuals with CVD is directly compared (n=16). 10 ug of LDL and HDL diluted 10-fold was used for the ELISA. O.D measurements for PCSK9 in LDL are below the limit of quantification (a). Standard curve O.D values are represented for reference (b).

[0160] FIG. 9. Apolipoprotein profile of plasma depleted of lipoproteins. ApoB containing lipoproteins were immunoprecipitated from plasma (n=14) and the resulting apolipoprotein profile was quantified by MRM-MS using heavy labelled peptide standards (a). HDL was immunodepleted from plasma using anti-human HDL specific IgY antibody columns (n=8). The apolipoprotein profile after HDL depletion was quantified by MRM-MS MRM-MS using heavy labelled peptide standards and compared to matched non-depleted plasma (b).

[0161] FIG. 10. PCSK9 association with lipoproteins. The level of PCSK9 upon lipoproteins was determined through the use of a modified chemiluminescent ELISA (n=20, 8 time points). PCSK9 was first captured and then antibodies specific for APOB, LP(a) and

[0162] APOA1 were used to detect relative levels of PCSK9-lipoprotein association.

[0163] FIG. 11. HDL Proteome Correlations. A Pearson correlation matrix was generated for proteins quantifiable in every HDL sample across the cohort (n=191, 66 proteins including PCSK9) and a hierarchical cluster analysis was conducted upon the resulting matrix, being represented in heat map form.

[0164] FIG. 21. HDL Proteome Reactome Analysis. The HDL proteome as defined by label-free mass spectrometry was analysed using the open-source Reactome platform (reactome.org). Pathways enriched were ranked based on p-value and the 15 most significant pathways are graphically represented.

[0165] FIG. 13. PCSK9 is significantly increased in female HDL, as measured by label-free MS. Females from the CVD cohort used in this study had a significantly increased level of PCSK9 bound to HDL, when measured by label-free MS. Significance was determined by the Mann Whitney U test, with Benjamini Hochberg correction.

[0166] FIG. 14. HDL Proteome Correlations. A Pearson correlation matrix was generated for the core HDL proteome as measured by label-free discovery-based mass spectrometry, including only proteins that were quantifiable in every sample across the cohort (n=191, 66 proteins which includes PCSK9). Proteins correlating with APOA1 (a) and Apo(a) (b) are represented, protein correlations are ranked 1 to 65, representing the strength of correlation.

[0167] FIG. 15. Characterisation of the HDL lipidome by high resolution mass spectrometry. 365 lipid species were quantified in the plasma and HDL samples of the cohort. The sum of each lipid in a respective class was taken and the percentage contribution to the plasma and HDL lipidome was calculated (a, b).

[0168] FIG. 16. Post-prandial PCSK9 kinetics. Postprandial plasma samples were obtained from 20 individuals at 8, hourly time points. The circulating PCSK9 levels were measured by ELISA, alongside clinical measurement of triacylglycerides (TAGs) (a). The relationship between the levels of circulating PCSK9 and TAGs is represented as a linear regression (b). HDL was then immuno-precipitated from postprandial plasma samples and PCSK9 content was measured by ELISA in 8 individuals at 3 time points (c). Significance was determined using the repeated measure one-way ANOVA test, with Benjamini Hochberg FDR correction.

[0169] FIG. 17. PCSK9 is enriched in small-HDL. The PCSK9 content of pooled HDL2 (1.063-1.125 g/mL) and HDL3 (1.125-1.210 g/mL) subfractions, obtained from a commercial source, was determined using an anti-PCSK9 ELISA. 10 ug of both HDL2 and HDL3 was used as the input for this assay and each subfraction was run in triplicate.

[0170] FIG. 18. PCSK9 is associated with small-HDL and ApoC3. NMR lipoprotein analysis and targeted apolipoprotein profiling was conducted in the Bruneck study (n=656). Plasma PCSK9 levels were correlated with the apolipoprotein profiles as measured by targeted MS with authentic heavy standards.

[0171] FIG. 19. HDL-bound PCSK9 modulation during the postprandial response.

[0172] (A) Postprandial plasma samples from 20 individuals at 8, hourly time points (validation cohort) were assessed for PCSK9 and HDL-TG content. (B) NMR-based lipoprotein analysis was conducted over the postprandial time course, and the particle concentration of small (S.HDL), medium (M.HDL), large (L.HDL) and extra-large (XL.HDL) HDL are shown. (C) Quantitative label-free proteomics was conducted upon HDL immuno-isolated from postprandial plasma samples (n=8, 3 times points), and significantly changing protein clusters over 8 h are represented graphically. Significance was determined using the non-parametric Friedman test with Dunn's correction, * p<0.05, ** p<0.005, p<0.0005, **** p<0.0001.

[0173] FIG. 20. HDL facilities PCSK9 uptake and multimerisation.

[0174] (A) PCSK9 and reconstituted HDL (rHDL) were co-incubated prior to HDL immuno-isolation to demonstrate the interaction between rHDL and PCSK9. PCSK9 alone was HDL immuno-isolated as a negative control. LLOD, lower limit of detection. (B) To determine whether HDL can influence PCSK9 cellular uptake, HepG2 cells were treated with His-tagged PCSK9 (5 pg/mL), rHDL or ultracentrifuge-isolated HDL (ucHDL) (25 pg/mL) or a combination of rHDL or ucHDL and HIS-tagged PCSK9 for 6 h prior to immunoblot analysis. (C) Densitometry analysis of three independent replicates using ucHDL are represented. Significance was determined using a t-test with Welch's correction, **** p<0.0001. (D) Recombinant HIS-tagged PCSK9 at a concentration of 1 ug/mL was incubated in the presence of an increasing concentration of rHDL or ucHDL for 24 h at 37° C. The control lane represents PCSK9 incubated without lipoproteins at 4° C. for 24 h. Immunoblot analysis was then conducted, with equal amounts of PCSK9 loading for each sample. Total protein stain is used to visualise ApoA1.

[0175] FIG. 21. HDL facilities PCSK9-mediated LDLR degradation.

