A Novel Small Molecule 1,2,3,4,6-penta-O-galloyl-α-D-glucopyranose Mimics the Antiplatelet Actions of Insulin

BACKGROUND: We have shown that 1,2,3,4,6-penta-O-galloyl-α-D-glucopyranose (α-PGG), an orally effective hypoglycemic small molecule, binds to insulin receptors and activates insulin-mediated glucose transport. Insulin has been shown to bind to its receptors on platelets and inhibit platelet activation. In this study we tested our hypothesis that if insulin possesses anti-platelet properties then insulin mimetic small molecules should mimic antiplatelet actions of insulin. PRINCIPAL FINDINGS: Incubation of human platelets with insulin or α-PGG induced phosphorylation of insulin receptors and IRS-1 and blocked ADP or collagen induced aggregation. Pre-treatment of platelets with α-PGG inhibited thrombin-induced release of P-selectin, secretion of ATP and aggregation. Addition of ADP or thrombin to platelets significantly decreased the basal cyclic AMP levels. Pre-incubation of platelets with α-PGG blocked ADP or thrombin induced decrease in platelet cyclic AMP levels but did not alter the basal or PGE(1) induced increase in cAMP levels. Addition of α-PGG to platelets blocked agonist induced rise in platelet cytosolic calcium and phosphorylation of Akt. Administration of α-PGG (20 mg kg(-1)) to wild type mice blocked ex vivo platelet aggregation induced by ADP or collagen. CONCLUSIONS: These data suggest that α-PGG inhibits platelet activation, at least in part, by inducing phosphorylation of insulin receptors leading to inhibition of agonist induced: (a) decrease in cyclic AMP; (b) rise in cytosolic calcium; and (c) phosphorylation of Akt. These findings taken together with our earlier reports that α-PGG mimics insulin signaling suggest that inhibition of platelet activation by α-PGG mimics antiplatelet actions of insulin

α-PGG inhibited collagen-induced phosphorylation of Akt.

<p>Washed human platelets were stimulated with collagen (1.0 µg mL⁻¹) in the presence or absence of α-PGG (10 µM). Lysis buffer was added to samples at 6 min to terminate reactions. Total Akt and p-Akt were visualized after PAGE and Western blotting as described in the Experimental Procedures. The β-actin was used as a loading control.</p

Administration of α-PGG inhibited <i>ex vivo</i> platelet aggregation induced by ADP or collagen.

<p>A, ADP or B, collagen was added to platelet-rich plasma, prepared from murine blood drawn at 30 min after oral administration of α-PGG (20 mg kg⁻¹) or vehicle, to induce aggregation. The aggregation tracings are representative of three experiments.</p

Insulin or α-PGG induced phosphorylation of insulin receptors and IRS-1.

<p>Washed human platelets were incubated with insulin (100 nM) or α-PGG (10 µM) for five and ten minutes. The reactions were stopped by adding lysis buffer and the total and phosphorylated insulin receptors (A) and total IRS-1 and phosphorylated IRS-1 (B) were visualized after immuno-precipitation and Western blotting. The phosphorylation of insulin receptors and IRS-1 was quantified by densitometry.</p

α-PGG inhibited thrombin induced rise in cytosolic calcium.

<p>Changes in cytosolic calcium were quantified in Fura2/AM loaded platelets. Platelets were incubated with α-PGG (3 or 10 µM) prior to stimulation with thrombin (0.1 U mL⁻¹) and changes in calcium levels were recorded by fluorescence spectrometry as described in Experimental Procedures. The results are reported as means ± SE (n = 4).</p

α-PGG inhibited ADP- or thrombin-induced lowering of cyclic AMP.

<p>ADP (10 µM), thrombin (0.1 U ml⁻¹) or PGE₁ (1 µM) induced changes in platelet cyclic AMP (pmoles/10⁸ platelets) levels were quantified in the presence or absence of α-PGG (10 µM) using enzyme-linked assay kits as described in Experimental Procedures. ADP and thrombin decreased basal cyclic AMP levels by 24% (*p<0.03) and 22% (*p<0.02) respectively. α-PGG blocked ADP and thrombin induced decrease in cyclic AMP levels.</p

α-PGG inhibited thrombin induced secretion from the α- and dense-granules and platelet aggregation.

<p>Washed human platelets were stimulated with thrombin (0.1 U mL⁻¹) in the presence or absence of α-PGG and expression of P-selectin (A) and secretion of ATP (B) and platelet aggregation (C) was monitored as detailed in Experimental Procedures. The results are reported as means ± SD for P-selectin expression (n = 3). ATP secretion and aggregation tracings are representative of three experiments.</p