Platelets are small circulating blood cells that play essential roles in the maintenance of haemostasis via blood clotting. However, they also play critical roles in the regulation of innate immune responses. Inflammatory receptors, specifically Toll-like receptor (TLR)-4, have been reported to modify platelet reactivity. A plethora of studies have reported controversial functions of TLR4 in the modulation of platelet function using various chemotypes and preparations of its ligand, lipopolysaccharide (LPS). The method of preparation of LPS may explain these discrepancies however this is not fully understood. Hence, to determine the impact of LPS on platelet activation, we used ultrapure preparations of LPS from Escherichia coli (LPSEC), Salmonella minnesota (LPSSM), and Rhodobacter sphaeroides (LPSRS) and examined their actions under diverse experimental conditions in human platelets. LPSEC did not affect platelet activation markers such as inside-out signalling to integrin αIIbβ3 or P-selectin exposure upon agonist-induced activation in platelet-rich plasma or whole blood whereas LPSSM and LPSRS inhibited platelet activation under specific conditions at supraphysiological concentrations. Overall, our data demonstrate that platelet activation is not largely influenced by any of the ultrapure LPS chemotypes used in this study on their own except under certain conditions.
Flavonoids exert innumerable beneficial effects on cardiovascular health including the reduction of platelet activation, and thereby, thrombosis. Hence, flavonoids are deemed to be a molecular template for the design of novel therapeutic agents for various diseases including thrombotic conditions. However, the structure-activity relationships of flavonoids with platelets is not fully understood. Therefore, this study aims to advance the current knowledge on structure-activity relationships of flavonoids through a systematic analysis of structurally-related flavones. Here, we investigated a panel of 16 synthetic flavones containing hydroxy or methoxy groups at C-7,8 positions on the A-ring, with a phenyl group or its bioisosteres as the B-ring, along with their thio analogues possessing a sulfur molecule at the 4th carbon position of the C-ring. The antiplatelet efficacies of these compounds were analysed using human isolated platelets upon activation with cross-linked collagen-related peptide by optical aggregometry. The results demonstrate that the hydroxyl groups in flavonoids are important for optimum platelet inhibitory activities. In addition, the 4-C=O and B ring phenyl groups are less critical for the antiplatelet activity of these flavonoids. This structure-activity relationship of flavonoids with the modulation of platelet function may guide the design, optimisation and development of flavonoid scaffolds as antiplatelet agents.
Isorhapontigenin is a polyphenolic compound found in Chinese herbs and grapes. It is a methoxylated analogue of a stilbenoid, resveratrol, which is well-known for its various beneficial effects including anti-platelet activity. Isorhapontigenin possesses greater oral bioavailability than resveratrol and has also been identified to possess anti-cancer and anti-inflammatory properties. However, its effects on platelet function have not been reported previously. In this study, we report the effects of isorhapontigenin on the modulation of platelet function. Isorhapontigenin was found to selectively inhibit ADP-induced platelet aggregation with an IC50 of 1.85 μM although it displayed marginal inhibition on platelet aggregation induced by other platelet agonists at 100 μM. However, resveratrol exhibited weaker inhibition on ADP-induced platelet aggregation (IC50 > 100 μM) but inhibited collagen induced platelet aggregation at 50 μM and 100 μM. Isorhapontigenin also inhibited integrin αIIbβ3 mediated inside-out and outside-in signalling and dense granule secretion in ADP-induced platelet activation but interestingly, no effect was observed on α-granule secretion. Isorhapontigenin did not exert any cytotoxicity on platelets at the concentrations of up to 100 μM. Furthermore, it did not affect haemostasis in mice at the IC50 concentration (1.85 μM). In addition, the mechanistic studies demonstrated that isorhapontigenin increased cAMP levels and VASP phosphorylation at Ser157 and decreased Akt phosphorylation. This suggests that isorhapontigenin may interfere with cAMP and PI3K signalling pathways that are associated with the P2Y12 receptor. Molecular docking studies emphasised that isorhapontigenin has greater binding affinity to P2Y12 receptor than resveratrol. Our results demonstrate that isorhapontigenin has selective inhibitory effects on ADP-stimulated platelet activation possibly via P2Y12 receptor.