Abstract 11593: Thrombus Formation Under Flow is Inhibited by the Mechanosensitive Cation Channel Blockers GsMTx-4 Peptide and Gadolinium Chloride
Introduction: Ion channels contribute significantly to platelet physiology, including control of Ca2+ as a second messenger. Although there is a clear link between platelet activation and shear stress, no study has examined the functional relevance of mechanosensitive (MS) ion channels in platelets or their precursors, megakaryocytes.
Hypothesis: Human platelets express MS cation channels such as Piezo1, which contribute to platelet function and the development of thrombosis.
Methods: Thrombus formation and [Ca2+]i (intracellular Ca2+) responses were studied by confocal microscopy using the indicators DiOC6 and Fluo-3, respectively, under physiological levels of shear stress in a parallel-plate flow chamber. Aggregation and [Ca2+]iresponses to collagen and thrombin in washed human platelets were assessed by light transmission aggregometry and Fura-2 ratiometric measurements. GsMTx-4 (2.5μM) and gadolinium chloride (GdCl3) (30μM) were used as MS cation channel inhibitors, targeting Piezo1 and TRPC6 in platelets. Piezo1 mRNA and protein expression were studied in platelets and the megakaryocytic cell line Meg-01.
Results: Both inhibitors significantly inhibited thrombus formation on a collagen surface under normal arterial shear rate (average thrombus volume was reduced from 48132.7±7739.4μm3 to 13478.9±2744.8μm3 by GdCl3 (n=6, P<0.01) and to 25125.9±6433.5μm3 (n=6, P<0.05) by GsMTx-4). GsMTx-4 also caused a reduction in thrombin-induced platelet aggregation (to 53.6±14.0% of control, n=4, P<0.05), however GdCl3did not affect platelet shape change or aggregation under static conditions. Pre-treatment with either GsMTx-4 or GdCl3 did not reduce calcium influx in response to TRPC6 stimulation with 60μM OAG, suggesting a MS role for Piezo1, which was found at the mRNA transcript and protein level in platelets and Meg-01 cells. Meg-01 cells showed shear rate-dependent increases in [Ca2+]ilevels; at a shear rate of 3989.2s-1, F/F0 increased to 1.38±0.05 from 1.00±0.00 baseline (n=53 cells, P<0.0001), a response which was diminished by GsMTx-4, and by chelation of extracellular Ca2+ by EGTA.
Conclusions: We provide evidence for the first time for a functional contribution of MS cation channels in platelet physiology and thrombosis.
Author Disclosures: Z. Ilkan: None. J.R. Wright: None. L. Francescut: None. A.H. Goodall: None. M.P. Mahaut-Smith: None.
- © 2015 by American Heart Association, Inc.