PCSK9 Association With Lipoprotein(a)

Abstract

Rationale: Lipoprotein(a) [Lp(a)] is a highly atherogenic low-density lipoprotein-like particle characterized by the presence of apoprotein(a) [apo(a)] bound to apolipoprotein B. Proprotein convertase subtilisin/kexin type 9 (PCSK9) selectively binds low-density lipoprotein; we hypothesized that it can also be associated with Lp(a) in plasma.

Objective: Characterize the association of PCSK9 and Lp(a) in 39 subjects with high Lp(a) levels (range 39-320 mg/dL) and in transgenic mice expressing either human apo(a) only or human Lp(a) (via coexpression of human apo(a) and human apolipoprotein B).

Methods and results: We show that PCSK9 is physically associated with Lp(a) in vivo using 3 different approaches: (1) analysis of Lp(a) fractions isolated by ultracentrifugation; (2) immunoprecipitation of plasma using antibodies to PCSK9 and immunodetection of apo(a); (3) ELISA quantification of Lp(a)-associated PCSK9. Plasma PCSK9 levels correlated with Lp(a) levels, but not with the number of kringle IV-2 repeats. PCSK9 did not bind to apo(a) only, and the association of PCSK9 with Lp(a) was not affected by the loss of the apo(a) region responsible for binding oxidized phospholipids. Preferential association of PCSK9 with Lp(a) versus low-density lipoprotein (1.7-fold increase) was seen in subjects with high Lp(a) and normal low-density lipoprotein. Finally, Lp(a)-associated PCSK9 levels directly correlated with plasma Lp(a) levels but not with total plasma PCSK9 levels.

Conclusions: Our results show, for the first time, that plasma PCSK9 is found in association with Lp(a) particles in humans with high Lp(a) levels and in mice carrying human Lp(a). Lp(a)-bound PCSK9 may be pursued as a biomarker for cardiovascular risk.

Keywords: lipids and lipoprotein metabolism; lipoprotein(a); low-density lipoprotein; proprotein convertase subtilisin/Kexin type 9.

Figure 1. Characterization of PCSK9 association with Lp(a)

(A) lipoprotein isolation via iodixanol-based gradient ultracentrifugation. (B) agarose gel electrophoresis of LDL and Lp(a) fractions isolated as described in A, followed by neutral lipid staining. (C) Reducing gel immunoblot of LDL and Lp(a) fractions isolated as described in A. (D) Non-reducing PAGE of LDL and Lp(a) fractions isolated as described in A, followed by Coomassie protein staining (upper panel) and immunoblot (lower panel). (E) Immuno-precipitation of apo(a) using anti-PCSK9 antibody from plasma of subjects with one (left panel) or two (right panel) visible apo(a) isoforms. (F) Immuno-precipitation of apo(a) (upper panel) and apoB (lower panel) using anti-PCSK9 antibody from plasma of Lp(a) (left panel), Lp(a)-LBS mutant (middle panel), and apo(a) transgenic mice (right panel).

Figure 2. Quantitative assessments of PCSK9 levels and correlations in subject with high Lp(a) levels

(A) Linear regression analysis of LDL-C and PCSK9 levels, adjusted to age, sex, and interaction with lipid-lowering medications. Blue regression analysis (r0, p0) = subjects without lipid-lowering medication, red regression analysis (r1, p1) = subjects on lipid-lowering medication. (B) Linear regression analysis of Lp(a) and PCSK9 levels, adjusted to age and sex. (C) Linear regression analysis of PCSK9 levels and the average number of kringle IV-2 repeats, adjusted to age and sex. (D) Linear regression analysis of LDL-C and apoB levels, adjusted to age and sex. (E) Linear regression analysis of LDL-C and Lp(a) levels, adjusted to age, sex, and interaction with lipid-lowering medications. Blue regression analysis (r0, p0) = without lipid-lowering medication, red regression analysis (r1, p1) = on lipid-lowering medication. For all panels, blue = subjects without lipid-lowering medication, red = subjects on lipid-lowering medication, black regression lines represents the combined regression of both blue and red dots and indicate no interaction with lipid-lowering medications.

Figure 3. Association of PCSK9 with Lp(a) in subjects with high Lp(a) levels

(A) Illustration of the ELISA method used to detect Lp(a)-bound PCSK9. (B) Linear regression analysis of levels of Lp(a) and Lp(a)-bound PCSK9, adjusted for age and sex. (C) Linear regression analysis of levels of PCSK9 and Lp(a)-bound PCSK9, adjusted for age and sex. (D) Illustration of the ELISA method used to detect apoB-associated PCSK9 in LDL and Lp(a) fractions. (E) Fold increase in levels of Lp(a)-bound PCSK9 over LDL-bound PCSK9, measured as apoB-bound PCSK9 (ELISA) and normalized for apoB level in each fraction. (F) Percentage of apoB-associated PCSK9 in plasma of subjects with high Lp(a) levels vs. those with low Lp(a) levels (groups matched for plasma LDL-C levels). All panels: blue = subjects without lipid-lowing medication, red = subjects on lipid-lowering medication, black regression lines represent the combined regression of both blue and red dots and indicate no interaction with lipid-lowering medications.