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Technical Brief

Nonmuscle Myosin IIA Regulates Platelet Contractile Forces Through Rho Kinase and Myosin Light-Chain Kinase

[+] Author and Article Information
Shirin Feghhi

Department of Mechanical Engineering,
University of Washington,
Seattle, WA 98195
e-mail: shfeghhi@uw.edu

Wes W. Tooley

Department of Mechanical Engineering,
University of Washington,
Seattle, WA 98195
e-mail: westooley@gmail.com

Nathan J. Sniadecki

Department of Mechanical Engineering,
University of Washington,
Seattle, WA 98195
e-mail: nsniadec@uw.edu

Manuscript received October 7, 2015; final manuscript received August 15, 2016; published online September 1, 2016. Assoc. Editor: Mohammad Mofrad.S. Feghhi and W. W. Tooley performed the experiments and analyzed the data, and S. Feghhi and N. J. Sniadecki designed the research and wrote the manuscript.

J Biomech Eng 138(10), 104506 (Sep 01, 2016) (4 pages) Paper No: BIO-15-1497; doi: 10.1115/1.4034489 History: Received October 07, 2015; Revised August 15, 2016

Platelet contractile forces play a major role in clot retraction and help to hold hemostatic clots against the vessel wall. Platelet forces are produced by its cytoskeleton, which is composed of actin and nonmuscle myosin filaments. In this work, we studied the role of Rho kinase, myosin light-chain kinase, and myosin in the generation of contractile forces by using pharmacological inhibitors and arrays of flexible microposts to measure platelet forces. When platelets were seeded onto microposts, they formed aggregates on the tips of the microposts. Forces produced by the platelets in the aggregates were measured by quantifying the deflection of the microposts, which bent in proportion to the force of the platelets. Platelets were treated with small molecule inhibitors of myosin activity: Y-27632 to inhibit the Rho kinase (ROCK), ML-7 to inhibit myosin light-chain kinase (MLCK), and blebbistatin to inhibit myosin ATPase activity. ROCK inhibition reduced platelet forces, demonstrating the importance of the assembly of actin and myosin phosphorylation in generating contractile forces. Similarly, MLCK inhibition caused weaker platelet forces, which verifies that myosin phosphorylation is needed for force generation in platelets. Platelets treated with blebbistatin also had weaker forces, which indicates that myosin's ATPase activity is necessary for platelet forces. Our studies demonstrate that myosin ATPase activity and the regulation of actin–myosin assembly by ROCK and MLCK are needed for the generation of platelet forces. Our findings illustrate and explain the importance of myosin for clot compaction in hemostasis and thrombosis.

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Figures

Grahic Jump Location
Fig. 1

Platelet aggregates on arrays of microposts. (a) Scanning electron microscopy image of platelets on an array of microposts with pseudocoloring (green). (b) Fluorescent image of a platelet aggregate on microposts. Forces are measured by analyzing the deflection of the microposts. Green: actin and red: microposts.

Grahic Jump Location
Fig. 2

Platelet contractile forces are myosin-dependent. (a) Fluorescent image of a platelet aggregate on microposts treated with 10 μM Y-2732. (b) Platelets forces are reduced by Y-27632 concentration. (c) Fluorescent image of a platelet aggregate on microposts treated with 5 μM ML-7. (d) Platelets forces are reduced by ML-7 concentration. (e) Fluorescent image of a platelet aggregate on microposts treated with 10 μM blebbistatin. (f) Platelets forces are reduced by blebbistatin concentration. Green: actin and red: microposts.

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