Abstract 433: Examination of Factors Affecting the Association of PCSK9 With Low-Density Lipoprotein Particles in Human Plasma
Rationale: We have previously shown that a substantial proportion of plasma PCSK9 (30-40%) is associated with LDL particles in normolipidemic subjects. Cellular assays show that LDL-bound PCSK9 is less active for binding to cell surface LDLRs. Therefore, the ability of circulating PCSK9 to direct LDLR degradation in liver could be regulated by plasma LDL levels. In addition, LDL subspecies may have altered abilities in binding PCSK9. We have mapped the LDL binding region to a short stretch of amino acids (aa 31-52) in the PCSK9 prodomain. It is unknown whether a common loss-of-function PCSK9 mutation (R46L) within this region affects LDL binding.
Objective: To determine whether plasma PCSK9 distribution (LDL-bound versus unbound) is affected in hypercholesterolemic subjects. To further characterize the interaction of PCSK9 and LDL, we investigated the interaction of PCSK9 with two subspecies of LDL - large, buoyant LDL (LBLDL; d=1.019-1.044 g/ml) and small, dense LDL (SDLDL; d=1.044-1.063 g/ml). Additionally, we investigated the effect of the R46L PCSK9 mutation on the LDL binding affinity of PCSK9.
Methods and Results: We used flotation ultracentrifugation in Optiprep density gradients to fractionate human plasma samples followed by immunoprecipitation and western blot to quantify PCSK9 distribution in LDL and non-LDL fractions. In a pilot study, the proportion of total plasma PCSK9 in the LDL fraction was increased from 38±5% to 57±3% (N=6) in hypercholesterolemic subjects (LDL>4.9mM, TG<2.3mM) versus normal controls (LDL<3 mM, TG<2.3mM). Saturation binding assays showed that SDLDL bound PCSK9 with lower affinity (Kd = 361.9 nM) than LDLDL (Kd = 263.9 nM). Competition binding assays determined that recombinant purified PCSK9-R46L secreted from HEK293 cells did not bind to isolated LDL with significantly altered affinity compared to wild-type PCSK9.
Conclusion: Our preliminary results indicate that plasma PCSK9 distribution is altered in hypercholesterolemia, with an increased proportion of total PCSK9 bound to LDL particles. Our in vitro results suggest that circulating small, dense LDL may bind more poorly to PCSK9 than larger LDL subspecies.
Author Disclosures: M. Golder: None. S. Sarkar: None. T. Kosenko: None. R. McPherson: None. T.A. Lagace: None.
- © 2014 by American Heart Association, Inc.