Mapping of a Minimal Apolipoprotein(a) Interaction Motif Conserved in Fibrin(ogen) β- and γ-Chains*

Lipoprotein(a) (Lp(a)) is a major independent risk factor for atherothrombotic disease in humans. The physiological function(s) of Lp(a) as well as the precise mechanism(s) by which high plasma levels of Lp(a) increase risk are unknown. Binding of apolipoprotein(a) (apo(a)) to fibrin(ogen) and other components of the blood clotting cascade has been demonstrated in vitro, but the domains in fibrin(ogen) critical for interaction are undefined. We used apo(a) kringle IV subtypes to screen a human liver cDNA library by the yeast GAL4 two-hybrid interaction trap system. Among positive clones that emerged from the screen, clones were identified as fibrinogen β- and γ-chains. Peptide-based pull-down experiments confirmed that the emerging peptide motif, conserved in the carboxyl-terminal globular domains of the fibrinogen β and γ modules specifically interacts with apo(a)/Lp(a) in human plasma as well as in cell culture supernatants of HepG2 and Chinese hamster ovary cells, ectopically expressing apo(a)/Lp(a). The influence of lysine in the fibrinogen peptides and of lysine binding sites in apo(a) for the interaction was evaluated by binding experiments with apo(a) mutants and a mutated fibrin(ogen) peptid. This confirmed the lysine binding sites in kringle IV type 10 of apo(a) as the major fibrin(ogen) binding site but also demonstrated lysine-independent interactions.

[1]  C. J. Halfman,et al.  The relationship between the effect of lysine analogues and salt on the conformation of lipoprotein(a). , 2000, Biochemistry.

[2]  G. Utermann,et al.  Molecular basis of congenital lp(a) deficiency: a frequent apo(a) 'null' mutation in caucasians. , 1999, Human molecular genetics.

[3]  H. Hobbs,et al.  Lipoprotein(a): intrigues and insights. , 1999, Current opinion in lipidology.

[4]  R. Lawn,et al.  Fibrinogen deficiency reduces vascular accumulation of apolipoprotein(a) and development of atherosclerosis in apolipoprotein(a) transgenic mice. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[5]  T. Ugarova,et al.  Identification of a Novel Recognition Sequence for Integrin αMβ2 within the γ-chain of Fibrinogen* , 1998, The Journal of Biological Chemistry.

[6]  R. Willette,et al.  Factor XIIIa cross-links lipoprotein(a) with fibrinogen and is present in human atherosclerotic lesions. , 1998, Circulation research.

[7]  P. Harpel,et al.  Demonstration of covalent binding of lipoprotein(a) [Lp(a)] to fibrin and endothelial cells. , 1998, Biochemistry.

[8]  J. Morrisett,et al.  Lipoprotein(a) in plasma, arterial wall, and thrombus from patients with aortic aneurysm , 1997, Clinical genetics.

[9]  R. Doolittle,et al.  Crystal structures of fragment D from human fibrinogen and its crosslinked counterpart from fibrin , 1997, Nature.

[10]  G. Baier,et al.  Novel interaction of apolipoprotein(a) with beta-2 glycoprotein I mediated by the kringle IV domain. , 1997, Blood.

[11]  E. Rubin,et al.  Modification of apolipoprotein(a) lysine binding site reduces atherosclerosis in transgenic mice. , 1997, The Journal of clinical investigation.

[12]  A. Noma,et al.  Immunolocalization of Lipoprotein(a) in Wounded Tissues , 1997, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[13]  E. Davie,et al.  Crystal structure of a 30 kDa C-terminal fragment from the γ chain of human fibrinogen , 1997 .

[14]  O. Klezovitch,et al.  Evidence that the fibrinogen binding domain of Apo(a) is outside the lysine binding site of kringle IV-10: a study involving naturally occurring lysine binding defective lipoprotein(a) phenotypes. , 1996, The Journal of clinical investigation.

[15]  B. Nordestgaard,et al.  Specific accumulation of lipoprotein(a) in balloon-injured rabbit aorta in vivo. , 1996, Circulation research.

[16]  J. Eaton,et al.  Molecular determinants of acute inflammatory responses to biomaterials. , 1996, The Journal of clinical investigation.

[17]  O. Klezovitch,et al.  Lys and fibrinogen binding of wild-type (Trp72) and mutant (Arg72) human apo(a) kringle IV-10 expressed in E coli and CHO cells. , 1996, Arteriosclerosis, thrombosis, and vascular biology.

[18]  S. Meredith,et al.  Ligand-induced conformational change of lipoprotein(a). , 1996, Biochemistry.

[19]  F. Kronenberg,et al.  A pentanucleotide repeat polymorphism in the 5' control region of the apolipoprotein(a) gene is associated with lipoprotein(a) plasma concentrations in Caucasians. , 1995, The Journal of clinical investigation.

[20]  H. Robenek,et al.  Kringle 4 of human apolipoprotein[a] shares a linear antigenic site with human catalase. , 1995, Journal of lipid research.

[21]  V. Armstrong,et al.  Identification of Two Functionally Distinct Lysine-binding Sites in Kringle 37 and in Kringles 3236 of Human Apolipoprotein(a) (*) , 1995, The Journal of Biological Chemistry.

