Introduction Directory UMM :Data Elmu:jurnal:A:Atherosclerosis:Vol154.Issue1.Jan2001:

Atherosclerosis 154 2001 23 – 29 Platelet HDL 3 binding sites are not related to integrin a IIb b 3 GPIIb – IIIa Javier Pedren˜o a, , Conxita de Castellarnau b , Lluı´s Masana a a Facultat de Medicina, Uni6ersitat Ro6ira i Virgili, Unitat de Recerca en Lı´pids i Arteriosclerosi, Sant Llorenc¸ 21 Reus, Tarragona, Spain b Institut de Recerca del Hospital de la Santa Creu i Sant Pau de Barcelona, Barcelona, Spain Received 1 October 1999; received in revised form 14 February 2000; accepted 18 February 2000 Abstract Early studies considered that fibrinogen receptor glycoprotein [GP] IIb – IIIa or platelet integrin a IIb b 3 is the binding site for low-density lipoprotein LDL and high-density lipoprotein type 3 HDL 3 . Recent data, however, do not support the hypothesis that the binding of LDL to human intact resting platelets is related to integrin a IIb b 3 . In this study we present evidence that platelet integrin a IIb b 3 is also not involved in the interaction of HDL 3 and human intact resting platelets. Firstly, specific ligands for platelet integrin a IIb b 3 , such as fibrinogen, vitronectin, von Willebrand factor and fibronectin, were unable to inhibit the binding of HDL 3 to intact resting platelets. Secondly, the HDL 3 binding characteristics K d and B max values, the activation of protein kinase C PKC and the inhibition of thrombin-induced inositoltriphosphate IP 3 formation and calcium Ca 2 + mobilization mediated by HDL 3 particles were similar in platelets from control subjects and patients with type I and type II Glanzmann’s thrombasthenia, which are characterized by total and partial lack of GPIIb – IIIa and fibrinogen, respectively. In contrast, nitrosylation of tyrosine residues of HDL 3 by tetranitromethane fully abolished both the ability of particles to interact with its specific binding sites and the functional effects. Thirdly, polyclonal antibodies against the GPIIb – IIIa complex edu-3 and 5B12, human antiserums against platelet alloantigens anti-Bak aB and anti-PL A12 , anti-integrin subunits anti-a V and anti-b 3 , and a wide panel of monoclonal antibodies mAbs against well-known epitopes of GPIIb M3, M4, M5, M6, M8 and M95-2b and GPIIIa P23-7, P33, P37, P40, and P97 did not affect the binding of HDL 3 particles to human intact resting platelets. Overall results show that neither the GPIIb – IIIa complex nor GPIIb or GPIIIa individually are the membrane binding proteins for HDL 3 on intact resting platelets. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords : Platelets; HDL 3 ; Glanzmann’s thrombasthenia; Integrin a IIb b 3 www.elsevier.comlocateatherosclerosis

1. Introduction

Elevated serum cholesterol is widely accepted as a systemic thrombogenic risk factor in thrombus forma- tion on ruptured atherosclerotic plaques [1]. Platelet reactivity is enhanced by low density lipoproteins LDL, whereas high density lipoproteins HDL seem to have the opposite effect [2,3]. Specific platelet LDL and HDL receptors coupled to phosphatidylinositol hydrolysis have been implicated in the aggregatory response mediated by lipoproteins [4 – 6]. Advances in characterizing the platelet LDL receptor [7 – 10] and in understanding both the mechanisms of action and the effects on platelet function have been rapid [11 – 13]. In contrast, little is known about the platelet HDL recep- tor [14,15]. Early studies argued that platelet HDL 3 and LDL binding sites could be related to integrin a IIb b 3 . Koller et al. [16], using ligand and western blotting techniques, identified platelet membrane GPs such as GPIIb 136 kDa or a IIb subunit and GPIIIa 92 kDa or b 3 subunit, and also the intact GPIIb – IIIa complex, as the main GPs implicated in the binding of HDL 3 . Overall, these results indicated that fibrinogen and platelet lipoprotein binding sites for LDL and HDL 3 could be related via platelet integrin a IIb b 3 . However, several studies have recently demonstrated that GPIIb – Portions of this work were presented at the 11th International Symposium on Atherosclerosis, Paris, France, October 1997, and published in abstract form Atherosclerosis, 1997;138:184 – 185. Corresponding author. Tel.: + 34-977-759370; fax: + 34-977- 759322. E-mail address : jpfmcs.urv.es J. Pedren˜o. 0021-915001 - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S0021-91500000442-1 IIIa apparently does not act as a platelet LDL binding site [7,12,13,17] and that other receptors, as CD36 [18,19] are implicated in the LDL-platelet interaction. Overall, these new data provide conflicting results, and further studies are needed to explore the implication of GPIIb – IIIa in platelet HDL 3 binding. In fact, HDL 3 binding characteristics clearly differ from those of fibrinogen binding [4 – 6]. Moreover, rather than induc- ing inhibition, HDL 3 particles did not alter the binding of fibrinogen to activated platelets [7]. The aim of this study was to investigate the hypothetical involvement of platelet integrin a IIb b 3 on the binding of HDL 3 to intact human resting platelets.

2. Materials and methods