Aurin Tricarboxylic Acid Inhibits Platelet Adhesion to Collagen by Binding to the 509-695 Disulphide Loop of von Willebrand Factor and Competing with Glycoprotein Ib

Abstract

Aurin tricarboxylic acid (ATA) is known to inhibit ristocetin-induced platelet agglutination but not arachidonic acid-, epinephrine- or ADP-induced aggregation. Its capacity to abolish human von Willebrand factor (vWF)-platelet interactions was further investigated by measurement of platelet adhesion to collagen, platelet agglutination tests and binding studies. In flowing blood using parallel-plate perfusion chambers and human collagen, ATA inhibited platelet adhesion to completion in a dose-dependent manner only at the highest shear rate tested (2,600 s-1). It was without effect at 100 and 650 s-1. ATA completely abolished vWF-dependent platelet agglutination induced by ristocetin, botrocetin and asialo-vWF, respectively. 125I-vWF binding to ristocetin- and botrocetin-treated platelets, to heparin and to sulfatides as well as 125I-botrocetin binding to vWF was competitively inhibited by ATA. By contrast, binding of 125I-vWF to collagen was not affected. To further localize the domain of vWF interacting with ATA, experiments of inhibition of binding of selected 125I-monoclonal antibodies (MoAbs) to immobilized vWF by ATA were performed. Our data led to the conclusion that: 1) the interaction of ATA with vWF involves sequences of the A1 disulphide loop of vWF (residues 509-695) and close epitopes which interact with GPIb and 2) the inhibition of platelet adhesion by ATA occurs only at a high shear rate where vWF is known to play a key role. Thus ATA, which blocks the vWF/GPIb pathway by interfering with vWF and not with platelets, is a potential tool in preventing the early stages of thrombosis.