Mapping densely packed GPIIb/IIIa receptors in murine blood platelets with expansion microscopy

Abstract

Interrogating small platelets and their densely packed, highly abundant receptor landscape is key to understand platelet clotting. Blot clots can save lives when stopping blood loss after an injury, but also kill when blocking a major vessel. The highly abundant and densely distributed GPIIb/IIIa receptors are one reason why the underlying key distributions and interactions, in particular the relevance of integrin clustering, are not fully understood. Such dense receptor scenarios are difficult to assess even by super-resolution fluorescence microscopy. Here, we quantify various receptor interactions, and demonstrate that expansion microscopy can pinpoint such challenging interactions where conventional methods fail in such dense 3D scenarios with highly abundant receptors. We successfully combine dual-color expansion and confocal microscopy with colocalization analysis and assess platelet receptor organization without the need of a super-resolution microscope. We reveal that GPIIb/IIIa receptors are organized in pre-formed clusters in resting platelets - a pattern that orchestrates platelet clotting. We show that 4x expansion is most straight-forward for platelet imaging, while 10x expansion provides highest precision which turned out to be absolutely necessary for the most difficult of the scenarios described here.