Redox regulation of morphology, cell stiffness, and lectin-induced aggregation of human platelets

Abstract

Redox regulation and carbohydrate recognition are potent molecular mechanisms which can contribute to platelet aggregation in response to various stimuli. The purpose of this study is to investigate the relationship between these mechanisms and to examine whether cell surface glycocalyx and cell stiffness of human platelets are sensitive to the redox potential formed by glutathione. To this end, human platelets were treated with different concentrations (0.05 μM to 6 mM) and ratios of reduced or oxidized glutathione (GSH or GSSG), and platelet morphological, mechanical, and functional properties were determined using conventional light microscopy, atomic force microscopy, and lectin-induced cell aggregation analysis. It was found that lowering the glutathione redox potential changed platelet morphology and increased platelet stiffness as well as modulated nonuniformly platelet aggregation in response to plant lectins with different carbohydrate-binding specificity including wheat germ agglutinin, Sambucus nigra agglutinin, and Canavalia ensiformis agglutinin. Extracellular redox potential and redox buffering capacity of the GSSG/2GSH couple were shown to control the availability of specific lectin-binding glycoligands on the cell surface, while the intracellular glutathione redox state affected the general functional ability of platelets to be aggregated independently of the type of lectins. Our data provide the first experimental evidence that glutathione as a redox molecule can affect the mechanical stiffness of human platelets and induce changes of the cell surface glycocalyx, which may represent a new mechanism of redox regulation of intercellular contacts.

Comparison of Platelet Ultrastructure and Elastic Properties in Thrombo-Embolic Ischemic Stroke and Smoking Using Atomic Force and Scanning Electron Microscopy

“…The Young’s modulus for the fixed and dehydrated control platelets were in the range of 30–130 MPa (megapascal), which is comparable to results found in similar studies [20] and approximately a thousand times higher than living “wet” platelets, 1–50 kPa (kilopascal) [5].…”

Myeloperoxidase modulates human platelet aggregation via actin cytoskeleton reorganization and store-operated calcium entry

“…AFM measurements were performed as previously described (Shamova et al, 2011). Freshly washed platelets (2.5×10 8 cells/ml) in PBS containing 1 mM CaCl 2 and 0.5 mM MgCl 2 were incubated with or without MPO for 5 minutes and fixed for 30 minutes in 1.5% glutaraldehyde in PBS.…”

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

“…This softening was significantly (p = 0.046) less compared to platelets without pretreatment, which softened by 39 % (▶ Figure 2 G, Thrombin). In contrast, the addition of cytochalasin D to platelets pretreated with thrombin still resulted in a substantial and rapid softening of the platelets (▶ Figure 4 B Figure 2, available online at www.thrombosis-online.com) without the need of labelling (11), direct mechanical contact (12), or fixation (13,14), which are required for currently used methods. However, SICM is restricted to adhered platelets, which might become preactivated owing to the contact with the culture plate surface.…”

“…The elastic modulus of activated platelets had been estimated by AFM as about 1-50 kPa (40) and 10 kPa (32), which is in good agreement with the values found here. Measurements on fixed or even dried platelets (13,14) are of limited applicability, since they give elastic moduli more than three orders of magnitude larger than those of live platelets.…”

“…The underlying cytoskeletal remodelling processes in platelets (9) and their mechanical interaction with the environment (10) are still poorly understood, because current imaging methods are hampered by inherent limitations for morphological and mechanical assessment of dynamic platelet function. For instance, fluorescence microscopy may influence the mechanics of platelet cytos-keleton (11), atomic force microscopy (AFM) may trigger platelet activation owing to the direct mechanical contact with the scanning tip (12), and electron microscopy requires fixation, drying, and metal coating of the platelets (13,14).…”