Regulation of Platelet Functions by Targeting STAT3 with a g-Quartet Oligonucleotide.

Collagen exposed on the subendothelial matrix supports platelet adhesion and aggregation at the site of vessel injury. Its interaction with the platelet membrane receptor GP V/FcRγ chain initiates intracellular signals that recruit the hematopoietic tyrosine kinase Syk to immunoreceptor tyrosine activation motifs (ITAMs) within the FcRγ cytoplasmic tail, leading a cascade of intracellular signal events that eventually activate platelets. In the present study, we demonstrated that collagen and the collagen-related peptide (CRP) stimulation also dose-dependently (2.5–10 mg/ml, 10 min) phosphorylated the signal transducers and activators of transcription 3 (STAT3) at the tyrosine residue 705 (Tyr705) in platelets, indicating that STAT3 participates in collagen-induced platelet activation, likely through a non-transcriptional mechanism. To test this hypothesis, we determined whether a STAT3-blocking G-rich oligonucleotide (T40214, GGGCGGGCGGGCGGGC) blocks platelet activation and aggregation induced by collagen or CRP. T40214 was designed based on G-rich DNA sequences, which were originally identified in the telomeres DNA to have the ability to form the protein-binding rigid G-quartet structures. It was formed through four guanine (G) bases into cyclic Hoogsteen H-bonding. NMR determined the tertiary structure of T40214 as two stacked identical G-quartets in the center and two G-C-G-C loop domains on the top and bottom, respectively. It has previously been shown to inhibit STAT3 phosphorylation in cancer cell lines and to induce tumor apoptosis in vivo. T40214 was delivered into platelets using nano-particle polyethylenimine (PEI) as the carrier. The treatment of washed platelets with T40214 inhibited STAT3 phosphorylation induced by collagen (10 mg/ml, 10 min), while the scramble oligonucleotides did not. Incubation of washed platelets and platelets-rich plasma (PRP) with T40214 inhibited collagen-induced platelets aggregation. Finally, thrombus formation on immobilized collagen was significantly reduced when whole blood treated with T40214 (10ug/ml) under a wall shear stress of 30 dyn/cm². Results demonstrate a novel role of STAT3 in regulating the collagen-induced intracellular signal and platelet functions. The STAT3-blocking G-quartet offers a new class of potential platelet-regulating reagents derived from screening tertiary structure of the G-rich DNA sequences that form protein-binding G-quartets.