Abstract
Human platelets aggregate at sites of blood vessel damage in response to a rise in their cytosolic calcium concentration. Controlling these cytosolic calcium rises would provide a method to inhibit platelet activation and prevent the unwanted blood clots that causes heart attack and strokes. Previously we have predicted that calcium accumulation within the lumen of an infolded portion of the platelet plasma membrane called the open canalicular system (OCS) is essential for maintaining this cytosolic calcium rise. Due to its nanometer dimensions of the OCS, it has been difficult to measure or interfere with the predicted luminal calcium accumulation. Here we utilise iron oxide magnetic nanoparticles coated with the known calcium chelator, citrate, to create calcium-binding nanoparticles. These were used to assess whether an OCS calcium store plays a role in controlling the dynamics of human platelet activation and aggregation. We demonstrate that citrate-coated nanoparticles are rapidly and selectively uptaken into the OCS of activated human platelets, where they act to buffer the accumulation of calcium there. Treatment with these calcium-binding nanoparticles reduced thrombin-evoked cytosolic calcium rises, and slowed platelet aggregation and clot retraction in human platelets. In contrast, nanoparticles that cannot bind calcium have no effect. This study demonstrates that the OCS acts as a key source of calcium for maintaining cytosolic calcium rises and accelerating platelet aggregation, and that calcium-binding nanoparticles targeted to the OCS could provide an anti-platelet therapy to treat patients at risk of suffering heart attacks or strokes.
Highlights
Human platelets are small, discoid, anucleate cells that orchestrate blood clotting at sites of blood vessel injury [1]
We provide the first direct evidence that the open canalicular system (OCS) plays a key role in regulating Ca2+ signalling in human platelets, and that calcium-binding nanoparticles targeted to the OCS could be used to create novel anti-thrombotic drugs to prevent blood clotting in patients at risk of heart attacks and strokes
2.1 Characterisation of the physical properties of the. Prior to assessing their effect on human platelets, the physical and chemical properties of the Iron oxide nanoparticles (IONPs) used in this study were characterised with transmission electron microscopy (TEM) and dynamic light scattering-electrophoretic light scattering (DLS-ELS)
Summary
Discoid, anucleate cells that orchestrate blood clotting at sites of blood vessel injury [1]. As citric acid is an effective Ca2+-chelating agent that is commonly used in blood anti-coagulants [16], we hypothesized that citrate-coated IONPs could be utilized to create Ca2+ nanochelators that could enter the OCS lumen and buffer thrombin-evoked rises here This would allow us to test the hypothesis that increases in Ca2+ concentration within the OCS are important in regulating human platelet activation. We produce calcium-binding IONPs and assess their ability to inhibit thrombin-evoked Ca2+ signalling and platelet activation Through these experiments, we provide the first direct evidence that the OCS plays a key role in regulating Ca2+ signalling in human platelets, and that calcium-binding nanoparticles targeted to the OCS could be used to create novel anti-thrombotic drugs to prevent blood clotting in patients at risk of heart attacks and strokes
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