Binding of Recombinant Apolipoprotein(a) to Human Platelets and Effect on Platelet Aggregation

C. Martínez; J. Rivera; S. Loyau; J. Corral; R. González-Conejero; M. L. Lozano; V. Vicente; E. Anglés-Cano

doi:10.1055/s-0037-1615654

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2001; 85(04): 686-693
DOI: 10.1055/s-0037-1615654

Review Articles

Schattauer GmbH

Binding of Recombinant Apolipoprotein(a) to Human Platelets and Effect on Platelet Aggregation

C. Martínez

¹Unit of Hematology and Clinical Oncology, University General Hospital, Murcia, Spain

,

J. Rivera

¹Unit of Hematology and Clinical Oncology, University General Hospital, Murcia, Spain

,

S. Loyau

²INSERM U.143, Hemostasis and Vascular Biology Research Unit, Hôpital de Bicêtre, Bicêtre, France

,

J. Corral

¹Unit of Hematology and Clinical Oncology, University General Hospital, Murcia, Spain

,

R. González-Conejero

¹Unit of Hematology and Clinical Oncology, University General Hospital, Murcia, Spain

,

M. L. Lozano

¹Unit of Hematology and Clinical Oncology, University General Hospital, Murcia, Spain

,

V. Vicente

¹Unit of Hematology and Clinical Oncology, University General Hospital, Murcia, Spain

,

E. Anglés-Cano

²INSERM U.143, Hemostasis and Vascular Biology Research Unit, Hôpital de Bicêtre, Bicêtre, France

› Author Affiliations

Abstract

Summary

The interaction of lipoprotein(a) [Lp(a)] with platelets is not well defined, particularly with regards to the individual contribution of the protein components of Lp(a), the apo B-100 and the apolipoprotein apo(a). This study investigated the binding of different recombinant apo(a) [r-apo(a)] isoforms, to human platelets and its effect on platelet aggregation. Scatchard analysis of saturation binding experiments demonstrated that human platelets display a single class of high affinity r-apo(a) binding sites (71 ± 46 molec./platelet, Kd = 5.6 ± 2.0 nmol/L). Platelet activation with strong agonists (thrombin, arachidonic acid) increased 2- to 10-fold the r-apo(a) binding, without affecting the affinity. Competition assays showed that the binding sites are highly specific for r-apo(a) and Lp(a). At high concentration t-PA could also bind to the r-apo(a) binding sites. By contrast, neither fibrinogen nor plasminogen inhibited to the r-apo(a) binding. The lysine analogue EACA inhibits the binding of r-apo(a) to platelets, thus suggesting the involvement of lysine residues in that interaction. Moreover, the r-apo(a) binding to platelets is unlikely mediated by GPIIb/IIIa-attached fibrin since it is not affected by platelet treatment with either LJ-CP8, a monoclonal antibody that specifically blocks fibrinogen binding to GPIIb/IIIa, nor GPRP, an inhibitor of fibrin polymerisation. Finally, we show that the distinct recombinant apo(a) proteins, as well as native Lp(a), promote an aggregation response of platelets to otherwise subaggregant doses of arachidonic acid. This proaggregant effect of r-apo(a) is dependent on its binding to platelets since it requires a minimum incubation time, and it is prevented by EACA at concentration inhibiting the r-apo(a)-platelet interaction.

These results suggest that the prothrombotic action of Lp(a) may be in part mediated by modulating the platelet function through the interaction of its apo(a) subunit with a specific receptor at the platelet surface.

