The Kringle V-protease domain is a fibrinogen binding region within Apo(a).
Lp(a) binds directly to fibrin and competes for the interaction of plasminogen with this substrate. This competition may play a role in the proatherothrombogenic consequences of high Lp(a) levels. Previous studies by us and others showed that apo(a) Kringle IV-10 competes for the interaction of Lp(a) with plasmin-treated fibrinogen. However, kringle IV-10 cannot account for the entire high affinity interaction of Lp(a) with fibrinogen. Therefore, we tested the hypothesis that the apo(a) kringle V protease-like domain (KV-PD) could interact with plasmin-treated fibrinogen. We cloned the apo(a) KV-PD region from a human liver cDNA library. Fusion apo(a) KV-PD was expressed in COS 7 cells and purified from the conditioned media. Western blotting of the apo(a) KV-PD protein revealed two bands migrating with apparent molecular weights of 45K and 48K. When fusion apo(a) KV-PD was treated with O-glycosidase and neuraminidase, the higher molecular weight band disappeared suggesting that the apo(a) KV-PD was O-glycosylated. Apo(a) KV-PD bound to plasmin-treated fibrinogen in a dose-dependent fashion. An EC50 of 3.9+/-0.2 microM was determined for this interaction. Treatment of the apo(a) KV-PD with O-glycosidase did not significantly affect its ability to bind to plasmin-treated fibrinogen. In addition, apo(a) KV-PD competed for the binding of 125I-Lp(a) to plasmin-treated fibrinogen. An IC50 of 7.90+/-0.95 microM was obtained. Our data suggest that the KV-PD of apo(a) shares binding sites on plasmin-treated fibrinogen with Lp(a) and also may participate in the interaction of the Lp(a) particle with plasmin-treated fibrinogen.
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The Scripps Research Institute, Department of Vascular Biology, La Jolla, California 92037, USA.
Madison
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Miles
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D001054:Apolipoproteins A / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D001667:Binding, Competitive; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D005340:Fibrinogen / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D018082:Kringles / Q000502:physiology*; D017270:Lipoprotein(a) / Q000378:metabolism; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D001054:Apolipoproteins A; D017270:Lipoprotein(a); D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D005340:Fibrinogen