Platelet Expression of a Genetically Encoded Calcium Indicator Reveals Platelet Activation Gradients in Real Time during Thrombus Formation in Vivo

Abstract

Introduction: Recent studies have demonstrated that platelet activation during hemostatic thrombus formation in vivo is heterogeneous in time and space. We have previously shown that in response to a penetrating injury in the microcirculation platelet accumulation and activation are driven by gradients of soluble agonists emanating from the site of injury. These gradients result in a characteristic thrombus architecture in which a core of fully activated platelets that have released their alpha granules is overlaid by a shell of less activated platelets that frequently embolize. While dependent on ADP/P2Y12 signaling, the extent and dynamics of platelet activation in the shell region remain poorly understood. Here, we used intravital imaging of platelet cytosolic calcium concentration to examine platelet activation in real time following vascular injury in vivo.Methods: We generated transgenic mice expressing a genetically encoded calcium indicator (GCaMP3) specifically in megakaryocytes and platelets by crossing PF4-Cre mice with Ai38 mice carrying the GCaMP3 transgene. GCaMP3 fluorescence was visualized in platelets following laser-induced injury in mouse cremaster arterioles using spinning disk confocal intravital microscopy.Results: Rapid and dynamic changes in GCaMP3 fluorescence were observed during platelet accumulation following vascular injury. Platelets immediately adjacent to the site of injury exhibited a rapid and sustained increase in cytosolic calcium. Peak cytosolic calcium levels in the core region occurred within the first minute post-injury, prior to peak P-selectin expression. Platelets in the shell region were characterized by dynamic changes in cytosolic calcium at the level of individual platelets, with the appearance of transient “waves” of calcium signaling propagating among groups of platelets. At the population level, cytosolic calcium concentration was substantially lower in the shell region as compared to the core.Conclusions: Expression of a genetically encoded calcium indicator in platelets allows for the visualization of platelet activation events in real time in vivo. Platelets within the core region of a thrombus exhibit a rapid and sustained increase in cytosolic calcium concentration, indicating robust activation. In contrast, platelets in the shell region exhibit transient increases in cytosolic calcium indicating weak activation, consistent with their increased likelihood of embolization. The examination of platelet calcium signaling in vivo provides a sensitive readout of platelet activation that will shed new light on mechanisms regulating hemostasis and thrombosis, and may be useful in assessing the impact of anti-thrombotic agents on platelet function. Research support was provided by the American Heart Association and National Heart, Lung and Blood Institute. DisclosuresStalker:Medicines Company: Research Funding. Brass:Medicines Company: Research Funding.