Definition of the structural elements in plasminogen required for high-affinity binding to apolipoprotein(a): a study utilizing surface plasmon resonance.
Lipoprotein(a) [Lp(a)] is suggested to link atherosclerosis and thrombosis owing to the similarity between the apolipoprotein(a) [apo(a)] moiety of Lp(a) and plasminogen. Lp(a) may interfere with tPA-mediated plasminogen activation in fibrinolysis, thereby generating a hypercoaguable state in vivo. The present study employed surface plasmon resonance (SPR) to examine the binding interaction between plasminogen and a physiologically relevant, 17-kringle recombinant apo(a) species [17K r-apo(a)] in real time. Native, intact Glu(1)-plasminogen bound to apo(a) with substantially higher affinity (K(D) approximately 0.3 microM) compared to a series of plasminogen fragments (K1-5, K1-3, K4, K5P, and tail domain) that interacted weakly with apo(a) (K(D) > 50 microM). Treatment of Glu(1)-plasminogen with citraconic anhydride (a lysine modification reagent) completely abolished binding to wild-type 17K r-apo(a), whereas citraconylated 17K r-apo(a) decreased binding to wild-type Glu(1)-plasminogen by approximately 50%; inhibition of binding was also observed using the lysine analogue epsilon-aminocaproic acid. Whereas native Glu(1)-plasminogen exhibited monophasic binding to 17K r-apo(a), truncated Lys(78)-plasminogen exhibited biphasic binding. Altering Glu(1)-plasminogen from its native, closed conformation (in chloride buffer) to an open conformation (in acetate buffer) also yielded biphasic isotherms. These SPR data are consistent with a two-state kinetic model in which a conformational change in the plasminogen-apo(a) complex may occur following the initial binding event. Differential binding kinetics between Glu(1)-/Lys(78)-plasminogen and apo(a) may explain why Lp(a) is a stronger inhibitor of tPA-mediated Glu(1)-plasminogen activation compared to Lys(78)-plasminogen activation.
Hancock
Mark A
MA
Department of Biochemistry, Queen's University, Kingston, Ontario, Canada K7L 3N6.
Spencer
Craig A
CA
Koschinsky
Marlys L
ML
D000818:Animals; D001054:Apolipoproteins A / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D002478:Cells, Cultured; D006224:Cricetinae; D004789:Enzyme Activation; D018698:Glutamic Acid / Q000378:metabolism; D008024:Ligands; D008239:Lysine / Q000378:metabolism; D010958:Plasminogen / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D020349:Surface Plasmon Resonance
D001054:Apolipoproteins A; D008024:Ligands; D018698:Glutamic Acid; D010958:Plasminogen; D008239:Lysine