Download PDF
Thromb Haemost 2001; 86(05): 1229-1237
DOI: 10.1055/s-0037-1616056
Review Article
Schattauer GmbH

The Kringle V-Protease Domain Is a Fibrinogen Binding Region within Apo(a)

Song Xue
1   The Scripps Research Institute, Department of Vascular Biology, La Jolla, California, USA
,
Edwin L. Madison   *
1   The Scripps Research Institute, Department of Vascular Biology, La Jolla, California, USA
,
Lindsey A. Miles
1   The Scripps Research Institute, Department of Vascular Biology, La Jolla, California, USA
› Author AffiliationsThis work was supported in part by The National Institutes of Health, Grant HL38272, HL45934, HL31950 and by Grant-In-Aid #9650636 from the American Heart Association. Dr. Xue is the recipient of an Individual National Research Service Award HL09721.
Further Information

Publication History

Received 22 August 2000

Accepted after revision 04 June 2001

Publication Date:
13 December 2017 (online)

    Permissions and Reprints

Summary

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 μM 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 μM 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.

Keywords

Apo(a) - kringle - fibrinogen

* Dr. Madison’s present address is: Edwin L. Madison, Ph. D., Director, Molecular Biology, Corvas International, Inc., 3030 Science Park Road, San Diego, CA 92121


 

