Endo-chip laser-induced thrombus formation; a vessel-on-chip model for in vitro testing of antithrombotic agents Open Access

1. Laser-induced thrombus formation in the Endo-chip provides a humanized model for the ex vivo study of responses to endothelial injury.

2. The laser injury Endo-chip model offers a fast tool for testing the efficacy of antithrombotic agents.

Mouse models, such as the intravital laser-induced model of thrombus formation, are commonly used for mechanistic and preclinical studies in thrombosis. However, their translational value is limited by species differences. Few in vitro models incorporate laser-induced vascular injury. Here, we developed an endothelialized microfluidic device, the Endo-chip, to model thrombus formation in response to laser injury. Citrated blood was treated with IXA4, an endoplasmic reticulum (ER) stress inducer, or with antithrombotic agents including anti-tissue factor antibody (5G9), antiplatelet drugs (Abciximab, aspirin) and anticoagulants (argatroban, heparin). Fluorescently labelled antibodies (to platelets, fibrin, von Willebrand factor (vWF) or Annexin V and Cal520, were added to the blood. Following recalcification at 10 mM, blood was perfused through the Endo-chip at a venous shear rate of 100 s^-1 or 800 s^-1. Endothelial injury was induced with a 355 nm laser pulse producing a focal 10 μm injury and phosphatidylserine exposure on endothelial cells within ∼1 cell diameter from the injury site. Annexin V ve+ endothelial cells expressed tissue factor (TF) and released vWF supporting localized platelet and fibrin deposition. The thrombus formed a tear-drop morphology aligned with flow incorporating vWF with increasing shear. IXA4 enhanced platelet cytoplasmic calcium. Platelet accumulation was inhibited by Abciximab, but not aspirin whereas coagulation inhibitors (5G9, argatroban, heparin) markedly reduced thrombus formation. These findings support that the Endo-chip laser injury model incorporates key features of thrombus formation after endothelial injury and provides a humanized, in vitro, alternative or auxiliary to mouse models for preclinical and antithrombotic drug development studies.