[0176] (A) To determine the ability of HDL to effect PCSK9 action, HepG2 cells were treated with His-tagged PCSK9 (1 pg/mL), ucHDL (50 pg/mL) or a combination of ucHDL and His-tagged PCSK9 for 6 h in the presence of actinomycin D (5 pg/mL), prior to immunoblot analysis. Actinomycin D prevents the compensatory increase in LDLR upon treatment with ucHDL or rHDL without altering the PCSK9 response. (B) Densitometry analysis of three independent replicates are represented, significance was determined using a t-test with Welch's correction, ** p<0.005. (C) The same co-incubation experiment was repeated by isolating the membrane protein fraction through cell-surface biotinylation and NeutrAvidin agarose enrichment, TfR: Transferrin receptor. (D) A working model of the relationship between HDL and PCSK9 is represented. PCSK9 is lost from HDL during postprandial lipaemia, while HDL can positively modulate the uptake and multimerisation of PCSK9, resulting in the enhanced degradation of the LDLR.

EXAMPLES

Example 1—NMR Lipoprotein Profiling Identifies PCSK9 Association with HDL

[0177] NMR enables the determination of lipoprotein concentrations, alongside their respective lipid content and particle size (17,23,24). We conducted NMR lipoprotein analysis and ELISA measurement of PCSK9 in plasma samples from the community-based prospective Bruneck cohort (year 2000 evaluation, n=668) to determine the relationship between circulating PCSK9 levels and lipoprotein characteristics as measured by NMR. As expected, PCSK9 positively associated with the particle number (P) and lipid content (L) of all circulating VLDL, IDL and LDL particles (FIG. 1). However, PCSK9 revealed a surprisingly strong positive association with the particle number of S-HDL (Pearson correlation=0.26, p<0.001).

Example 2—PCSK9 is Predominantly Associated with HDL

[0178] Previous reports have suggested that PCSK9 binds to LDL (21) and Lp(a) (22).

[0179] To determine whether PCSK9 is predominantly associated with APOB-containing lipoproteins or HDL, we performed immunodepletion experiments for APOB and APOA1 from human plasma (n=14)

[0180] Immunodepletion of APOB resulted in a 99% reduction in APOB (FIG. 29). As expected, a similar depletion efficiency was observed for Lp(a), an LDL particle that carries apolipoprotein(a) as an additional protein component.

[0181] After APOB depletion, the plasma concentration of PCSK9 was reduced by less than 20%, suggesting that contrary to current assumption most circulating PCSK9 is not LDL or Lp(a)-associated (FIG. 2b). In fact, PCSK9 was below the limit of quantification by ELISA in isolated LDL from human plasma (n=16) (FIG. 8).

[0182] Next, APOA1 was immuno-depleted (n=8), again resulting in a 99% reduction of APOA1, confirming a highly efficient depletion (FIG. 2c). APOA2, another abundant HDL protein, showed a similar reduction. Notably, PCSK9 plasma levels revealed an over 90% reduction upon HDL depletion (FIG. 2d).

[0183] Full plasma apolipoprotein profiles post-lipoprotein depletions are shown in FIG. 9. Efficient HDL depletion and PCSK9 enrichment in the HDL-fraction were further confirmed by immunoblotting (FIG. 2e, ). Furthermore, the determination of PCSK9 association with lipoproteins through an independent antibody-based apolipoprotein capture method revealed a much greater abundance of PCSK9 associated with APOA1, when compared to APOB and apolipoprotein(a) (FIG. 10). Thus, PCSK9 is predominantly associated with HDL rather than LDL or Lp(a).

Example 3—the HDL Proteome in Patients with CVD

[0184] Further interrogation of the HDL proteome was conducted in 172 patients with different CVD (Table 1), namely: [0185] microvascular angina, [0186] stable coronary artery disease (CAD), [0187] myocardial infarction (MI) and [0188] stable CAD with percutaneous coronary intervention (PCI)

TABLE-US-00002 TABLE 1 Cohort Patient Characteristics for HDL isolation Microvascular PCI with p- Angina Stable-CAD MI follow up value n (% Total) 18 (10.5) 66 (38.4) 56 (32.6) 32 (18.6) Age(±SD) 57.6 ± 10.93 64.43 ± 14.65 65.45 ± 9.05 62.76 ± 9.23 0.05 Males (%)  6 (33.3) 43 (65.2) 31 (55.4) 24 (75.0) 0.06 Current Smoker (%) 1 (5.9)  9 (13.6) 16 (30.8)  4 (12.5) 0.07 History of Diabetes(%)  2 (12.5) 17 (25.8) 16 (31.3)  5 (15.6) 0.45 Statin Use 11 (61.1) 62 (93.9) 13 (30.2) 29 (93.5) <0.001

[0189] For the last subgroup, a 6-month follow-up post-PCI was included (n=32). Initially, discovery-based mass spectrometry (MS) was used to obtain a comprehensive overview of the proteome. To determine inter-protein relationships only proteins quantifiable in all HDL samples were retained. This core HDL proteome of 66 proteins includes PCSK9 and is represented in table form based on functionality (n=191, FIG. 3). A Pearson correlation matrix was generated for these 66 proteins, revealing the distinct clusters of HDL-associated proteins (FIG. 11).

[0190] Similar to APOAI, PCSK9 showed remarkable stability in abundance across HDL samples (FIG. 4a). In contrast, key drivers of the variation in the HDL proteome were inflammatory-related proteins, such as acute-phase proteins serum amyloid A1 and serum amyloid A2. The main pathways returned by Reactome analysis on the HDL proteome in CVD patients were related to lipoproteins, interferon signalling, platelet and neutrophil degranulation, fibrin clotting and complement activation (FIG. 12).

[0191] To validate the quantitative accuracy of the MS measurement, PCSK9 was measured by ELISA. MS and ELISA-based quantification of PCSK9 in HDL were highly correlated (r=0.76, n=165, FIG. 4b). A weaker correlation was observed when comparing circulating versus HDL-bound levels of PCSK9 (r=0.56, n=161, FIG. 4c). Thus, HDL-bound PCSK9 might provide additional information.

