The Solution Phase Interaction between Apolipoprotein(a) and Plasminogen Inhibits the Binding of Plasminogen to a Plasmin-Modified Fibrinogen Surface

Abstract

In the present study, we assessed the binding of recombinant forms of apolipoprotein(a) [r-apo(a)] to plasminogen. Apo(a)-plasminogen interactions were demonstrated to be lysine-dependent, as they were abolished by the addition of epsilon-aminocaproic acid. Binding of r-apo(a) and plasma-derived Lp(a) to Glu-plasminogen was assessed in solution using a mutant form of recombinant plasminogen [Plg(S741C)] labeled at the active site with 5'-(iodoacetamido)fluorescein. High-affinity binding of apo(a) to plasminogen was observed with the 17-kringle r-apo(a) (Kd = 20.1 +/- 3.3 nM) as well as with plasma-derived Lp(a) (Kd = 5.58 +/- 0.08 nM). Binding studies using various truncated and mutant forms of r-apo(a) demonstrated that sequences within apo(a) kringle IV types 2-9 and the strong lysine binding site (LBS) in apo(a) kringle IV type 10 are not required for high-affinity binding to plasminogen. In all cases, the binding stoichiometry for the apo(a)-plasminogen interaction was determined to be 1:1. Binding data obtained using a 17-kringle r-apo(a) derivative lacking the protease-like domain (17KDeltaP; Kd = 3158 +/- 138 nM) indicate that sequences within the protease-like domain of apo(a) mediate its interaction with LBS in plasminogen. We determined that r-apo(a) and plasminogen bind to distinct sites on plasmin-modified fibrinogen with the concentration of plasminogen binding sites exceeding the concentration of r-apo(a) sites by a factor of 10. Furthermore, r-apo(a) is capable of inhibiting the binding of plasminogen to plasmin-modified fibrinogen surfaces, an effect which we show is attributable to the formation of a solution phase apo(a)/plasminogen complex which exhibits a greatly reduced affinity for plasminogen binding sites on plasmin-modified fibrinogen. The results of this study provide new insights into the mechanism by which apo(a) and Lp(a) may inhibit fibrinolysis, thus contributing to the atherothrombotic risk associated with this lipoprotein.

Lipoprotein (a): truly a direct prothrombotic factor in cardiovascular disease?

“…The strong LBSs in KIV 10 , as well as the KV, are required for both inhibitory activities, and additional sequences within KIV 5 -KIV 8 are also required to inhibit plasminogen activation. Although the exact role of each of these domains has not been uncovered, it is likely that apo(a), by virtue of its complex modular structure, makes a series of contacts with fibrin, plasminogen, and, possibly, tPA to effect its antifibrinolytic functions (55).…”

Mechanistic insights into Lp(a)-induced IL-8 expression: a role for oxidized phospholipid modification of apo(a)

“…Plasmids encoding a 17 kringle (17K)-containing form of recombinant apo(a) [17K r-apo(a)], as well as 17K apo(a) with a mutant lysine binding site in KIV 10 (17K ⌬ LBS10) (containing an Asp → Ala substitution at amino acid position 57 that abolishes the strong LBS in the KIV 10 domain), and 17K apo(a) lacking the KV domain (17K ⌬ V) were constructed and expressed in human embryonic kidney (HEK 293) cells, as previously described ( 12,27,28 ). All r-apo(a) species were purifi ed from the conditioned medium (CM) of stably expressing cell lines by lysine-Sepharose (GE Healthcare) affi nity chromatography, as previously reported ( 12,28 ).…”

“…All r-apo(a) species were purifi ed from the conditioned medium (CM) of stably expressing cell lines by lysine-Sepharose (GE Healthcare) affi nity chromatography, as previously reported ( 12,28 ). Purifi ed proteins were assessed for purity by SDS-PAGE followed by silver staining, and the absence of endotoxin was verifi ed using the ToxinSensor chromogenic LAL endotoxin assay kit (GenScript) as per the manufacturer's protocol.…”

Quantitative Evaluation of the Contribution of Weak Lysine-binding Sites Present within Apolipoprotein(a) Kringle IV Types 6–8 to Lipoprotein(a) Assembly

“…Purification of Recombinant Apo(a) Variants-All r-apo(a) variants used in this study, with the exception of KIV 7 and KIV 8 , were purified from the conditioned medium of stably expressing 293 cell lines using lysine-Sepharose chromatography as previously described (24). Protein concentrations for all r-apo(a) derivatives were obtained by absorbance measurements at 280 nm (corrected for Rayleigh scattering) using previously determined extinction coefficients (25).…”

“…7 or KIV 8 were assembled in the pET16b vector and expressed in E. coli, as described under "Experimental Procedures." Panel B, 17K, 17K KIV6 E56G, 17K KIV7 E56G, 17K KIV8 E56G, 17K KIV6/7 -E56G, and 17K KIV7/8 E56G r-apo(a) derivatives were purified using lysine-Sepharose chromatography (24). To assess protein integrity and purity, purified r-apo(a) derivatives (5 g) corresponding to 17K (1), 17K KIV6 E56G (2), 17K KIV7 E56G (3), 17K KIV8 E56G (4), 17K KIV6/7 E56G (5), and 17K KIV7/8 E56G (6) were subjected to SDS-PAGE under non-reducing (NR) and reducing (R) and stained with Coomassie Blue.…”