Platelet Immobilization on Supported Phospholipid Bilayers for Single Platelet Studies

Abstract

The worldwide cardiovascular disease (CVD) epidemic is of grave concern. A major role in the etiology of CVDs is played by the platelets (thrombocytes). Platelets are anuclear cell fragments circulating in the blood. Their primary function is to catalyze clot formation, limiting traumatic blood loss in the case of injury. The same process leads to thrombosis in the case of CVDs, which are commonly managed with antiplatelet therapy. Platelets also have other, nonhemostatic functions in wound healing, inflammation, and tissue regeneration. They play a role in the early stages of atherosclerosis and the spread of cancer through metastases. Much remains to be learned about the regulation of these diverse platelet functions under physiological and pathological conditions. Breakthroughs in this regard are expected to come from single platelet studies and systems approaches. The immobilization of platelets at surfaces is advantageous for developing such approaches, but platelets are activated when they come in contact with foreign surfaces. In this work, we develop and validate a protocol for immobilizing platelets on supported lipid bilayers without activation due to immobilization. Our protocol can therefore be used for studying platelets with a wide variety of surface-sensitive techniques.

Supported lipid bilayer platforms to probe cell mechanobiology

“…By comparing Notch activation in cells on fluid, nonfluid, and rigid surfaces, Narui and Salaita identified that the Notch/Delta pathway is mechanosensitive and responds nonlinearly to ligand fluidity [41]. More recently, it was shown that platelets prefer to adhere to crowded membranes [90]. A nonfluid interface for cell adhesion was also recently fabricated by covalently linking ECM proteins to fluid lipids [91].…”

Atomic Force Microscopy-Based Force Spectroscopy and Multiparametric Imaging of Biomolecular and Cellular Systems

“…Several methods have been established to guide the attachment of cells to AFM supports, including adjustment of electrostatic interactions, 103 coating the support by lectins or extracellular matrix proteins, 104 mechanical trapping, 105 or biotinylation of cell surface proteins. 106 When choosing an immobilization strategy, the user should keep in mind that this might affect the native state, structure, or functionality of the cell. For example, concanavalin A is known to elicit changes in the functional state of mononuclear phagocytes and lymphocytes, 107,108 and the mechanical trapping of living cells can affect their mechanical properties.…”

“…It is also important to carefully choose the correct immobilization procedure for cells to avoid, alternatively, their detachment from the support and their functional impairment. Several methods have been established to guide the attachment of cells to AFM supports, including adjustment of electrostatic interactions, coating the support by lectins or extracellular matrix proteins, mechanical trapping, or biotinylation of cell surface proteins . When choosing an immobilization strategy, the user should keep in mind that this might affect the native state, structure, or functionality of the cell.…”

Optical uncaging of ADP reveals the early calcium dynamics in single, freely moving platelets

“…Anyway, any contact with artificial materials can activate platelets, for instance, they may undergo transformation into the spread form on the glass. In a recently proposed technique, biotinylated platelets were anchored onto phospholipid bilayer with streptavidin [21]. While this technique is promising, it implies the modification of platelet membrane, which might also influence the activation signaling.…”