  • References

  • 1 Loscalzo J. Lipoprotein(a). A unique risk factor for atherothrombotic disease. Arteriosclerosis 1990; 10 (Suppl. 05) 672-9.
    CrossrefPubMedGoogle Scholar
  • 2 Nordestgaard BG. The vascular endothelial barrier – selective retention of lipoproteins. Current Opinion In Lipidology 1996; 7 (Suppl. 05) 269-73.
    CrossrefPubMedGoogle Scholar
  • 3 Simionescu M, Simionescu N. Proatherosclerotic events: pathobiochemical changes occurring in the arterial wall before monocyte migration. Faseb Journal 1993; 7 (Suppl. 14) 1359-66.
    CrossrefPubMedGoogle Scholar
  • 4 Fless GM, Rolih CA, Scanu AM. Heterogeneity of human plasma lipoprotein (a). Isolation and characterization of the lipoprotein subspecies and their apoproteins. J Biol Chem 1984; 259 (Suppl. 18) 11470-8.
    PubMedGoogle Scholar
  • 5 McLean JW. et al. cDNA sequence of human apolipoprotein(a) is homo-logous to plasminogen. Nature 1987; 330 6144 132-7.
    CrossrefPubMedGoogle Scholar
  • 6 Eaton DL. et al. Partial amino acid sequence of apolipoprotein(a) shows that it is homologous to plasminogen. Proceedings of the National Academy of Sciences of the USA 1987; 84 (Suppl. 10) 3224-8.
    CrossrefPubMedGoogle Scholar
  • 7 Lucas MA, Fretto LJ, McKee PA. The binding of human plasminogen to fibrin and fibrinogen. J Biol Chem 1983; 258 (Suppl. 07) 4249-56.
    PubMedGoogle Scholar
  • 8 Wiman B, Wallen P. The specific interaction between plasminogen and fibrin. A physiological role of the lysine binding site in plasminogen. Thrombosis Research 1977; 10 (Suppl. 02) 213-22.
    CrossrefPubMedGoogle Scholar
  • 9 Whitaker AN. et al. The binding of glu- and lys-plasminogens to fibrin and their subsequent effects on fibrinolysis. Thrombosis Research 1980; 19 (Suppl. 03) 381-91.
    CrossrefPubMedGoogle Scholar
  • 10 Fleury V, Anglés-Cano E. Characterization of the Binding of Plasminogen to Fibrin Surfaces: The Role of Carboxy-Terminal Lysines. Biochemistry 1991; 30: 7630-8.
    CrossrefPubMedGoogle Scholar
  • 11 Loscalzo J. et al. Lipoprotein(a), fibrin binding, and plasminogen activation. Arteriosclerosis 1990; 10 (Suppl. 02) 240-5.
    CrossrefPubMedGoogle Scholar
  • 12 Miles L. et al. Interaction of Lipoprotein(a) with the Extracellular Matrix. Fibrinolysis and Proteolysis 1998; 12: 79-83.
    PubMedGoogle Scholar
  • 13 Rouy D. et al. Apolipoprotein(a) and plasminogen interactions with fibrin: a study with recombinant apolipoprotein(a) and isolated plasminogen fragments. Biochemistry 1992; 31 (Suppl. 27) 6333-9.
    CrossrefPubMedGoogle Scholar
  • 14 Harpel PC, Gordon BR, Parker TS. Plasmin catalyzes binding of lipoprotein (a) to immobilized fibrinogen and fibrin. Proceedings of the National Academy of Sciences of the USA 1989; 86 (Suppl. 10) 3847-51.
    CrossrefPubMedGoogle Scholar
  • 15 Xue S. et al. Comparison of the effects of Apo(a) kringle IV-10 and plasminogen kringles on the interactions of lipoprotein(a) with regulatory molecules. Thromb Haemost 1999; 81 (Suppl. 03) 428-35.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 16 Lou XJ. et al. Fibrinogen deficiency reduces vascular accumulation of apolipoprotein(a) and development of atherosclerosis in apolipoprotein(a) transgenic mice. Proc Natl Acad Sci USA 1998; 95 (Suppl. 21) 12591-5.
    CrossrefPubMedGoogle Scholar
  • 17 Lawn RM. et al. Atherogenesis in transgenic mice expressing human apolipoprotein(a). Nature 1992; 360 6405 670-2.
    CrossrefPubMedGoogle Scholar
  • 18 Palabrica TM. et al. Antifibrinolytic activity of apolipoprotein(a) in vivo: human apolipoprotein(a) transgenic mice are resistant to tissue plasminogen activator-mediated thrombolysis. Nature Medicine 1995; 1 (Suppl. 03) 256-9.
    CrossrefPubMedGoogle Scholar
  • 19 Huby T. et al. Characterization of the N-terminal and C-terminal domains of human apolipoprotein(a): relevance to fibrin binding. Biochemistry 1995; 34 (Suppl. 22) 7385-93.
    CrossrefPubMedGoogle Scholar
  • 20 LoGrasso PV, Cornell KS, Boettcher BR. Cloning, expression, and characterization of human apolipoprotein(a) kringle IV37. J Biol Chem 1994; 269 (Suppl. 34) 21820-7.
    PubMedGoogle Scholar
