title: Removal of plasma mature and furin-cleaved proprotein convertase subtilisin/kexin 9 by low-density lipoprotein-apheresis in familial hypercholesterolemia: development and application of a new assay for PCSK9.
abstract: BACKGROUND
  Proprotein convertase subtilisin/kexin 9 (PCSK9) is known to be a good target to decrease LDL cholesterol (LDL-C) and two forms of PCSK9, mature and furin-cleaved PCSK9, circulate in blood. However, it has not been clarified whether and how the levels of each PCSK9 are affected by LDL-apheresis (LDL-A) treatment, a standard therapy in patients with severe forms of familial hypercholesterolemia (FH).
  
  
  OBJECTIVE
  Our objective was to investigate the differences in LDL-A-induced reduction of mature and furin-cleaved PCSK9 between homozygous and heterozygous FH, and between dextran sulfate (DS) cellulose adsorption and double membrane (DM) columns and to clarify the mechanism of their removal.
  
  
  METHODS
  A sandwich ELISA to measure two forms of PCSK9s using monoclonal antibodies was developed. Using the ELISA, PCSK9 levels were quantified before and after LDL-A with DS columns in 7 homozygous and 11 heterozygous FH patients. A crossover study between the two column types was performed. The profiles of PCSK9s were analyzed after fractionation by gel filtration chromatography. Immunoprecipitation of apolipoprotein B (apoB) in FH plasma was performed.
  
  
  RESULTS
  Both mature and furin-cleaved PCSK9s were significantly decreased by 55-56% in FH homozygotes after a single LDL-A treatment with DS columns, and by 46-48% or 48-56% in FH heterozygotes after treatment with DS or DM columns. The reduction ratios of LDL-C were strongly correlated with that of PCSK9 in both FH homozygotes and heterozygotes. In addition, more than 80% of plasma PCSK9s were in the apoB-deficient fraction and a significant portion of mature PCSK9 was bound to apoB, as shown by immunoprecipitation.
  
  
  CONCLUSIONS
  Both mature and furin-cleaved PCSK9s were removed by LDL-A in homozygous and heterozygous FH either by binding to apoB or by other mechanisms. The ELISA method to measure both forms of plasma PCSK9 would be useful for investigating physiological or pathological roles of PCSK9.
authors.affiliation: Department of Molecular Innovation in Lipidology (M.H., Y.Y., M.H-S.), National Cerebral and Cardiovascular Center Research Institute, 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan; Advanced Medical Technology and Development Division (M.I., T.K., H.H.), BML, Inc., 1361-1 Matoba, Kawagoe, Saitama 350-1101, Japan; Department of Endocrinology and Metabolism (H.M., T.T., I.K.), National Cerebral and Cardiovascular Center, 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan; and Department of Cardiology (K.Y.), Kenporen Osaka Central Hospital, Umeda 3-3-30, Kita-ku, Osaka 530-0001, Japan.
authors.forename: Mika
authors.initials: M
authors.lastname: Hori
authors.forename: Mitsuaki
authors.initials: M
authors.lastname: Ishihara
authors.forename: Yumiko
authors.initials: Y
authors.lastname: Yuasa
authors.forename: Hisashi
authors.initials: H
authors.lastname: Makino
authors.forename: Koji
authors.initials: K
authors.lastname: Yanagi
authors.forename: Tamiko
authors.initials: T
authors.lastname: Tamanaha
authors.forename: Ichiro
authors.initials: I
authors.lastname: Kishimoto
authors.forename: Takeshi
authors.initials: T
authors.lastname: Kujiraoka
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Hattori
authors.forename: Mariko
authors.initials: M
authors.lastname: Harada-Shiba
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001055:Apolipoproteins B / Q000097:blood; D001781:Blood Component Removal / Q000379:methods*; D008078:Cholesterol, LDL / Q000097:blood; D018592:Cross-Over Studies; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D006801:Humans; D006938:Hyperlipoproteinemia Type II / Q000097:blood / Q000628:therapy*; D008297:Male; D008875:Middle Aged; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases / Q000032:analysis / Q000097:blood*; D012697:Serine Endopeptidases / Q000032:analysis / Q000097:blood*; D055815:Young Adult
chemical_list: D001055:Apolipoproteins B; D008078:Cholesterol, LDL; C472125:PCSK9 protein, human; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases; D012697:Serine Endopeptidases