Example 4—HDL-Bound PCSK9 in Patients with CVD

[0192] Circulating PCSK9 levels are influenced by sex and statin use (25,26). In our cohort of patients with CVD, plasma PCSK9 levels were similar between males and females (FIG. 5a). HDL-bound PCSK9, however, was significantly increased in females compared to males in the label-free MS analysis (FIG. 13), and a similar trend was observed using PCSK9 immunoassays (p=0.06) (FIG. 5b). In contrast, a highly significant increase in plasma PCSK9 levels in patients taking statins (p=0.0008) was mirrored in the HDL fraction but only with borderline significance (p=0.048) (FIG. 5c, d). Among the various CVD manifestations, patients with stable CAD had the highest concentrations of PCSK9. In patients with 6-month follow-up post-PCI (n=32), PCSK9 levels in both plasma and HDL were stable over time (FIGS. 5e, f). Patients with microvascular angina had lower levels of PCSK9 on HDL compared to patients with MI and stable CAD, a difference that was not evident in plasma (FIG. 5g, h).

Example 5—PCSK9 on HDL Correlates with PLTP and Complement Factors

[0193] PCSK9 on HDL showed a strong positive association with phospholipid transfer protein (PLTP), the key protein responsible for exchanging lipids between VLDL and LDL to mature HDL (27). Other proteins that were positively correlated with PSCK9 were proteins involved in complement formation (Clusterin—CLU, complement factor 9—C9) (FIG. 6a). Lastly, strong clustering between APOB and LPA revealed the minor presence of Lp(a), a lipoprotein particle known to overlap in density with HDL(28). Proteins that strongly correlated with apolipoprotein(a) revealed an Lp(a) protein signature (FIG. 11, 14b). There was no correlation between apolipoprotein(a) and PCSK9 in the HDL proteome (FIG. 14b).

Example 6—Comparison to the HDL Lipidome in Patients with CVD

[0194] The strong positive association between PCSK9 and lipid metabolism related proteins prompted us to interrogate the associations of lipid species with PCSK9. Targeted quantitation of 365 lipid species was conducted in HDL from the entire cohort. Cholesterol esters (CE) and phosphatidylcholine (PC) were the most abundant lipid species in HDL, contributing approximately 90% of total lipid content (FIG. 15a, b). Another major constituent of the HDL lipidome were sphingomyelins (SM) (FIG. 15b).

[0195] Hierarchical cluster analysis on correlation matrices between the apolipoprotein profile and lipidome in HDL replicated the proteome-based clustering of PCSK9 with PLTP and clusterin but included apolipoprotein E (APOE) as well. When correlated with the HDL lipidome, this protein cluster revealed a strong positive association with SM (Pearson correlation=0.4, P<0.0001) (FIG. 6b).

Example 7—the Effect of Food Intake on Postprandial HDL Remodelling

[0196] Lastly, postprandial HDL remodelling was evaluated by proteomics. The test meal contained 50 g fat and 85 g carbohydrate (850 kcal, 15 g protein). A 50 g fat load has been shown to be the optimum quantity to discriminate between individual postprandial responses. A large cluster of inflammatory proteins increased in abundance upon HDL at the 4 hr time point before normalising to fasted levels (FIG. 7a, b, Cluster 2). In contrast, a postprandial reduction in HDL-bound PCSK9 was revealed by proteomics, alongside a reduction in APOA1 (FIG. 7a, b, Cluster 1).

[0197] PCSK9 measurements by immunoassays confirmed a reduction of circulating PCSK9 levels compared to the fasted state. This reduction coincided with peak postprandial lipaemia, within the first 5 hrs. Circulating PCSK9 and triglyceride levels reverted back to baseline concentrations at 8 hrs post-prandially (FIG. 16a, n=20, 8 time points). A highly significant, negative correlation was observed between the postprandial PCSK9 and triglyceride responses (r=−0.33, p=0.0001, FIG. 16b). The postprandial change in HDL-bound PCSK9 mirrored the changes observed in the circulation (FIG. 16c, n=8, 3 time points). These findings uncovered dynamic changes in PCSK9 during the postprandial remodelling of the HDL protein content.

[0198] The plasma reduction of PCSK9 was replicated in a second postprandial cohort, adhering to the same test meal (8 time points, n=20, FIG. 19A). Additionally, NMR-based lipoprotein analyses were conducted in the postprandial validation cohort and revealed triglyceride loading of HDL (FIG. 19A) as well as a significant reduction in the particle concentrations of S.HDL and M.HDL between 2-4 hours when lower PCSK9 levels were observed (FIG. 40). In contrast, L.HDL and XL.HDL remained unchanged (FIG. 190). Next, HDL was immuno-isolated over the postprandial time course at 0 h, 4 h and 8 h (n=8 each) and analysed by quantitative proteomics. Consistent with previous results, ApoA1 content of HDL was greatest at 8 h postprandially (FIG. 4C)..sup.15 In contrast, a cluster of inflammatory proteins increased in abundance upon HDL at 4 h postprandially, before returning to fasted levels. The postprandial reduction in HDL-bound PCSK9 as confirmed by proteomics was accompanied by a reduction of C apolipoproteins, including ApoC3.

Example 8—HDL Facilitates PCSK9-Mediated LDLR Degradation

[0199] Lastly, we interrogated whether HDL can alter PCSK9 function. Recombinant PCSK9 was capable of associating with rHDL in vitro (FIG. 20A). Treatment of cells with recombinant PCSK9 and either rHDL or ucHDL increased PCSK9 uptake compared to treatment with PCSK9 alone (FIG. 20B, C). In contrast, ucHDL but not rHDL was able to promote multimerisation of PCSK9 (1 μg/mL) at physiological concentrations (FIG. 50). Treatment of HepG2 cells with PCSK9 and ucHDL reduced cellular LDLR protein levels to a greater extent as compared to PCSK9 alone (FIG. 21A, B). This coincided with higher uptake and increased multimerisation of PCSK9 (FIG. 21A, B). Both total and cell membrane LDLR levels were reduced upon incubation of cells with PCSK9 and ucHDL (FIG. 21C).

[0200] The functional relationship between HDL and PCSK9 is schematically represented in FIG. 21D.

