Identification of Glycoprotein lib as the Lipoprotein (a)-Binding Protein on Platelets Lipoprotein(a) Binding Is Independent of an Arginyl-Glycyl-Aspartate Tripeptide Located in Apolipoprotein(a)

Lipoprotein(a) [Lp(a)] plays an important role in atherosclerosis. The amino acid sequence of apolipoprotein(a) [apo(a)] reveals an arginyl-glycyl-aspartate (RGD) tripeptide that is the consensus sequence for binding of adhesive plasma proteins of the fibrinolytic system, such as fibrinogen and von Willebrand factor, to the platelet membrane glycoprotein Ilb-IIIa (GPIIb-IIIa) complex. Therefore, we undertook the present study to further investigate the role of Lp(a) in hemostasis. Binding of I-Lp(a) to a single platelet membrane-associated protein (137±6 kD) comigrating with platelet GPIIb (140 kD) was found to be specific, saturable, and Ca independent. Binding of I-Lp(a) to resting human blood platelets was saturable, insensitive to temperature, and independent of the apo(a) isoform (B, SI through S3). Scatchard analysis revealed a Kd of 7.2± 1.8x10"' mol/L, with 729±313 Lp(a) molecules bound per platelet. Monoclonal anti-GPIIb IgG diminished Lp(a) binding by approximately 80%, monoclonal anti-GPIIb-IIIa IgG by 60%, and antiGPIIIa IgG by just 15%. I-Lp(a) binding was competitively The main physiological role of platelets in the hemostatic system is to provide the initial protection against hemorrhage after an interruption of vascular integrity. Thus, whenever the continuous interior layer of vascular endothelium is interrupted, platelets become attached to exposed subendothelium. The subsequent activation of platelets and their adhesion to the fibrin network are key elements in the formation of a thrombus' —a process that involves a variety of receptor-mediated steps. Conversion of platelet membrane glycoprotein lib (GPIIb) and GPIIIa from the latent state to the GPIIb-IIIa complex provides a specific binding site for adhesive plasma proteins of the fibrinolytic system such as fibrinogen, fibronectin, von WilleReceived September 23, 1993; revision accepted October 28, 1993. From the Department of Internal Medicine, Division of Endocrinology and Metabolism, Philipps University, Marburg, Germany (E.M., A.S.); the Institute of Medical Biochemistry, KarlFranzens University, Graz, Austria (E.M., A.I., G.M.K.); and the Heart Research Institute, Biochemistry Group, Sydney, Australia (W.S.). Presented in part at the Fourth Erfurt Conference on Platelets, June 1992, Erfurt, Germany, and published in abstract form {Platelets. 1993;4:105). Reprint requests to Dr Wolfgang Sattler, Karl-Franzens University, Institute of Medical Biochemistry, Harrachgasse 21, A-8010 Graz, Austria. inhibited to the same extent by either unlabeled Lp(a) or fibrinogen. Lowand high-density lipoproteins were much weaker competitors. A polyclonal antibody raised against the RGDGQSYRGT sequence of apo(a) was used to verify the presence of an RGD sequence in the different Lp(a) preparations investigated. However, two lines of evidence indicated that the RGD sequence is not the binding domain mediating Lp(a) binding to platelets. First, incubation of platelets with isolated RGD tripeptide did not influence Lp(a) binding. Second, coincubation of I-Lp(a) with anti-CRGDGQSYRGT IgG or anti-CRGDGQSYRGT IgG F(ab')2 fragments had no effect on the binding of I-Lp(a) to intact, resting platelets. These data strongly suggest that platelet GPIIb is the major Lp(a) binding protein, but binding of Lp(a) is apparently not mediated by the RGD peptide motif to GPIIb-IIIa. (Arterioscler Thromb. 1994;14:345-352.)