Keywords

Apolipoprotein(a) binding sites - platelet receptors - platelet aggregation

Full Text

References

References
1 Dahlen GH, Guyton JR, Attar M, Farmer JA, Kautz JA, Gotto AM. Association of levels of lipoprotein Lp(a), plasma lipids, and other lipoproteins with coronary artery disease documented by angiography. Circulation 1986; 74: 758-65.
2 Rhoads GG, Dahlen G, Berg K, Morton NE, Dannenberg AL. Lp(a) lipo-protein as a risk factor for myocardial infarction. J Am Med Assoc (JAMA) 1987; 256: 2540-4.
3 Seed M, Hoppichler F, Reaveley D, McCarthy S, Thompson GR, Boerwinkle E, Utermann G. Relation of serum lipoprotein(a) and apolipoprotein(a) phenotype to coronary heart disease in patients with familial hypercholesterolemia. New Engl J Med 1990; 322: 1494-9.
4 Pepin JM, O’Neil JA, Hoff H. Quantification of apo(a) and apoB in human atherosclerotic lesions. J Lipid Res 1993; 32: 317-27.
5 Hoff HF, O’Neil J, Smejkal GB, Yashiro A. Immunochemically detectable lipid-free apo(a) in plasma and in human atherosclerotic lesions. Chem Phys Lipids 1994; 67-8: 271-80.
6 Brunner C, Kraft HG, Utermann G, Muller HJ. Cys⁴⁰⁵⁷ of apolipoprotein(a) is essential for lipoprotein(a) assembly. Proc Natl Acad Sci USA 1993; 90: 11643-7.
7 McLean JW, Tomlinson JE, Kuang W-J, Eaton DL, Chen EY, Fless GM, Scanu AM, Lawn RM. cDNA sequence of human apolipoprotein(a) is homologous to plasminogen. Nature 1987; 330: 132-7.
8 Lackner C, Cohen JC, Hobbs HH. Molecular definition of the extreme size polymorphism in apolipoprotein(a). Hum Mol Genet 1993; 2: 933-40.
9 Ernst A, Helmhold M, Brunner C, Pethö-Schramm A, Armstrong VW, Müller HJ. Identification of two functionally lysine-binding sites in kringle 37 and kringles 32-36 of human apolipoprotein(a). J Biol Chem 1995; 270: 6227-34.
10 Li ZG, Gambino R, Fless GM, Copeland RA, Halfpenny AJ, Scanu AM. Expression and Purification of kringle-4-type-2 of human apolipoprotein(a) in Escherichia coli. Protein Expr Purif 1992; 3: 212-22.
11 Loscalzo J, Weinfeld M, Fless GM, Scanu AM. Lipoprotein(a) fibrin binding and plasminogen activation. Arteriosclerosis 1990; 10: 240-5.
12 Rouy D, Koschinsky ML, Fleury V, Chapman J, Anglés-Cano E. Apolipoprotein(a) and plasminogen interactions with fibrin: a study with recombinant apolipoprotein(a) and isolated plasminogen fragments. Biochemistry 1992; 31: 6333-9.
13 Miles LA, Fless GM, Levin EG, Scanu AM, Plow EF. A potential basis for the thrombotic risks associated with lipoprotein(a). Nature 1989; 339: 301-3.
14 Kang C, Durlach V, Soulat T, Fournier C, Anglés-Cano E. Lipoprotein(a) isoforms display differences in affinity for plasminogen-like binding to human mononuclear cells. Arterioscler Thromb Vasc Biol 1997; 17: 2036-43.
15 Hajjar KA, Gavish D, Breslow JL, Nachman RL. Lipoprotein(a) modulation of endothelial cell surface fibrinolysis and its potential role in atherosclerosis. Nature 1989; 339: 303-5.
16 Ezratty A, Simon DI, Loscalzo J. Lipoprotein(a) binds to human platelets and attenuates plasminogen binding and activation. Biochemistry 1993; 32: 4628-33.
17 Malle E, Ibovnik A, Steinmetz A, Kostner GM, Sattler W. Identification of glycoprotein IIb as the lipoprotein(a)binding protein on platelets. Lipoprotein(a) binding is independent of an arginyl-glycyl-aspartate tripeptide located in apolipoprotein(a). Arterioscler Thromb 1994; 14: 345-52.
18 Gries A, Gries M, Wurm H, Kenner T, Ijsseldijk M, Sixma JJ, Kostner GM. Lipoprotein(a) inhibits collagen-induced aggregation of thrombocytes. Arterioscler Thromb Vasc Biol 1996; 16: 648-55.
19 Malle E, Ibovnik A, Leis HJ, Kostner GM, Verhallen PFJ, Sattler W. Lysine modification of LDL or lipoprotein(a) by 4-hydroxynonenal or malondialdehyde decreases platelet serotonin secretion without affecting platelet aggregability and eicosanoid formation. Arterioscler Thromb Vasc Biol 1995; 15: 377-84.
20 Pedreño J, Fernández R, Cullaré C, Barceló A, Elorza MA, de Castellarnau C. Platelet integrin α_IIbβ₃ (GPIIb-IIIa) is not implicated in the binding of LDL to intact resting platelets. Arterioscler Thromb Vasc Biol 1997; 17: 156-63.
