BACKGROUND: Shape change and centralization of granules surrounded by a microtubular coil (internal contraction) are among the earliest morphologic changes observed following platelet activation. Myosin IIA contributes to initiation of platelet shape change, but its role in internal contraction has not been defined. OBJECTIVE: To define the contribution of myosin IIA to platelet internal contraction. METHODS: Aspirin-treated platelets suspended in calcium-free buffer were activated with a low concentration (25 nm) of the thromboxane A(2) analog U46619 which initiated shape change and internal contraction via a Rho kinase pathway. Shape change and internal contraction were assessed by aggregometry and transmission electron microscopy (TEM), and Rho activation and myosin regulatory light chain (MRLC) phosphorylation were studied concurrently. RESULTS AND CONCLUSIONS: Low-concentration blebbistatin (10 microm) inhibited internal contraction in the majority of platelets with minimal inhibition of shape change without significant suppression of MRLC phosphorylation. Higher blebbistatin concentrations (25-100 microm) produced concentration-dependent inhibition of aggregation, shape change, Rho activation, and MRLC phosphorylation. These data demonstrate: (i) direct platelet myosin IIA participation in internal contraction; and (ii) inhibition of Rho activation and MRLC phosphorylation by >10 microm blebbistatin.
BACKGROUND: Shape change and centralization of granules surrounded by a microtubular coil (internal contraction) are among the earliest morphologic changes observed following platelet activation. Myosin IIA contributes to initiation of platelet shape change, but its role in internal contraction has not been defined. OBJECTIVE: To define the contribution of myosin IIA to platelet internal contraction. METHODS:Aspirin-treated platelets suspended in calcium-free buffer were activated with a low concentration (25 nm) of the thromboxane A(2) analog U46619 which initiated shape change and internal contraction via a Rho kinase pathway. Shape change and internal contraction were assessed by aggregometry and transmission electron microscopy (TEM), and Rho activation and myosin regulatory light chain (MRLC) phosphorylation were studied concurrently. RESULTS AND CONCLUSIONS: Low-concentration blebbistatin (10 microm) inhibited internal contraction in the majority of platelets with minimal inhibition of shape change without significant suppression of MRLC phosphorylation. Higher blebbistatin concentrations (25-100 microm) produced concentration-dependent inhibition of aggregation, shape change, Rho activation, and MRLC phosphorylation. These data demonstrate: (i) direct platelet myosin IIA participation in internal contraction; and (ii) inhibition of Rho activation and MRLC phosphorylation by >10 microm blebbistatin.
Authors: Gauthami Jalagadugula; Guangfen Mao; Gurpreet Kaur; Lawrence E Goldfinger; Danny N Dhanasekaran; A Koneti Rao Journal: Blood Date: 2010-09-27 Impact factor: 22.113
Authors: Miguel Vicente-Manzanares; Xuefei Ma; Robert S Adelstein; Alan Rick Horwitz Journal: Nat Rev Mol Cell Biol Date: 2009-11 Impact factor: 94.444
Authors: Benjamin Aj Reddi; Samantha M Iannella; Stephanie N O'Connor; Adam M Deane; Scott R Willoughby; David P Wilson Journal: Intensive Care Med Exp Date: 2015-02-12