Department of Clinical Medicine, Macquarie University, Sydney 2109, Australia.
Department of Pathology, University of Sydney, Sydney 2050, Australia.
Thromb Res. 2018 Jul;167:104-112. doi: 10.1016/j.thromres.2018.05.016. Epub 2018 May 17.
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.
The study aim was to establish an in vitro parallel-plate flow chamber to test the efficacy of a pro-thrombotic conjugate targeting phosphatidylserine.
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.
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.
我们的目标是开发一种针对脑动静脉畸形（AVM）的血管靶向治疗方法。已将外翻磷脂酰丝氨酸确立为放射治疗 AVM 血管内皮的潜在生物标志物。我们假设磷脂酰丝氨酸可以在 AVM 放射手术后通过配体导向的血管靶向剂进行选择性靶向，以实现局部血栓形成和病理性 AVM 血管的快速闭塞。
本研究旨在建立一种体外平行板流动腔室，以测试靶向磷脂酰丝氨酸的促血栓形成缀合物的疗效。
通过凝血酶与磷脂酰丝氨酸靶向配体 annexin V 的赖氨酸-赖氨酸交联制备缀合物。脑微血管内皮细胞（endothelial cells）经照射（5、15 和 25 Gy）后，在含有全人血和缀合物（1.25 或 2.5 μg/mL）的平行板流动腔室中组装 1 或 3 天。通过荧光标记的血小板和纤维蛋白原的结合和聚集来评估荧光显微镜下的血栓形成。
在平行板流动装置中的剪切应力下，annexin V-凝血酶缀合物可诱导照射后的内皮细胞快速血栓形成（纤维蛋白沉积）。未缀合的非靶向凝血酶不会诱导纤维蛋白沉积。观察到辐射和缀合物剂量之间存在协同作用。在辐射（25 Gy）和缀合物（2.5 μg/mL）的最高联合剂量下，血栓形成最大。平行板流动系统为预测试促血栓形成的血管靶向剂提供了一种快速方法。这些发现验证了 annexin V-凝血酶缀合物向临床前研究的转化。
