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

Research output: Contribution to journalArticleResearchpeer-review

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.

LanguageEnglish
Pages104-112
Number of pages9
JournalThrombosis Research
Volume167
DOIs
Publication statusPublished - 1 Jul 2018

Fingerprint

Pulsatile Flow
Arteriovenous Malformations
Annexin A5
Phosphatidylserines
Thrombin
Blood Vessels
Thrombosis
Endothelial Cells
Brain
Fibrin
lysyllysine
Ligands
Radiation Dosage
Radiosurgery
Therapeutics
Confocal Microscopy
Fibrinogen
Endothelium
Blood Platelets
Biomarkers

Cite this

@article{ff14c829abce4fc08083bf25bede5cac,
title = "Stable thrombus formation on irradiated microvascular endothelial cells under pulsatile flow: pre-testing annexin V-thrombin conjugate for treatment of brain arteriovenous malformations",
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.",
keywords = "Arteriovenous malformation, Endothelial cells, Ionizing radiation, Phosphatidylserine, Thrombosis",
author = "S. Subramanian and Ugoya, {S. O.} and Z. Zhao and McRobb, {L. S.} and Grau, {G. E.} and V. Combes and Inglis, {D. W.} and Gauden, {A. J.} and Lee, {V. S.} and V. Moutrie and Santos, {E. D.} and Stoodley, {M. A.}",
year = "2018",
month = "7",
day = "1",
doi = "10.1016/j.thromres.2018.05.016",
language = "English",
volume = "167",
pages = "104--112",
journal = "Thrombosis Research",
issn = "0049-3848",
publisher = "Elsevier",

}

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, 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.

In: Thrombosis Research, Vol. 167, 01.07.2018, p. 104-112.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Stable thrombus formation on irradiated microvascular endothelial cells under pulsatile flow

T2 - Thrombosis Research

AU - Subramanian,S.

AU - Ugoya,S. O.

AU - Zhao,Z.

AU - McRobb,L. S.

AU - Grau,G. E.

AU - Combes,V.

AU - Inglis,D. W.

AU - Gauden,A. J.

AU - Lee,V. S.

AU - Moutrie,V.

AU - Santos,E. D.

AU - Stoodley,M. A.

PY - 2018/7/1

Y1 - 2018/7/1

N2 - 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.

AB - 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.

KW - Arteriovenous malformation

KW - Endothelial cells

KW - Ionizing radiation

KW - Phosphatidylserine

KW - Thrombosis

UR - http://www.scopus.com/inward/record.url?scp=85047240809&partnerID=8YFLogxK

U2 - 10.1016/j.thromres.2018.05.016

DO - 10.1016/j.thromres.2018.05.016

M3 - Article

VL - 167

SP - 104

EP - 112

JO - Thrombosis Research

JF - Thrombosis Research

SN - 0049-3848

ER -