  • 21 Klezovitch O, Scanu AM. Lys and fibrinogen binding of wild-type (Trp72) and mutant (Arg72) human apo(a) kringle IV-10 expressed in E coli and CHO cells. Arterioscler Thromb Vasc Biol 1996; 16 (Suppl. 03) 392-8.
    CrossrefPubMedGoogle Scholar
  • 22 Scanu AM. et al. Rhesus monkey lipoprotein(a) binds to lysine Sepharose and U937 monocytoid cells less efficiently than human lipoprotein(a). Evidence for the dominant role of kringle 4 (37). J Clin Invest 1993; 91 (Suppl. 01) 283-91.
    CrossrefPubMedGoogle Scholar
  • 23 Scanu AM. et al. A single point mutation (Trp72—>Arg) in human apo(a) kringle 4-37 associated with a lysine binding defect in Lp(a). Biochimica et Biophysica Acta 1994; 1227 1-2 41-5.
    CrossrefPubMedGoogle Scholar
  • 24 Klezovitch O, Edelstein C, Scanu AM. 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. J Clin Invest 1996; 98 (Suppl. 01) 185-91.
    CrossrefPubMedGoogle Scholar
  • 25 Ernst A. et al. Identification of two functionally distinct lysine-binding sites in kringle 37 and in kringles 32-36 of human apolipoprotein(a). J Biol Chem 1995; 270 (Suppl. 11) 6227-34.
    CrossrefPubMedGoogle Scholar
  • 26 Chenivesse X. et al. Expression of a recombinant kringle V of human apolipoprotein(a): antibody characterization and species specificity. Protein Expression & Purification 1996; 8 (Suppl. 02) 145-50.
    PubMedGoogle Scholar
  • 27 Chang Y. et al. Structure and ligand binding determinants of the recombinant kringle 5 domain of human plasminogen. Biochemistry 1998; 37 (Suppl. 10) 3258-71.
    CrossrefPubMedGoogle Scholar
  • 28 McCance SG, Menhart N, Castellino FJ. Amino acid residues of the kringle-4 and kringle-5 domains of human plasminogen that stabilize their interactions with omega-amino acid ligands. J Biol Chem 1994; 269 (Suppl. 51) 32405-10.
    PubMedGoogle Scholar
  • 29 Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 1977; 74 (Suppl. 12) 5463-7.
    CrossrefPubMedGoogle Scholar
  • 30 Zhang X-P, Kamata T, Yokoyama K, Puzon-McLaughlin W, Takada Y. Specific interaction of the recombinant disintegrin-like domain of MDC-15 (Metargidin, ADAM-15) with integrin αvβ3. J Biol Chem 2001; 273 (Suppl. 13) 7345-50.
    PubMedGoogle Scholar
  • 31 Deutsch DG, Mertz ET. Plasminogen: purification from human plasma by affinity chromatography. Science 1970; 170 (Suppl. 962) 1095-6.
    CrossrefPubMedGoogle Scholar
  • 32 Miles LA, Dahlberg CM, Plow EF. The cell-binding domains of plasminogen and their function in plasma. J Biol Chem 1988; 263 (Suppl. 24) 11928-34.
    PubMedGoogle Scholar
  • 33 Shepherd J, Bedford DK, Morgan HG. Radioiodination of human low density lipoprotein: a comparison of four methods. Clinica Chimica Acta 1976; 66 (Suppl. 01) 97-109.
    CrossrefPubMedGoogle Scholar
  • 34 Miles LA. et al. Interaction of Lp(a) with plasminogen binding sites on cells. Thromb Haemost 1995; 73 (Suppl. 03) 458-65.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 35 Fraker PJ, Speck Jr JC. Protein and cell membrane iodinations with a sparingly soluble chloroamide, 1,3,4,6-tetrachloro-3a,6a-diphrenylglycoluril. Biochem Biophys Res Commun 1978; 80 (Suppl. 04) 849-57.
    CrossrefPubMedGoogle Scholar
  • 36 Scanu AM, Edelstein C. Kringle-dependent structural and functional polymorphism of apolipoprotein (a). Biochimica et Biophysica Acta 1995; 1256 (Suppl. 01) 1-12.
    CrossrefPubMedGoogle Scholar
  • 37 Bonen DK. et al. Expression of a recombinant apolipoprotein(a) in HepG2 cells. Evidence for intracellular assembly of lipoprotein(a). J Biol Chem 1997; 272 (Suppl. 09) 5659-67.
    CrossrefPubMedGoogle Scholar
  • 38 Sangrar W, Marcovina SM, Koschinsky ML. Expression and characterization of apolipoprotein(a) kringle IV types 1, 2 and 10 in mammalian cells. Protein Engineering 1994; 7 (Suppl. 05) 723-31.
    CrossrefPubMedGoogle Scholar
  • 39 Makino K, Furbee JW, Scanu AM, Fless GM. Effect of glycation on the properties of lipoprotein(a). Arterioscler Thromb Vasc Biol 1995; 15 (Suppl. 03) 385-91.
    CrossrefPubMedGoogle Scholar
  • 40 Hughes SD. et al. Lipoprotein(a) vascular accumulation in mice. In vivo analysis of the role of lysine binding sites using recombinant adenovirus. J Clin Invest 1997; 100 (Suppl. 06) 1493-500.
    CrossrefPubMedGoogle Scholar