Example 9—Discussion of Examples 1-8

[0201] The data presented in this study provide the first proteomics evidence that PCSK9 is found upon HDL. Combining findings from a prospective, community-based study with findings in isolated HDL from CVD patients and in healthy volunteers during the postprandial phase, it demonstrates that the majority of circulating PCSK9 is associated with HDL, overturning the prevailing assumption that PCSK9 is bound to APOB-carrying lipoprotein particles such as LDL and Lp(a). Instead, HDL acts as reservoir of PCSK9 with higher levels in patients on statins and release during postprandial hyperlipidemia.

[0202] Plasma PCSK9 levels correlate to small HDL. The combination of NMR lipoprotein profiling alongside quantitative PCSK9 measurement in a prospective community-based cohort revealed the relationships between PCSK9 and lipoprotein subpopulations. The positive correlation of PCSK9 with the particle number and lipid content of small-HDL (S-HDL-P/L) was similar to the strength of correlation seen with VLDL and LDL. The correlation analyses also revealed a strong positive association between PCSK9 and the triglyceride content of HDL, a component of HDL recently associated with CVD-risk (17).

[0203] For validation, measurements of PCSK9 were performed in isolated small, dense HDL (HDL3) and larger, less dense HDL (HDL2), confirming an enrichment of PCSK9 within HDL3 (FIG. 21).

[0204] Next, PCSK9 measurements were correlated with plasma apolipoprotein measurements by targeted mass spectrometry (MS) (FIG. 18).sup.13. PCSK9 plasma levels showed a surprisingly strong correlation with C apolipoproteins, in particular with ApoC3, an inhibitor of lipoprotein lipase, the enzyme primarily responsible for the hydrolysis of plasma triglycerides (FIG. 18).sup.24.

[0205] PCSK9 is actually associated with HDL. Previous publications suggested that PCSK9 was circulating partly in association with LDL and Lp(a), with reports suggesting up to 40% of PCSK9 to be LDL-associated (21,22). However, in our study circulating PCSK9 levels were only reduced by <20% upon APOB and Lp(a) removal. Through the use of immuno-depletion technologies for APOA1, the major apolipoprotein of HDL, we demonstrated a striking reduction of plasma PCSK9 levels, suggesting a predominant interaction of PCSK9 with HDL rather than LDL and Lp(a). The presence of PCSK9 upon HDL was further confirmed in isolated HDL in two separate large-scale human cohorts, in which HDL was isolated either through ultracentrifugation or immuno-isolation and consistently detected by MS. In contrast, Romagnuolo et al were unable to demonstrate in vitro binding between isolated Lp(a) and PCSK9, an association that has only been observed in patients with extremely high levels of Lp(a) (22,32). Furthermore, the in vitro study first identifying PCSK9 to associate with LDL also could not detect PCSK9 in LDL isolated from normolipidemic human plasma, an observation deemed due to salt concentration and high centrifugal force causing a loss of PCSK9 from LDL during isolation (21). Our data concur with the latter interpretation as the measured content of PCSK9 upon HDL isolated through ultracentrifugation compared to that measured in immuno-isolated HDL was almost 10-fold lower (33). Lastly, human lipoprotein apheresis studies determined a 50% reduction in PCSK9 upon the removal of 77% and 89% of LDL and Lp(a) respectively, however a significant 18% reduction in HDL was also present (34,35). Our data would suggest that HDL removed during apheresis was a contributor to the PCSK9 reduction observed.

[0206] HDL as an endogenous reservoir of PCSK9. The core function of PCSK9, and the rationale for therapeutic targeting, is its downregulation of hepatic LDLR surface expression, thereby raising circulating levels of atherogenic VLDL, IDL and LDL particles (18,20,36,37). PCSK9 not only regulates the LDLR through binding to the extracellular region of this receptor but can also control LDLR degradation within the cell (38). PCSK9 has also been shown to regulate the production of triglyceride-rich lipoproteins in both the liver and intestine, through an LDLR-dependent and independent manner (39-42).

[0207] Without wishing to be bound by theory, and in light of the findings herein it could be envisaged that HDL acts as a PCSK9 reservoir in the circulation and that internalisation of HDL could deliver a pool of PCSK9 to the intracellular environment, that can then influence the pathways outlined above. Alternatively, differential PCSK9 compartmentalisation could modulate its activity.

[0208] Furthermore, unlike LDL that is thought to inhibit PCSK9 function upon the LDLR.sup.21, our data suggest, that at physiological concentrations of PCSK9 and HDL, HDL promotes the multimerisation of PCSK9 in a dose-dependent manner. The multimeric state of PCSK9 has previously been associated with its LDLR degrading capabilities, therefore a varying ratio of LDL and HDL within the human circulation could determine the activity of PCSK9 (Fan et al. supra).

[0209] Postprandial changes in PCSK9 compartmentalisation. Of the limited human postprandial studies to date that investigate the PCSK9 response, our study is the first to reveal PCSK9 to be significantly reduced in the circulation postprandially, with previous reports highlighting a trend of reduction (43,44). The change in PCSK9 was mirrored when analysing its abundance in the HDL fraction. Intriguingly, although no change in APOA1 was detected when analysing the plasma over this postprandial time course, a reduction was observed in the APOA1 content of HDL, a change mimicking that of PCSK9. The change in APOA1 content of HDL, independent of total HDL variation, would suggest a subpopulation remodelling of HDL over this postprandial period. Previously, NMR-based lipoprotein analysis of plasma over the postprandial response in humans revealed a reduction in small-HDL, versus a concomitant increase in medium-HDL particle number. It was also revealed in this study that women had a greater shift in HDL subpopulation redistribution when compared to men, a sex difference that may be apparent in our postprandial study, particularly due to the known effects of gender on circulating PCSK9 (25,45,46).

[0210] Associations of lipid and inflammatory HDL proteins with PCSK9. PCSK9 abundance strongly correlated with lipid and complement-related proteins, including PLTP and CLU respectively. PLTP has been shown to be able to directly bind PCSK9, in vitro, emphasising the validity of the positive correlation seen between the two proteins within the HDL proteome in this study (47). Besides PLTP and CLU, PCSK9 was also associated with APOE in respect to the HDL lipidome pointing towards an involvement of PCSK9 in the lipid modelling of HDL.

