A Quantitative Immunoassay for the Lysine-Binding Function of Lipoprotein(a): Application to Recombinant Apo(a) and Lipoprotein(a) in Plasma

• Published in: Arteriosclerosis, thrombosis, and vascular biology Vol. 16; no. 5; pp. 656 - 664
• Main Authors: Hoover-Plow, Jane L, Boonmark, Nataya, Skocir, Pamela, Lawn, Richard, Plow, Edward F
• Format: Journal Article
• Language: English
• Published: Philadelphia, PA American Heart Association, Inc 01-05-1996; Hagerstown, MD Lippincott
• Subjects: Amino Acid Sequence; Apolipoproteins A - blood; Apolipoproteins A - genetics; Base Sequence; Binding Sites; Biological and medical sciences; Blood coagulation. Blood cells; Fundamental and applied biological sciences. Psychology; General aspects, investigation methods, hemostasis, fibrinolysis; Humans; Immune Sera; Immunoassay; Lipoprotein(a) - blood; Lipoprotein(a) - isolation & purification; Lipoprotein(a) - metabolism; Lysine - metabolism; Molecular and cellular biology; Molecular Sequence Data; Mutagenesis, Site-Directed; Oligonucleotide Probes - genetics; Recombinant Proteins; Human; Transfection; Lysine; Binding site; Lipoprotein; Apolipoproteins; In vitro
• ISSN: 1079-5642; 1524-4636
• DOI: 10.1161/01.ATV.16.5.656

Apo(a), the unique apoprotein of lipoprotein(a) (Lp[a]), can express lysine-binding site(s) (LBS). However, the LBS activity of Lp(a) is variable, and this heterogeneity may influence its pathogenetic properties. An LBS-Lp(a) immunoassay has been developed to quantitatively assess the LBS function of Lp(a). Lp(a) within a sample is captured with an immobilized monoclonal antibody specific for apo(a), and the captured Lp(a) is reacted with an antibody specific for functional LBS. The binding of this LBS-specific antibody is then quantified by using an alkaline phosphatase-conjugated disclosing antibody. The critical LBS-specific antibody was raised to kringle 4 of plasminogen. When applied to plasma samples, the LBS activity of Lp(a) ranged from 0% to 100% of an isolated reference Lp(a); the signal corresponded to the percent retention of Lp(a) on a lysine-Sepharose column but did not correlate well with total Lp(a) levels in plasma. Mutation of residues in the putative LBS in the carboxy-terminal kringle 4 repeat (K4-37) in an eight-kringle apo(a) construct resulted in marked but not complete loss of activity in the LBS-Lp(a) immunoassay. These data suggest that this kringle is the major but not the sole source of LBS activity in apo(a). The LBS-Lp(a) immunoassay should prove to be a useful tool in establishing the role of the LBS in the pathogenicity of Lp(a). (Arterioscler Thromb Vasc Biol. 1996;16:656-664.)