[22]  L. Curtiss,et al.  Interaction of Lp(a) with Plasminogen Binding Sites on Cells , 1995, Thrombosis and Haemostasis.

[23]  B. Furie,et al.  Antifibrinolytic activity of apolipoprotein(a) in vivo: Human apolipoprotein(a) transgenic mice are resistant to tissue plasminogen activator-mediated thrombolysis , 1995, Nature Medicine.

[24]  G. Kostner,et al.  Structural requirements of apo-a for the lipoprotein-a assembly. , 1994, The Biochemical journal.

[25]  J. Lee,et al.  A single point mutation (Trp72-->Arg) in human apo(a) kringle 4-37 associated with a lysine binding defect in Lp(a). , 1994, Biochimica et biophysica acta.

[26]  K. Zatloukal,et al.  High-level expression of various apolipoprotein(a) isoforms by "transferrinfection": the role of kringle IV sequences in the extracellular association with low-density lipoprotein. , 1994, Biochemistry.

[27]  M. Koschinsky,et al.  Carboxyl‐terminal truncation of apolipoproteinB‐100 inhibits lipoprotein(a) particle formation , 1994 .

[28]  R. Lawn,et al.  Activation of transforming growth factor-β is inhibited in transgenic apolipoprotein(a) mice , 1994, Nature.

[29]  G. Utermann,et al.  Cys4057 of apolipoprotein(a) is essential for lipoprotein(a) assembly. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[30]  M. Koschinsky,et al.  Apolipoprotein(a) and plasminogen interactions with fibrin: a study with recombinant apolipoprotein(a) and isolated plasminogen fragments. , 1992, Biochemistry.

[31]  J. Morrisett,et al.  A structural assessment of the apo[a] protein of human lipoprotein[a] , 1992, Proteins.

[32]  S. Fields,et al.  The two-hybrid system: a method to identify and clone genes for proteins that interact with a protein of interest. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[33]  J. Loscalzo,et al.  Tissue-type plasminogen activator binds to and is inhibited by surface-bound lipoprotein(a) and low-density lipoprotein. , 1991, Biochemistry.

[34]  D. Rifkin,et al.  Lipoprotein (a) inhibits the generation of transforming growth factor beta: an endogenous inhibitor of smooth muscle cell migration , 1991, The Journal of cell biology.

[35]  E. Boerwinkle,et al.  Molecular basis of apolipoprotein (a) isoform size heterogeneity as revealed by pulsed-field gel electrophoresis. , 1991, The Journal of clinical investigation.

[36]  D. Rouy,et al.  Lipoprotein(a) impairs generation of plasmin by fibrin-bound tissue-type plasminogen activator. In vitro studies in a plasma milieu. , 1991, Arteriosclerosis and thrombosis : a journal of vascular biology.

[37]  H. Hoff,et al.  Quantification of apo[a] and apoB in human atherosclerotic lesions. , 1991, Journal of lipid research.

[38]  M. Koschinsky,et al.  Apolipoprotein(a) size heterogeneity is related to variable number of repeat sequences in its mRNA. , 1990, Biochemistry.

[39]  J. Breslow,et al.  Lipoprotein(a) modulation of endothelial cell surface fibrinolysis and its potential role in atherosclerosis , 1989, Nature.

[40]  A. Scanu,et al.  A potential basis for the thrombotic risks associated with lipoprotein(a) , 1989, Nature.

[41]  B. Gordon,et al.  Plasmin catalyzes binding of lipoprotein (a) to immobilized fibrinogen and fibrin. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[42]  E. Chen,et al.  cDNA sequence of human apolipoprotein(a) is homologous to plasminogen , 1987, Nature.

[43]  G. Utermann,et al.  Lp(a) glycoprotein phenotypes. Inheritance and relation to Lp(a)-lipoprotein concentrations in plasma. , 1987, The Journal of clinical investigation.

[44]  J. Butler,et al.  The immunochemistry of sandwich ELISAs. II. A novel system prevents the denaturation of capture antibodies. , 1986, Immunology letters.

[45]  R. B. Merrifield,et al.  An SN2 deprotection of synthetic peptides with a low concentration of hydrofluoric acid in dimethyl sulfide: evidence and application in peptide synthesis , 1983 .

[46]  L. Patthy,et al.  Location of plasminogen-binding sites in human fibrin(ogen). , 1983, Biochemistry.

[47]  R. Laursen,et al.  Studies on the lysine-binding sites of human plasminogen. The effect of ligand structure on the binding of lysine analogs to plasminogen. , 1980, European journal of biochemistry.

[48]  Ivan Damjanov,et al.  Controlled synthesis of HBsAg in a differentiated human liver carcinoma-derived cell line , 1979, Nature.

[49]  E. Janus,et al.  Frequency Distributions of Apolipoprotein(a) Kringle IV Repeat Alleles and Their Effects on Lipoprotein(a) Levels in Caucasian, Asian, and African Populations: The Distribution of Null Alleles Is Non-Random , 1996, European journal of human genetics : EJHG.

[50]  E. Angles-cano,et al.  Overview on fibrinolysis: plasminogen activation pathways on fibrin and cell surfaces. , 1994, Chemistry and physics of lipids.

[51]  S. Pizzo,et al.  Lipoprotein(a) inhibition of plasminogen activation by tissue-type plasminogen activator. , 1990, Thrombosis research.