Abstract 11593: Thrombus Formation Under Flow is Inhibited by the Mechanosensitive Cation Channel Blockers GsMTx-4 Peptide and Gadolinium Chloride

Abstract

Introduction: Ion channels contribute significantly to platelet physiology, including control of Ca 2+ 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 [Ca 2+ ] i (intracellular Ca 2+ ) responses were studied by confocal microscopy using the indicators DiOC 6 and Fluo-3, respectively, under physiological levels of shear stress in a parallel-plate flow chamber. Aggregation and [Ca 2+ ] i responses 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 (GdCl 3 ) (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±7739.4μm 3 to 13478.9±2744.8μm 3 by GdCl 3 (n=6, P<0.01) and to 25125.9±6433.5μm 3 (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 GdCl 3 did not affect platelet shape change or aggregation under static conditions. Pre-treatment with either GsMTx-4 or GdCl 3 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 [Ca 2+ ] i levels; at a shear rate of 3989.2s -1 , F/F 0 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 Ca 2+ by EGTA. Conclusions: We provide evidence for the first time for a functional contribution of MS cation channels in platelet physiology and thrombosis.