Imaging the elastic modulus of human platelets during thrombininduced activation using scanning ion conductance microscopy

Johannes Rheinlaender; Sebastian Vogel; Jan Seifert; Marc Schächtele; Oliver Borst; Florian Lang; Meinrad Gawaz; Tilman E. Schäffer

doi:10.1160/TH14-05-0414

Thrombosis and Haemostasis, Inhaltsverzeichnis

Thromb Haemost 2015; 113(02): 305-311
DOI: 10.1160/TH14-05-0414

Cellular Haemostasis and Platelets

Schattauer GmbH

Imaging the elastic modulus of human platelets during thrombininduced activation using scanning ion conductance microscopy

Johannes Rheinlaender^†

¹Institute of Applied Physics, Eberhard Karls University Tübingen, Germany

,

Sebastian Vogel^†

²Department of Cardiology and Cardiovascular Diseases, Eberhard Karls University Tübingen, Germany

,

Jan Seifert

¹Institute of Applied Physics, Eberhard Karls University Tübingen, Germany

,

Marc Schächtele

¹Institute of Applied Physics, Eberhard Karls University Tübingen, Germany

,

Oliver Borst

²Department of Cardiology and Cardiovascular Diseases, Eberhard Karls University Tübingen, Germany

,

Florian Lang

³Department of Physiology, Eberhard Karls University Tübingen, Germany

,

Meinrad Gawaz^‡

²Department of Cardiology and Cardiovascular Diseases, Eberhard Karls University Tübingen, Germany

,

Tilman E. Schäffer^‡

¹Institute of Applied Physics, Eberhard Karls University Tübingen, Germany

› Institutsangaben

Abstract

Summary

Platelet activation plays a critical role in haemostasis and thrombosis. It is well-known that platelets generate contractile forces during activation. However, their mechanical material properties have rarely been investigated. Here, we use scanning ion conductance microscopy (SICM) to visualise morphological and mechanical properties of live human platelets at high spatial resolution. We found that their mean elastic modulus decreases during thrombin-induced activation by about a factor of two. We observed a similar softening of platelets during cytochalasin D-induced cytoskeleton depolymerisation. However, thrombin-induced temporal and spatial modulations of the elastic modulus were substantially different from cytochalasin D-mediated changes. We thereby provide new insights into the mechanics of haemostasis and establish SICM as a novel imaging platform for the ex vivo investigation of the mechanical properties of live platelets.

Keywords

Thrombin, stiffness - scanning ion conductance microscopy - SICM - cytochalasin D