[0211] PCSK9 also strongly correlated with known regulators of the complement cascade within the HDL proteome (48). CLU inhibits the formation of the membrane attack complex through the interruption of C9/C5b-C8 and C5b-C7 complex formation, respectively (49). PCSK9 positively associated with C9 within the HDL proteome, further suggesting its role in the regulation of the complement cascade. The possible interaction between PCSK9 and PLTP upon HDL is intriguing in this respect due to the arising role of PCSK9 in the immune response, particularly in the clearance of pathogen (50,51). Postprandial remodelling of the HDL proteome also saw a large cluster of protein changes related to the complement cascade, highlighting an inflammatory process implicated in postprandial lipaemia that could involve PCSK9.

[0212] Conclusions. This study for the first time provides proteomic evidence that HDL is the main carrier of PCSK9 in the circulation, and that this association is dynamic during the human postprandial response.

[0213] In the light of the results presented herein it is plausible that PCSK9 released from HDL, a facilitator of PCSK9-mediated LDLR degradation, may bind ApoB-containing lipoproteins that are known to inhibit PCSK9 function, and therefore control the hepatic uptake of triglycerides in the postprandial phase.

[0214] Our study for the first time reveals that HDL is capable of modulating the LDLR-degrading capacity of PCSK9 in vitro. In an HepG2 cell system the addition of PCSK9 with HDL (isolated by ultracentrifugation) led to a greater reduction in LDLR protein levels when compared to cells treated with PCSK9 alone, in both whole cell lysates and membrane fractions (FIG. 21). This positive regulation of PCSK9 function could be attributed to the observed promotion of uptake and multimerisation of PCSK9 when HDL was present, when compared to cell treatment with PCSK9 alone (FIG. 21B).

[0215] It has previously been shown that LDL is capable of negatively regulating the LDLR-degrading capacity of PCSK9. PCSK9-driven LDLR degradation was shown to be dose-dependently inhibited by LDL. These authors reveal also that the presence of LDL with PCSK9 reduced PCSK9 uptake by the cells, in comparison to cells treated with PCSK9 alone (FIGS. 6 and 7 within the paper cited).sup.21.

[0216] In the light of the results herein, it is plausible that that there are opposing roles between HDL and LDL in the regulation of LDLR degradation by PCSK9. PCSK9 can bind both HDL and LDL within the human circulation and therefore the amount of PCSK9 within each lipoprotein fraction could dictate the overall stimulatory or inhibitory effect upon PCSK9 function.

[0217] It follows that the ratio between the amount of PCSK9 within LDL (inhibited PCSK9) and the amount of PCSK9 within HDL (active PCSK9) may be used as an overall measure of PCSK9 activity and therefore may provide an additional measure of cardiovascular risk in a given individual, particularly given the fact that total levels of PCSK9 have proved inconclusive as an independent predictor of atherosclerotic risk.sup.52. Furthermore, the use of this ratio measure of PCSK9 activity could be used to determine the predicted benefit of anti-PCSK9 therapy in a given individual and to also assess the response to therapy.

Methods for Examples 1-9

[0218] Bruneck Cohort

[0219] The Bruneck Study is a community-based, prospective survey of the epidemiology and pathogenesis of atherosclerosis and cardiovascular disease (1,2). At the 1990 baseline evaluation, the study population comprised an age- and sex-stratified random sample of all inhabitants of Bruneck (125 men and 125 women from each of the fifth through eighth decades of age, all White). In the present study, citrate plasma samples from the 2000 (n=668) follow-up were analysed. These samples were drawn after an overnight fast and 12 hours of abstinence from smoking. During the 2000 follow-up, citrate plasma was prepared by single centrifugation and aliquots were immediately stored at −80° C.

[0220] NMR

[0221] NMR-based lipoprotein profiling was conducted using the commercial Nightingale Health assay (Nightingale Health Ltd). This metabolic profiling platform enables the quantification of 14 lipoprotein subclasses defined as follows: extremely large-VLDL (>75 nm), five subclasses of VLDL (average particle diameter of 64.0 nm, 53.6 nm, 44.5 nm, 36.8 nm and 31.3 nm), intermediate density lipoprotein (IDL) (28.6 nm), three LDL subclasses (25.5 nm, 23.0 nm and 18.7 nm) and lastly four HDL subclasses (14.3 nm, 12.1 nm, 10.9 nm and 8.7 nm). The particle number of each lipoprotein subclass is quantified alongside lipid content including; phospholipids, cholesterol, free cholesterol, cholesterol esters and triglycerides. This NMR-based platform has been used previously in multiple epidemiological studies, where detailed technological information can be found(3-7).

[0222] Enzyme-Linked Immunosorbent Assay

[0223] Human PCSK9 concentrations were measured using the DuoSet ELISA Development kit (DY3888, R&D Systems) and the corresponding DuoSet Ancillary Reagent Kit 2 (R&D Systems) according to the manufacturer's instructions. Absorbance at 450 nm was measured on a plate reader (Tecan Infinite 200 Pro) using 570 nm as a reference wavelength. Concentrations were calculated using a 4-parameter logistic (4-PL) fit. Plasma was diluted 1:100, using 1 ul of plasma per sample, whereas 10 ug of HDL protein was diluted in 100 ul reagent diluent for this assay.

