Confocal Real-Time Analysis of Cutaneous Platelet Recruitment during Immune-Complex-Mediated Inflammation

Platelets preserve vascular integrity during immune complex‒mediated skin inflammation by preventing neutrophil-provoked hemorrhage. However, the single-cell dynamics of this hemostatic process have never been studied in real-time. To monitor the onset of thrombocytopenia-associated hemorrhages and analyze platelet recruitment, we developed a confocal microscopy‒based video-imaging platform for the dorsal skinfold chamber in living mice. For ultrastructural analysis of recruited platelets, we correlated our imaging approach with serial block-face scanning electron microscopy. We found that bleeding events were transient and occurred preferentially at vascular sites, which were repeatedly penetrated by extravasating neutrophils. Hemorrhage only resumed when previously affected sites were again breached by yet another neutrophil. In non-thrombocytopenic mice, we observed that neutrophil extravasation provoked the recruitment of single platelets to the vessel wall, which required platelet immunoreceptor tyrosine-based activation motif receptors glycoprotein VI and C-type-lectin-like receptor 2. Recruited platelets were found to spread across the endothelial barrier and some even across the basement membrane while retaining their granules. Thus, by visualizing the spatiotemporal dynamics of thrombocytopenia-associated bleeding and platelet recruitment on a single-cell level and in real-time, we provide further insights into how platelets preserve vascular integrity during immune complex‒mediated skin inflammation.