Stable thrombus formation on irradiated microvascular endothelial cells under pulsatile flow

pre-testing annexin V-thrombin conjugate for treatment of brain arteriovenous malformations

S. Subramanian, S. O. Ugoya, Z. Zhao, L. S. McRobb, G. E. Grau, V. Combes, D. W. Inglis, A. J. Gauden, V. S. Lee, V. Moutrie, E. D. Santos, M. A. Stoodley*

*Corresponding author for this work

Research output: Contribution to journalArticle

2 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)104-112
Number of pages9
JournalThrombosis Research
Volume167
DOIs
Publication statusPublished - 1 Jul 2018

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Keywords

  • Arteriovenous malformation
  • Endothelial cells
  • Ionizing radiation
  • Phosphatidylserine
  • Thrombosis

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