Stable thrombus formation on irradiated microvascular endothelial cells under pulsatile flow: Pre-testing annexin V-thrombin conjugate for treatment of brain arteriovenous malformations. Issue 167 (July 2018)

• Record Type: Journal Article
• Authors: Subramanian, S.; Ugoya, S.O.; Zhao, Z.; McRobb, L.S.; Grau, G.E.; Combes, V.; Inglis, D.W.; Gauden, A.J.; Lee, V.S.; Moutrie, V.; Santos, E.D.; Stoodley, M.A.
• Is Part Of: Thrombosis research. Issue 167(2018)
• Page Start: 104
• Page End: 112
• Publication Date: 2018-07
• Subjects: Arteriovenous malformation -- Endothelial cells -- Ionizing radiation -- Phosphatidylserine -- Thrombosis; Thrombosis -- Periodicals; 616.135
• DOI: 10.1016/j.thromres.2018.05.016
• Languages: English
• ISSNs: 0049-3848

Abstract

Abstract: Background: Our goal is to develop a vascular targeting treatment for brain arteriovenous malformations (AVMs). Externalized phosphatidylserine has been established as a potential biomarker on the endothelium of irradiated AVM blood vessels. We hypothesize that phosphatidylserine could be selectively targeted after AVM radiosurgery with a ligand-directed vascular targeting agent to achieve localized thrombosis and rapid occlusion of pathological AVM vessels. Objective: The study aim was to establish an in vitro parallel-plate flow chamber to test the efficacy of a pro-thrombotic conjugate targeting phosphatidylserine. Methods: Conjugate was prepared by Lys-Lys cross-linking of thrombin with the phosphatidylserine-targeting ligand, annexin V. Cerebral microvascular endothelial cells were irradiated (5, 15, and 25 Gy) and after 1 or 3 days assembled in a parallel-plate flow chamber containing whole human blood and conjugate (1.25 or 2.5 μg/mL). Confocal microscopy was used to assess thrombus formation after flow via binding and aggregation of fluorescently-labelled platelets and fibrinogen. Results and conclusions: The annexin V-thrombin conjugate induced rapid thrombosis (fibrin deposition) on irradiated endothelial cells under shear stress in the parallel-plate flow device. Unconjugated, non-targeting thrombin did not induce fibrin deposition. A synergistic interaction between radiation and conjugate dose was observed. Thrombosis was greatest at the highest combined doses of radiation (25 Gy) and conjugate (2.5 μg/mL). The parallel-plate flow system provides a rapid method to pre-test pro-thrombotic vascular targeting agents. These findings validate the translation of the annexin V-thrombin conjugate to pre-clinical studies. Highlights: Pro-thrombotic vascular targeting agents were tested under parallel-flow. Radiation-primed endothelial cells externalized the target phosphatidylserine. Targeted annexin V-thrombin conjugate increased thrombus formation dose-dependently. Targeting radiation markers may be a new approach to brain AVM treatment.