Stable thrombus formation on irradiated microvascular endothelial cells using vascular targeting agents under pulsatile flow: pro-thrombotic treatment for brain arteriovenous malformations
thesisposted on 2022-03-29, 00:34 authored by Sinduja Subramanian
Background: Brain arteriovenous malformations (AVMs) are a leading cause of intracranial haemorrhage in children and young adults. Limitations of current treatment approaches leave one third of AVM patients at risk. The overall goal of this work is to develop a radiation-guided vascular targeting treatment for AVMs. Externalised phosphatidylserine and alpha-crystallin B (CRYAB) have been identified as potential biomarkers on the endothelium of irradiated AVM blood vessels. It was hypothesised that these molecules could be selectively targeted after AVM radiosurgery with ligand-directed pro-thrombotic vascular targeting agents (VTAs) to achieve localised thrombosis and rapid occlusion of pathological AVM vessels. Pre-testing of novel VTAs may be more economically and ethically performed in vitro. Aim 1: To establish an in vitro parallel-plate flow chamber system using whole blood for VTA testing. Aim 2: To develop and test the efficacy of pro-thrombotic VTAs targeting phosphatidylserine and CRYAB on irradiated endothelial cells under flow. Methods: Conjugates were prepared by Lys-Lys cross-linking of thrombin with annexin V targeting phosphatidylserine, or with an anti-CRYAB antibody. Cerebral microvascular endothelial cells were irradiated (5 Gy, 15 Gy, and 25 Gy) and after 1 or 3 days assembled in a parallel-plate flow chamber containing whole human blood and conjugate (1.25 μg/mL or 2.5 μg/mL). Confocal microscopy was used to assess thrombus formation after flow via binding and aggregation of fluorescently-labelled platelets or fibrinogen. Results and conclusions: Both annexin V-thrombin and anti-CRYAB-thrombin VTAs induced rapid thrombosis on irradiated endothelial cells under flow. Thrombosis was greatest at the highest combined doses of radiation and conjugate. Annexin V-thrombin induced moderate thrombus stimulation even at the lowest radiation dose. These molecules and their developed VTAs appear suitable for further testing in animal models. The parallel-plate flow chamber proved to be a simple and cost-effective method to pre-test pro-thrombotic vascular targeting agents prior to preclinical studies.