Ligand capture and activation of human platelets at monolayer modified gold surfaces

Abstract

Blood platelet adhesion is crucial in dictating haemocompatibility of medical implants and in platelet capture in diagnostics. Understanding the role of platelet activation in dictating platelet adhesion at chemically modified interfaces is important but relatively unexplored. Using scanning electron microscopy and confocal fluorescence microscopy a quantitative assessment of capture of blood platelets at self-assembled monolayers and mixed monolayers (SAMs) on gold as a function of the activation status of the platelets was conducted. Single and mixed monolayers were prepared using thiol-functionalized arginine-glycine-aspartic acid (RGD), C-Ahx-GRGDS (Ahx = aminohexanoic acid linker), thiolated poly(ethylene)glycol (PEG-COOH) and 1-octanethiol. When incubated with suspensions of resting platelets, RGD promoted platelet adhesion compared to bare or alkanethiol modified gold. Increasing the alkanethiol ratio in the deposition solution decreased the extent of platelet adhesion. Platelet adhesion increased approximately 3 fold at PEG-COO- modified surfaces compared to RGD-alone. Platelets adhered to RGD or mixed RGD : alkane SAM surfaces were found to be captured in their resting state. In contrast, platelets captured at PEG-COO- SAM surfaces were activated by these substrates. The effect of treating platelets with the chemical activators, Mn²⁺ or DTT or the physiological activator, thrombin, on the capture efficiency and activation at RGD modified surfaces was also investigated. Mn²⁺ treated platelets presented similar adhesion to untreated platelets, while surprisingly DTT yielded a very significant decrease in platelet adhesion. And, any platelets that were captured, were in a resting state. Thrombin activated platelets were captured with similar efficiencies as untreated platelets. However, the platelets captured were fully activated. The distinction between capture of chemically and physiologically activated platelet is interesting and likely to originate from differences in the conformation of the integrin induced by each process. Finally, platelet adhesion to each surface could be reversed by incubation with a solution of linear or cyclical RGD or PEG-COO- for the RGD and PEGCOO- surfaces respectively. The specificity of platelet removal confirmed that platelet adhesion at RGD surfaces is occurring through integrin–RGD interactions.