Carotid artery thrombosis assay

Ferric chloride induced carotid artery thrombosis assay was performed as an independent monitor of the role of vitronectin in thrombus formation^52,53. Mice were anaesthetised by intraperitoneal injection of a mixture of ketamine and xylazine and a cannula tube was prepared in the left jugular vein for the administration of Nembutal (to maintain anaesthesia) and antibodies⁴¹. The right common carotid artery was then exposed and platelets were labelled by infusion of Dylight 649 conjugated anti-CD42b antibody (0.1 μg g⁻¹ body weight)(Emfret Analytics). Thrombus formation was initiated by applying Whatman filter paper (1 mm by 3 mm) pre-soaked in ferric chloride (6%) solution to the carotid artery for 1 min. The carotid artery was then washed and superfused with pre-warmed (37 °C) and aerated (5% CO₂, 95% N₂) bicarbonate buffered saline⁴¹. Platelet accumulation and thrombus formation was monitored under an intravital fluorescent microscope and the time to complete blood flow occlusion was recorded. In all cases, the time to complete vessel occlusion was determined only after the vessel had remained closed for 20 min.