Stable thrombus formation on irradiated microvascular endothelial cells under pulsatile flow: Pre-testing annexin V-thrombin conjugate for treatment of brain arteriovenous malformations

Subramanian, S; Ugoya, SO; Zhao, Z; McRobb, LS; Grau, GE; Combes, V; Inglis, DW; Gauden, AJ; Lee, VS; Moutrie, V; Santos, ED; Stoodley, MA

Stable thrombus formation on irradiated microvascular endothelial cells under pulsatile flow: Pre-testing annexin V-thrombin conjugate for treatment of brain arteriovenous malformations

Subramanian, S Ugoya, SO Zhao, Z McRobb, LS Grau, GE Combes, V

Inglis, DW Gauden, AJ Lee, VS Moutrie, V Santos, ED Stoodley, MA

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Publication Type:: Journal Article
Citation:: Thrombosis Research, 2018, 167 pp. 104 - 112
Issue Date:: 2018-07-01

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Field	Value	Language
dc.contributor.author	Subramanian, S	en_US
dc.contributor.author	Ugoya, SO	en_US
dc.contributor.author	Zhao, Z	en_US
dc.contributor.author	McRobb, LS	en_US
dc.contributor.author	Grau, GE	en_US
dc.contributor.author	Combes, V https://orcid.org/0000-0003-2178-3596	en_US
dc.contributor.author	Inglis, DW	en_US
dc.contributor.author	Gauden, AJ	en_US
dc.contributor.author	Lee, VS	en_US
dc.contributor.author	Moutrie, V	en_US
dc.contributor.author	Santos, ED	en_US
dc.contributor.author	Stoodley, MA	en_US
dc.date.available	2020-05-25T19:16:28Z
dc.date.issued	2018-07-01	en_US
dc.identifier.citation	Thrombosis Research, 2018, 167 pp. 104 - 112	en_US
dc.identifier.issn	0049-3848	en_US
dc.identifier.uri	http://hdl.handle.net/10453/125233
dc.description.abstract	© 2018 Elsevier Ltd 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.	en_US
dc.relation.ispartof	Thrombosis Research	en_US
dc.relation.isbasedon	10.1016/j.thromres.2018.05.016	en_US
dc.subject.classification	Cardiovascular System & Hematology	en_US
dc.subject.mesh	Brain	en_US
dc.subject.mesh	Endothelial Cells	en_US
dc.subject.mesh	Humans	en_US
dc.subject.mesh	Arteriovenous Malformations	en_US
dc.subject.mesh	Thrombosis	en_US
dc.subject.mesh	Annexin A5	en_US
dc.title	Stable thrombus formation on irradiated microvascular endothelial cells under pulsatile flow: Pre-testing annexin V-thrombin conjugate for treatment of brain arteriovenous malformations	en_US
dc.type	Journal Article
utslib.citation.volume	167	en_US
utslib.for	1103 Clinical Sciences	en_US
pubs.embargo.period	Not known	en_US
pubs.organisational-group	/University of Technology Sydney
pubs.organisational-group	/University of Technology Sydney/Faculty of Science
pubs.organisational-group	/University of Technology Sydney/Faculty of Science/School of Life Sciences
utslib.copyright.status	open_access
pubs.publication-status	Published	en_US
pubs.volume	167	en_US

Abstract:

© 2018 Elsevier Ltd 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.

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http://hdl.handle.net/10453/125233