Abstract 18777: Glucagon-like Peptide-1 Inhibits Thrombin-induced Human Platelet Aggregation

Abstract

Background: Studies of glycemic control in diabetics receiving glucagon-like peptide-1 (GLP-1)-targeted drugs have suggested reduced incidences of cardiovascular events over 6-12 months. Mechanisms underlying this surprisingly rapid effect are not known.

Methods &

Results: To investigate potential anti-thrombotic effects of GLP-1 and related analogs, we tested GLP-1(7-36)NH2, the most abundant endogenous GLP-1, and the commercially available GLP-1 analog exenatide in thrombin-induced platelet aggregation assays using freshly isolated gel-filtered human platelets (3x108/ml) in normoglycemic conditions (5.5mM glucose). Incubation with GLP-1 (10-10 to 10-6 M) or exenatide (5x10-10 to 10-8 M) for 15 min at 37°C delayed and reduced platelet aggregation as compared to PBS-treated controls. GLP-1 (10^-9M) resulted in a 60% decrease in aggregation 1 min after thrombin stimulation. To investigate whether this effect may be mediated by a GLP-1 receptor (R), whole cell lysates (40 µg) from gel-filtered human platelets and the human megakaryocyte (MGK) cell line MEG-01 were probed with a polyclonal anti- GLP-1R Ab (1:1250; Santa Cruz). Although Western blot showed bands (∼56 kD) consistent with the known GLP-1R in both platelets and MEG-01 cells, GAPDH-normalized expression levels of GLP-1R were ∼13 and ∼45 fold lower in these cells than in human pancreas. Given the limited specificity of Abs raised against GPCRs, RT-PCR was performed on RNA from MEG-01 cells with primers designed to yield full-length human GLP-1R cDNA. PCR products were cloned and sequenced, revealing inserts identical to GLP-1R. As this receptor is known to be adenylate cyclase-coupled, cAMP assays were also performed (EIA kit, Cayman Chemicals). MEG-01 cells incubated for 15 min with 10-7 M GLP-1 showed a ∼60% increase in intracellular cAMP, an inhibitor of platelet activation, as compared to untreated controls; while the cAMP response to prostacyclin PGI2 was much greater (positive control; 16 fold).

Conclusion: Human platelets and MGK express low levels of GLP-1R. While GLP-1 induced only small increases in cAMP in MGK, both GLP-1 and exenatide inhibited thrombin-induced platelet aggregation. These data suggest that GLP-1-targeted drugs may possess anti-platelet effects.