4D Intravital Imaging Studies Identify Platelets As the Predominant Cellular Procoagulant Surface in Hemostasis

Recent intravital imaging studies suggest that the vasculature is the predominant procoagulant surface during thrombotic plug formation. Similar studies investigating the interplay between endothelial cells (ECs), platelets, and coagulation/fibrinolytic factors during hemostatic plug formation are missing. We here describe a novel four-dimensional (4D) imaging platform to visualize and quantify hemostatic plug components with high spatiotemporal resolution. Fibrin accumulation following laser-induced endothelial ablation was observed at the endothelium-platelet plug interface. Fibrin levels were governed by the antagonistic balance between coagulation and fibrinolytic pathways. Phosphatidylserine (PS) was first detected in close physical proximity to the fibrin ring but then quickly spread across the endothelium. Impaired PS exposure in cyclophilinD^-/- mice resulted in a significant reduction in fibrin accumulation. Adoptive transfer studies demonstrated a predominant role for platelets as a procoagulant surface during hemostatic plug formation, and fibrin accumulation was impaired by antiplatelet therapy (APT). Inhibition of fibrinolysis by tranexamic acid (TXA) led to increased fibrin accumulation in WT mice, but not in cyclophilinD^-/- or WT mice treated with antiplatelet drugs. These studies indicate a predominant role of platelets as a procoagulant surface during hemostatic plug formation, and demonstrate that impaired procoagulant activity due to antiplatelet therapy is not reversed by TXA treatment.