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Referenzen

References
1 Nurden AT. Platelets, inflammation and tissue regeneration. Thromb Haemost 2011; 105 (Suppl. 01) S13-33.
2 Ruggeri ZM. Platelets in atherothrombosis. Nat Med 2002; 08: 1227-1234.
3 Cohen I. The contractile system of blood platelets and its function. Methods Achiev Exp Pathol 1979; 09: 40-86.
4 Fox JE. The platelet cytoskeleton. Thromb Haemost 1993; 70: 884-893.
5 De Cristofaro R, De Candia E. Thrombin Domains: Structure, Function and Interaction with Platelet Receptors. J Thromb Thrombolysis 2003; 15: 151-163.
6 Hartwig JH. The Platelet Cytoskeleton. In: Platelets. Third Ed.. Academic Press; 2013. pp. 145-168.
7 Aslan JE, McCarty OJT. Rho GTPases in platelet function. J Thromb Haemost 2013; 11: 35-46.
8 Casella JF, Flanagan MD, Lin S. Cytochalasin D inhibits actin polymerisation and induces depolymerisation of actin filaments formed during platelet shape change. Nature 1981; 293: 302-305.
9 Bearer EL, Prakash JM, Li Z. Actin dynamics in platelets. In: Int Rev Cytol. Academic Press; 2002. pp. 137-182.
10 Weisel JW. Enigmas of Blood Clot Elasticity. Science 2008; 320: 456-457.
11 Walch M, Ziegler U, Groscurth P. Effect of streptolysin O on the microelasticity of human platelets analyzed by atomic force microscopy. Ultramicroscopy 2000; 82: 259-267.
12 Fritz M, Radmacher M, Gaub HE. In vitro activation of human platelets triggered and probed by atomic force microscopy. Exp Cell Res 1993; 205: 187-190.
13 Shamova EV, Gorudko IV, Drozd ES. et al. Redox regulation of morphology, cell stiffness, and lectin-induced aggregation of human platelets. Eur Biophys J 2011; 40: 195-208.
14 Du Plooy JN, Buys A, Duim W. et al. Comparison of Platelet Ultrastructure and Elastic Properties in Thrombo-Embolic Ischemic Stroke and Smoking Using Atomic Force and Scanning Electron Microscopy. PLoS ONE 2013; 08: e69774.
15 Hansma PK, Drake B, Marti O. et al. The scanning ion-conductance microscope. Science 1989; 243: 641-643.
16 Korchev YE, Bashford CL, Milovanovic M. et al. Scanning ion conductance microscopy of living cells. Biophys J 1997; 73: 653-658.
17 Zhang Y, Liu X, Liu L. et al. Contact- and agonist-regulated microvesiculation of human platelets. Thromb Haemost 2013; 110: 331-339.
18 Alesutan I, Seifert J, Pakladok T. et al. Chorein Sensitivity of Actin Polymerisation, Cell Shape and Mechanical Stiffness of Vascular Endothelial Cells. Cell Physiol Biochem 2013; 32: 728-742.
19 Sánchez D, Johnson N, Li C. et al. Noncontact measurement of the local mechanical properties of living cells using pressure applied via a pipette. Biophys J 2008; 95: 3017-3027.
20 Pellegrino M, Pellegrini M, Orsini P. et al. Measuring the elastic properties of living cells through the analysis of current-displacement curves in scanning ion conductance microscopy. Pflug Arch Eur J Phy 2012; 464: 307-316.
21 Schäffer TE. Nanomechanics of Molecules and Living Cells with Scanning Ion Conductance Microscopy. Anal Chem 2013; 85: 6988-6994.
22 Potter CM, Schobesberger S, Lundberg MH. et al. Shape and compliance of en-dothelial cells after shear stress in vitro or from different aortic regions: scanning ion conductance microscopy study. PLoS One 2012; 07: e31228.
23 Lab MJ, Bhargava A, Wright PT. et al. The scanning ion conductance microscope for cellular physiology. Am J Physiol Heart Circ Physiol 2013; 304: H1-H11.
24 Rheinlaender J, Schäffer TE. Mapping the mechanical stiffness of live cells with the scanning ion conductance microscope. Soft Matter 2013; 09: 3230-3236.
25 Rheinlaender J, Geisse NA, Proksch R. et al. Comparison of scanning ion conductance microscopy with atomic force microscopy for cell imaging. Langmuir 2011; 27: 697-704.
26 Gawaz M, Neumann FJ, Dickfeld T. et al. Activated platelets induce monocyte chaemotactic protein-1 secretion and surface expression of intercellular adhesion molecule-1 on endothelial cells. Circulation 1998; 98: 1164-1171.
27 Limpert E, Stahel WA, Abbt M. Log-normal distributions across the sciences: Keys and clues. BioScience 2001; 51: 341-352.
28 Rotsch C, Braet F, Wisse E. et al. AFM imaging and elasticity measurements on living rat liver macrophages. Cell Biol Int 1997; 21: 685-696.
29 Cross SE, Jin Y-S, Rao J. et al. Nanomechanical analysis of cells from cancer patients. Nat Nanotechnol 2007; 02: 780-783.
30 Limpert E, Stahel WA. Problems with using the normal distribution - and ways to improve quality and efficiency of data analysis. PLoS ONE 2011; 06: e21403.
31 Fox JEB, Phillips DR. Inhibition of actin polymerisation in blood platelets by cy-tochalasins. Nature 1981; 292: 650-652.
32 Lam WA, Chaudhuri O, Crow A. et al. Mechanics and contraction dynamics of single platelets and implications for clot stiffening. Nat Mater 2011; 10: 61-66.
33 Schwarz Henriques S, Sandmann R, Strate A. et al. Force field evolution during human blood platelet activation. J Cell Sci 2012; 125: 3914-3920.
34 Liang XM, Han SJ, Reems J-A. et al. Platelet retraction force measurements using flexible post force sensors. Lab Chip 2010; 10: 991-998.
35 Suzuki-Inoue K, Hughes CE, Inoue O. et al. Involvement of Src kinases and PLCy2 in clot retraction. Thrombosis Research 2007; 120: 251-258.
36 Vretenbrant K, Ramström S, Bjerke M. et al. Platelet activation via PAR4 is involved in the initiation of thrombin generation and in clot elasticity development. Thromb Haemost 2007; 97: 417-424.
37 Zhou L, Schmaier AH. Platelet Aggregation Testing in Platelet-Rich Plasma: Description of Procedures With the Aim to Develop Standards in the Field. Am J Clin Pathol 2005; 123: 172-183.
38 Redondo PC, Harper MT, Rosado JA. et al. A role for cofilin in the activation of store-operated calcium entry by de novo conformational coupling in human platelets. Blood 2006; 107: 973-979.
39 Rheinlaender J, Schäffer TE. Image formation, resolution, and height measurement in scanning ion conductance microscopy. J Appl Phys 2009; 105: 094905.
40 Radmacher M, Fritz M, Kacher CM. et al. Measuring the viscoelastic properties of human platelets with the atomic force microscope. Biophys J 1996; 70: 556-567.
41 Jen CJ, McIntire LV. The structural properties and contractile force of a clot. Cell Motility 1982; 02: 445-455..
42 Carr Jr. ME. Development of platelet contractile force as a research and clinical measure of platelet function. Cell Biochem Biophys 2003; 38: 55-78.
43 Tran R, Myers DR, Ciciliano J. et al. Biomechanics of haemostasis and thrombosis in health and disease: from the macro- to molecular scale. J Cell Mol Med 2013; 17: 579-596.
44 Escolar G, Diaz-Ricart M, Cases A. et al. Abnormal cytoskeletal assembly in platelets from uremic patients. Am J Pathol 1993; 143: 823-831.

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