G-Quartet Oligonucleotide T70923 Blocks GP Ib-VWF Mediated Platelet Aggregation under Static and Flow Conditions.

Hemostasis at site of the vessel injury is initiated by platelets being tethered to exposed subendothelium. The tethering events are mediated by interaction between subendothelial von Willebrand factor (VWF) and its platelet receptor GP Ib-IX-V complex. If the ligand-receptor interaction, when it occurs in intact vascular lumen or at the site of ruptured atherosclerotic plaques, results in life-threatening thrombosis found in patients with thrombotic thrombocytopenic purpura and myocardial infarction. The development of these thrombotic events suggests that the VWF-GP Ib interaction may be a potential target for therapeutic intervention. To explore such potentials, we have developed and examined the effects of a G-quartet oligonucleotide (T30923, GGGTGGGTGGGTGGGT) on the VWF-GP Ib interaction. G-rich DNA sequences, from which the G-quartets derive, were originally identified in the telomeres DNA and have the ability to form rigid G-quartet structures that interact with proteins. The T30923 structure was designed and determined by nuclear magnetic resonance to form an intramolecular G-quartet that is composed of two identical G-quartets in the center and two GTGT loops on the top and bottom of stacked G-quartets, respectively. Platelet aggregation was induced by four agonists: ristocetin, botrocetin, collagen, and ADP in the presence of either 20 mM of T30923 or the control oligonucleotide. We found that T30923 specifically and dose-dependently blocked platelet aggregation induced by ristocetin, but not by other agonists, with a maximal inhibition achieved at 20 mM. Computer-simulation based on the crystal structure of the VWF A1 domain and the N-terminal VWF-binding domain of GP Iba suggested that the G-quartet binds to multiple sites on VWF-A1. Since the action of ristocetin mimics that of fluid shear stress, we tested the ability of T30923 to block VWF-GP Ib interaction under flow condition. Shear-induced platelet aggregation at 100 dyn/cm² was blocked 59.3%±11.8% by 20 mM of T30923, but not a control oligonucleotide. Furthermore, the G-quartet T30923 blocked over 80% of platelet adhesion to immobilized VWF, but not collagen under a constant wall shear stress of 30 dyn/cm². These results demonstrate the potential of T30923 to serve as a VWF antagonist. Furthermore, alterations of DNA sequences and tertiary structure provide a large number of G-quartet variants to be generated and then screened for specific phenotypes. The approach may lead to a new class of potential G-quartet based antithrombotic agents.