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

Abstract

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 stress inducer, or with antithrombotic agents including antitissue factor antibody (5G9), antiplatelet drugs (abciximab, aspirin), and anticoagulants (argatroban, heparin). Fluorescently labeled antibodies (to platelets, fibrin, or von Willebrand factor [VWF]), annexin V or the calcium dye Cal520, were added to the blood. After recalcification at 10 mM, blood was perfused through the Endo-chip at a 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-positive endothelial cells expressed tissue factor and released VWF, supporting localized platelet and fibrin deposition. The thrombus formed a teardrop 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 studies and antithrombotic drug development.