21 Rand ML, Sangrar W, Hancock MA, Taylor DM, Marcovina SM, Packham MA, Koschinsky ML. Apolipoprotein(a) enhances platelet responses to the thrombin receptor-activating peptide SFLLRN. Arterioscler Thromb Vasc Biol 1998; 18: 1393-9.
22 Brunner C, Lobentanz EM, Petho-Schramm A, Ernst A, Kang C, Dieplinger H, Muller HJ, Utermann G. The number of identical kringle IV repeats in apolipoprotein(a) affects its processing and secretion by HepG2 cells. J Biol Chem 1996; 271: 32403-10.
23 Anglés-Cano E, Loyau S, Cardoso G, Couderc R, Gillery P. A novel kringle-4 number-based recombinant apo(a) standard for human apo(a) phenotyping. J Lipid Res 1999; 40: 354-9.
24 Deutsch D, Mertz ET. Plasminogen: purification from human plasma by affinity chromatography. Science 1970; 170: 203-9.
25 Kazal LA, Amsel S, Miller OP, Tocantins LM. The preparation and some properties of fibrinogen precipitated from human plasma with glycine. Proc Soc Exp Biol Med 1963; 98: 139-51.
26 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.
27 Grailhe P, Boyer-Neumann C, Haverkate F, Grimbergen J, Larrieu MJ, Anglés-Cano E. The mutation in fibrinogen Bicetre II (gamma Asn³⁰⁸ Lys) does not affect the binding of t-PA and plasminogen to fibrin. Blood Coagul Fibrinolysis 1993; 4: 679-87.
28 Laemmli UK. Cleavage of structural proteins during the assembly the head of bacteriophage T4. Nature 1970; 227: 680-5.
29 Mustard JF, Perry DW, Ardlie NG, Packham MA. Preparation of suspensions of washed platelets from humans. Br J Haematol 1972; 22: 193-204.
30 Fraker PJ, Speck JCJ. Protein and cell membrane iodinations with sparingly-soluble-chloroamide-1,3,4,6-tetrachloro-3^a,6^a-diphenylglycoluril. Biochem Biophys Res Commun 1978; 80: 849-57.
31 Munson PJ, Rodbard D. LIGAND: a versatile computerized approach for the characterization of ligand binding systems. Anal Biochem 1980; 107: 220-39.
32 McPherson GA. Analysis of radioligand binding experiments: a collection of computer programs for the IBM PC. J Pharmacol Methods 1985; 14: 213-28.
33 Klotz IM. Numbers of receptor sites from Scatchard graphs: facts and fantasies. Science 1982; 217: 1247-9.
34 Loscalzo J. Lipoprotein(a). A unique risk factor for atherothrombotic disease. Arteriosclerosis 1990; 10: 672-9.
35 Malle E, Sattler W. Platelets and the lipoproteins: native, modified and platelet modified lipoproteins. Platelets 1994; 5: 70-83.
36 Furman MI, Benoit SE, Barnard MR, Valeri CR, Borbone ML, Becker RC, Hechtman HB, Michelson AD. Increased platelet reactivity and circulating monocyte-platelet aggregates in patients with stable coronary artery disease. J Am Coll Cardiol 1998; 31: 352-8.
37 Felez J, Chanquia CJ, Fabregas P, Plow EF, Miles LA. Competition between plasminogen and tissue plasminogen activator for cellular binding sites. Blood 1993; 82: 2433-41.
38 Fless GM, Furbee Jr J, Snyder ML, Meredith SC. Ligand-induced conformational change of lipoprotein(a). Biochemistry 1996; 35: 2289-98.
39 Miles LA, Ginsberg MH, White JG, Plow EF. Plasminogen interactis with human platelets through two distinct mechanisms. J Clin Invest 1986; 77: 2001-9.
40 Adelman B, Rizk A, Hanners E. Plasminogen interactions with platelets in plasma. Blood 1988; 72: 1530-5.
41 Vaughan DE, Mendelsohn ME, Declerck PJ, Van Houtte E, Collen D, Loscalzo J. Characterization of the binding of human tissue-type plasminogen activator to platelets. J Biol Chem 1989; 264: 15869-74.
42 Watala C, Pietrucha T, Gwozdzinski K, Kralisz Cierniewski CS. Micro-environment changes in human blood platelet membranes associated with binding of tissue-type plasminogen activator. Eur J Biochem 1993; 215: 867-71.
43 Vassallo Jr RR, Kleber-Emmons T, Cichowski K, Brass LF. Structure-function relationships in the activation of platelet thrombin receptors by receptor-derived peptides. J Biol Chem 1992; 267: 6081-5.
44 Chao BH, Kalkunte S, Maraganore JM, Stone SR. Essential groups in synthetic agonist peptides for activation and the platelet thrombin receptor. Biochemistry 1992; 31: 6175-8.