[0224] Lipoprotein-associated PCSK9 was measured using an in-house sandwich ELISA as previously described(8). Briefly, microtiter 96-well plates were coated overnight at 4° C. with alirocumab (5 mg/mL at 40 mL/well). Excess material was washed off and the plates blocked with 1% tris-buffered saline/bovine serum albumin for 45 minutes. EDTA plasma was added at 1:50 dilution (40 mL/well) for 75 minutes to allow alirocumab to bind PCSK9. This dilution of plasma provided conditions, whereby a saturating and equal amount of PCSK9 was captured in each well. To detect apoB-100, Lp(a), or apoAI bound to PCSK9 (PCSK9-apoB, PCSK9-Lp(a), and PCSK9-apoAI, respectively) biotinylated goat antihuman apoB-100 antibody (Academy Biomedical Co, Houston, Tex.) at 1 mg/mL, biotinylated murine monoclonal antibody LPA4 at 1 mg/mL or biotinylated goat anti-human APOA1 at 0.8 ng/mL, respectively, were added. Alkaline phosphatase-conjugated to NeutrAvidin (Thermo Scientific, Waltham, Mass.) was added for 60 minutes. Lumi-Phos 530 (Lumigen, Inc, Southfield, Mich.) (25 mL/well) was added for 75 minutes and luminescence read on a Dynex luminometer (Chantilly Technologies, Chantilly, Va.). The results are reported as RLU in 100 ms after subtraction of background RLU (tris-buffered saline/bovine serum albumin blank).

[0225] APOB Depletion (Liposep)

[0226] APOB containing lipoproteins were depleted from plasma using the Liposep APOB-specific immunoprecipitation reagent according to the manufacturer's instructions (Sun Diagnostics, New Gloucester, Me., USA). Plasma and immunoprecipitation reagent were mixed at a 1:1 ratio and incubated at room temperature for 10 minutes, with occasional vortex mixing. Samples were then centrifuged at 10,000×g for 10 minutes and the APOB depleted supernatant was taken, without disturbing the pellet, with aliquots being immediately stored at −80° C.

[0227] HDL-Immunodepletion

[0228] HDL was immuno-depleted from plasma using human HDL-specific IgY affinity columns according to manufacturer's instructions (Genway Biotech, San Diego, Calif., USA). Briefly, 40 ul of plasma was diluted 10-fold in 360 ul TBS buffer (10 mM Tris, 150 mM NaCl, pH 7.4). Diluted plasma was then added to TBS equilibrated antibody beads and incubated at room temperature with end over end rotation for 15 minutes. Flow through, HDL-depleted plasma, was then collected through centrifugation at 500×g. The removal of non-specifically bound proteins from the antibody beads was achieved using 500 ul of wash buffer (TBS, 0.05% Tween-20) a total of 3 times. HDL was then stripped from the antibody beads by the addition of 500 ul stripping buffer (0.1M Glycine, pH 2.5), twice. The antibody columns were then regenerated using a series of stripping buffer wash steps, followed by the addition of neutralisation buffer (100 mM Tris-HCl, pH 8.0) and lastly the resuspension in 500 ul TBS containing 0.02% sodium azide for storage. Isolated HDL samples were further concentrated, due to the large isolation volume and stored at −80° C. until further processing.

[0229] MRM-based Proteomics

[0230] Targeted quantitation of plasma proteins was conducted using the commercially available Plasma Dive kit (Biognosys) (9). Plasma samples were processed according to the manufacturer's instructions. Briefly, 10 ul of plasma was denatured and reduced in 90 ul of denature buffer and alkylated by the addition of 16 ul alkylation solution. 3 ul of alkylated protein (approximately 20 ug) were spiked with authentic heavy peptide standards (the peptide standard for apoB-100 did not overlap with the proximal portion of apoB that would include both apoB-48 and apoB-100). An in-solution tryptic digestion (Pierce Porcine Tryspin, enzyme:protein=1:50, Thermo Fisher Scientific) was performed overnight at 37° C. with shaking. Digestion was stopped by the addition of 10 ul, 10% TFA. After solid-phase extraction with C18 cartridges (Bravo AssayMAP, Agilent Technologies), the eluted peptides were dried using a SpeedVac (Thermo Fisher Scientific, Woburn, Mass.) and resuspended in 40 ul of liquid chromatography solution.

[0231] The samples were analysed on an Agilent 1290 Infinity II liquid chromatography system (Agilent Technologies, Santa Clara, Calif.) interfaced to an Agilent 6495 Triple Quadrupole MS (Agilent Technologies). Both instruments were controlled by MassHunter Workstation software (version B.08.00). The samples (10 ul) were directly injected onto a 25-cm column (AdvanceBio Peptide Mapping, C18, 2.1 mm×250 mm, 2.7 um, 120 Å, Agilent Technologies) and separated over a 23-minute gradient at 350 ul/min. Data files were analysed using SpectroDive 8 (Biognosys). Every peak integration was manually checked. Q-value <0.01 (FDR <1%) was used. The absolute concentration was calculated using Light/Heavy peptide signal intensity and known heavy peptide concentration.

[0232] Immunoblotting

[0233] Laemmli sample buffer (4×) (62.5 mM Tris-HCL, pH 6.8, 10% glycerol, 1% SDS, 0.005% bromophenol blue and 10% 2-mercaptoethanol) or without 2-mercaptoethanol was mixed with protein samples and boiled at 95° C. for 10 minutes. Protein samples were separated using 4-12% bis-tris gradient gels (Thermo scientific) in MOPS SDS running buffer (Thermo Scientific) at 130V for 90 minutes. Gels were either stained for total protein using SimplyBlue Safe Stain (Thermo Fisher) or proteins were transferred onto nitrocellulose membranes in ice-cold transfer buffer (25 mM tris-base pH 8.3; 192 mM glycine; 20% methanol) at 350 mA for 2 hours. Ponceau S red staining was used to determine efficient transfer and equal loading before membranes were blocked in 5% fat-free milk powder in phosphate-buffered saline (PBS) containing 0.1% Tween-20 (PBST)(Sigma). Membranes were incubated in primary antibodies (Table 1) made to appropriate concentrations in 5% BSA in PBST overnight at 4° C. The membranes were then incubated in the appropriate light-chain specific peroxidase-conjugated secondary antibody (Table I) in 5% milk/PBST. Membranes were then washed for three times in PBST for 15 minutes. Western blots were developed using enhanced chemiluminescence (ECL) (GE Healthcare) on photographic films (GE Healthcare). Densitometry analysis was done using the ImageJ analysis software.

[0234] Patient Information HDL CVD Cohort

[0235] From 2006 to 2007, blood samples were prospectively taken from consecutive patients ≥18 years of age presenting either with acute myocardial infarction (MI, ST-segment elevation myocardial infarction or non-ST-segment elevation or with stable coronary artery disease (sCAD) at the Clinic of Cardiology, West German Heart Center, University Hospital Essen (patients with MI and with sCAD) and the Alfried Krupp Hospital Essen (patients with MI).

[0236] Blood Collection and Plasma Preparation

[0237] In the MI group, blood sampling was performed during percutaneous coronary artery intervention for the treatment of the myocardial infarction as soon as the patient was clinically stabilized via the inserted arterial sheath or via an inserted venous catheter. In the sCAD group, study samples were collected during a routine blood sampling from peripheral veins. If a percutaneous coronary angiography had been performed in this study group for disease evaluation, the blood collection was undertaken on the day following the angiography to rule out an acute phase reaction upon vascular manipulation. In all groups, 30 ml of blood was drawn into vacuum tubes containing 1.6 mg EDTA/mL (4.298 mM EDTA/L). Immediately after blood drawing, the vacuum tubes were placed on ice and stored at 4° C. until further processing. Plasma was generated by centrifugation (3000 rpm, 30 minutes, 4° C.), immediately recovered and frozen at −80° C.

[0238] Ultracentrifugation-Based Isolation of High-Density Lipoprotein

[0239] All experimental procedures were performed by an investigator blinded to patients' data. High-density lipoproteins were isolated by sequential density gradient ultracentrifugation according to their density (1.069-1.21 g/mL), following an established protocol(10,11). Protein concentration was determined in each sample by Bradford assay (Bio-Rad, USA).

[0240] Postprandial Study Information

[0241] Postprandial samples were analysed from a double-blinded, 3-armed, randomised controlled trial (trial registration; clinicaltrials.gov NCT03191513; approved by King's College London Research Ethics Committee (HR-16/17-4397)) in healthy adults (n=20; 10 men, 10 women) aged 58 (SD 6.4) years. Samples were selected following consumption of the control test meal only containing 50 g rapeseed oil (61% 18:1n-9cis; 19% 18:2n-6cis) fed in the form of a muffin and a milkshake (to deliver 897 kcal, 50 g fat, 18 g protein, 88 g carbohydrate), following an overnight fast, a 50 g fat load has been shown to be the optimum quantity to discriminate between individual postprandial responses. Venous blood samples were collected at hourly intervals 0-8 h postprandially for analysis of plasma. Triacylglycerol (TAG) concentrations were measured on a Siemens ADVIA 1800 using the ADVIA chemistry TG method based on the Fossati three-step enzymatic reaction with a Trinder endpoint. A second, postprandial validation cohort was assessed (n=20, 8 time points), adhering to the same study design and test meal outlined above and has been previously published..sup.10 Ethical approval for the study (ISRCTN20774126) was obtained from the relevant research ethics committees in the United Kingdom (NREC 08/H1101/122) and the Netherlands (MEC 09-3-009), and written informed consent was given by participants.

[0242] In-Solution Protein Digestion

[0243] HDL and Plasma samples were denatured by the addition of a final concentration of 6M urea and 2M thiourea and reduced by the addition of a final concentration of 10 mM DTT followed by incubation at 37° C. for 1 hour, 240 rpm. The samples were then cooled down to room temperature before being alkylated by the addition of a final concentration of 50 mM iodoacetamide followed by incubation in the dark for 30 minutes. Pre-chilled (−20° C.) acetone (10× volume) was used to precipitate the samples overnight at −20° C. Samples were centrifuged at 14000×g for 40 minutes at 4° C. and the supernatant subsequently discarded. Protein pellets were dried using a speed vac (Thermo Scientific, Savant SPD131DDA), resuspended in 0.1M TEAB buffer, pH 8.0, containing 0.02% ProteaseMax surfactant and mass spectrometry grade Trypsin/Lys-C (Promega Cooperation) (1:25 enzyme: protein) and digested overnight at 37° C., 240 rpm. Digestion was stopped by acidification with trifluoroacetic acid (TFA). Peptide samples were then purified by solid-phase extraction with C18 cartridges (Bravo AssayMAP, Agilent Technologies).

[0244] LC-MS/MS Analysis

[0245] The dried peptide samples for label free were reconstituted with 0.05% TFA in 2% ACN and separated by a nanoflow LC system (Dionex UltiMate 3000 RSLC nano). Samples were injected onto a nano-trap column (Acclaim® PepMap100 C18 Trap, 5 mm×300 um, 5 um, 100 Å), at a flow rate of 25 uL/min for 3 minutes, using 0.1% FA in H.sub.2O. The following nano-LC gradient was then run at 0.25 uL/min to separate the peptides: 0-10 min, 4-10% B; 10-75 min, 10-30% B; 75-80 min, 30-40% B; 80-85 min, 40-99% B; 85-89.8 min, 99% B; 89.8-90 min, 99-4% B; 90-120 min, 4% B; where A=0.1% FA in H.sub.2O, and B=80% ACN, 0.1% FA in H.sub.2O. The nano column (EASY-Spray PepMap® RSLC C18, 2 pm 100 Å, 75 um×50 cm), set at 40° C. was connected to an EASY-Spray ion source (Thermo Scientific). Spectra were collected from an Orbitrap mass analyser (Orbitrap Fusion™ Lumos Tribrid, Thermo Scientific) using full MS mode (resolution of 120,000 at 400 m/z) over the mass-to-charge (m/z) range 375-1500. Data-dependent MS2 scan was performed using Quadrupole isolation in Top Speed mode using CID activation and ion trap detection in each full MS scan with dynamic exclusion enabled.

[0246] MS Database Search and Analysis

[0247] Thermo Scientific Proteome Discoverer software (version 2.2.0.388) was used to search raw data files against the human database, (UniProtKB/Swiss-Prot version 2018_02, 20,400 protein entries) using Mascot (version 2.6.0, Matrix Science). The mass tolerance was set at 10 ppm for precursor ions and 0.8 Da for fragment ions. Trypsin was used as the digestion enzyme with up to two missed cleavages being allowed.

[0248] Carbamidomethylation of cysteines and oxidation of methionine residues were chosen as fixed and variable modifications, respectively. MS/MS-based peptide and protein identifications were validated with the following filters, a peptide probability of greater than 95.0% (as specified by the Peptide Prophet algorithm), a protein probability of greater than 99.0%, and at least two unique peptides per protein. Data was normalized to the total peptide amount to take into account variation in abundances between samples.

[0249] Biocrates Lipidomics

[0250] Lipidomics analysis was conducted using Biocrates AbsoluteIDQ p400 (Biocrates Life Sciences AG, Innsbruck, Austria) kits according to manufacturer's instructions. 10 ul of internal standard (ISTD) was added to each well of the kit plate, followed by 10 ul of sample, QC or blank mixture into their respective wells. The plate was then dried using a Positive Pressure-96 Processor (Waters) for 30 minutes before the addition of 50 ul phenylidothiocyanate (PITC) derivatization solution (5% PITC, 31.7% ethanol, 31.7% pyridine, in H.sub.2O) and was allowed to incubate at room temperature for 25 minutes. The plate was again dried using a pressure manifold and 300 ul of extraction buffer (5 mM ammonium acetate in methanol) was added to each well and incubated at room temperature, 450 rpm for 30 minutes. Lipid extracts were then collected by centrifugation, 500× g for 2 minutes. Extracts were then diluted in supplied FIA solvent and stored for no longer than overnight at 4° C. before analysis. Plasma and HDL lipid extracts were run by flow injection analysis (FIA), utilising the high resolution, accurate mass of a Q Exactive-Orbitrap MS coupled to a Vanquish Flex UHPLC system (Thermo Fisher), according to the manufacturer's specifications. Raw data was processed using the supplied MetIDQ software. Only Lipids that had a concentration greater than that of the limit of quantification were taken forward for analysis.

[0251] HepG2 Cell Culture

[0252] The human liver hepatocellular carcinoma cell line, HepG2 (ECACC 85011430), was used as in in vitro model of cellular cholesterol metabolism. Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM, Thermo Fisher Scientific) supplemented with 10% heat-inactivated foetal bovine serum (FBS), 2 mM L-glutamine and 1% penicillin/streptomycin (100 U/mL penicillin and 100 pg/mL streptomycin), at 37° C. in a humidified atmosphere of 95% air/5% CO.sub.2.

[0253] HepG2 Cell Treatments and Protein Isolation

[0254] For PCSK9 studies cells were seeded in 6-well plates at a density of 3×10.sup.5 per well, and the next day media was changed to DMEM containing 10% lipoprotein deficient serum (LPDS, Merck). 24 h later media was changed and supplemented with stated concentrations of PCSK9 (ACRO Biosystems, PC9-H5223), reconstituted HDL (rHDL, Genway), ultracentrifuge-isolated HDL (ucHDL, Merck), after a prior pre-incubation at 37° C. for 1 h to promote PCSK9-HDL interaction, and Actinomycin D (Sigma, A9415) for 6 h. Cellular proteins were isolated by the following; cells were washed twice in ice cold PBS to eliminate secreted protein contamination before the addition of cell lysis buffer (25 mM Tris-HCL, 110 mM NaCl, 2 mM EGTA, 5 mM EDTA, 1% Triton and 0.5% SDS) supplemented with protease inhibitor cocktail (Roche), at pH 7.4. Cells were detached through scraping in cell lysis buffer and full lysis achieved by sonication and lysates were incubated on ice for 30 minutes. Cellular debris was then pelleted by centrifugation, 10,000×g, for 10 minutes at 4° C. Protein concentration was measured using the BCA protein assay kit (Thermo Fisher).

[0255] Cell Surface Protein Isolation

[0256] Cell surface proteins were isolated using the Pierce membrane protein isolation kit (Thermo Fisher) according to the manufacturer's instructions. Cells were washed twice with ice-cold PBS before incubation with Sulfo-NHS-SS-Biotin dissolved in PBS (0.25 mg/mL) on an orbital shaker for 30 minutes at 4° C. Membrane protein labelling was stopped using provided quenching solution and cells were scraped and centrifuged at 500×g for 1 minute and resulting pellets were washed twice with ice-cold PBS. Cells were lysed in lysis buffer supplemented with protease inhibitor (Complete mini-EDTA free Protease inhibitor cocktail, Roche) and proteins were solubilised through sonication; clarified lysates were then incubated with NeutrAvidin agarose for 60 minutes with end-over-end rocking. Membrane proteins were eluted from the NeutrAvidin beads through the incubation with cell lysis buffer containing 50 mM DTT.

[0257] Statistics

[0258] Proteomic and Lipidomic datasets were initially filtered to keep only molecules with less than 50% missing values. The remaining missing values were imputed using KNN-Impute method with k equal to the minimum value of 10 and the minimum samples assigned to each of the examined phenotypes (12). The relative quantities of the quantified molecules were further scaled using log 10 transformation.

[0259] All statistical comparisons have been conducted using non-parametric tests. Mann-Whitney U test was used for comparisons between two phenotypes and Kruskal Wallis test for comparisons between more than two phenotypes(13,14). P-values were adjusted using Benjamini Hochberg adjustment for multiple testing keeping proteins with false discovery rate threshold of 5%(15).

[0260] Pearson correlation, hierarchical cluster analysis and visualisation was conducted in the open-source software Perseus(16). All other data visualisations were created in Graphpad Prism (Version, 7.00, GraphPad Software, La Jolla Calif. USA). All reactions were carried out in 96-well plate format when possible, and liquid handling was performed using a Bravo AssayMAP robot (Agilent Technologies, Santa Clara, Calif., USA).

REFERENCES FOR METHODS ONLY

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TABLE-US-00003 TABLE I Target Host Company, Catalogue Protein Species Application Number PCSK9 Sheep Immunoblotting (1:1000) R&D Systems, AF3888 APOA1 Rabbit Immunoblotting (1:1000) Abcam, ab52945 HRP-anti- Donkey Immunoblotting (1:1000) R&D Systems, HAF016 Sheep HRP-anti- Mouse Immunoblotting (1:5000) Jackson Immuno Rabbit Research, 211032171

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