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.

Computed tomography (CT) mixed density of traumatic extra-axial hemorrhages (TEH) or the 'swirl sign' has been reported to correlate with active bleeding found at craniotomy and poor outcome. This study was done to test the hypothesis that mixed density of TEH detected by third or fourth generation CT correlated with the type of bleeding or clinical outcome. All cases of TEH operated at Detroit Receiving Hospital from 1991-1997 were reviewed for type of bleeding (active vs. not active; arterial vs. venous); Glasgow Coma Scale (severe 1-8, not severe 9-15); and Glasgow Outcome Score (1-3 poor; 4,5 good). CT density (CTD) of 51 cases with specific written documentation of bleeding type were then independently reviewed (SKS and MHR) and classified into TEH with mixed or high density. Data was analyzed using corrected Chi Square analysis, Fischer's Exact Test and Pearson's Correlation (SPSS 6.0). The Pearson Chi Square probability for correlation follows: CTD vs. active bleeding, 0.21; CTD vs. arterial, 0.41; CTD vs. severity, 0.57; and CTD vs. outcome, 0.81. No other statistical analysis identified a significant correlation, thus the null hypothesis could not be rejected. CT mixed density was not found to be correlated by more than chance with bleeding type, injury severity or outcome. Surgeon inaccuracy in documentation of bleeding type and use of later generation CT may account for the discrepancy between this and previous studies. Nevertheless, we conclude clinical exam and other published CT criteria are better indicators of injury severity and outcome.

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Vascular targeting with pro-thrombotic antibody-conjugates is a promising biological treatment for brain arteriovenous malformations (bAVMs). However, targeted drug delivery relies on the identification of unique or overexpressed markers on the surface of a target cell. In the absence of inherent biological markers, stereotactic radiosurgery may be used to prime induction of site-specific and targetable molecular changes on the endothelial surface. To investigate lumen-accessible, endothelial targets induced by radiation, we combined Gamma knife surgery in an AVM animal model with in vivo biotin-labeling and comparative proteomics. Two proteins, αB-crystallin (CRYAB)—a small heat shock protein that normally acts as an intracellular chaperone to misfolded proteins—and activated leukocyte cell adhesion molecule CD166, were further validated for endothelial surface expression after irradiation. Immunostaining of endothelial cells in vitro and rat AVM tissue ex vivo confirmed de novo induction of CRYAB following irradiation (20 Gy). Western analysis demonstrated that CRYAB accumulated intracellularly as a 20 kDa monomer, but, at the cell surface, a novel 65 kDa protein was observed, suggesting radiation stimulates translocation of an atypical CRYAB isoform. In contrast, CD166 had relatively high expression in non-irradiated cells, localized predominantly to the lateral surfaces. Radiation increased CD166 surface exposure by inducing translocation from intercellular junctions to the apical surface without significantly altering total protein levels. These findings reinforce the dynamic molecular changes induced by radiation exposure, particularly at the cell surface, and support further investigation of radiation as a priming mechanism and these molecules as putative targets for focused drug delivery in irradiated tissue.

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IntroductionOne third of brain arteriovenous malformations (AVMs) are untreatable with current methods. Previous work has identified phosphatidylserine externalised in the endothelial plasma membrane after treatment with radiosurgery as a potential target for biological pro-thrombotic therapy. We hypothesise that treatment of AVMs with Gamma knife radiosurgery (GKS) and a vascular targeting agent with a thrombotic compound will cause localised thrombosis within the AVM vessels.MethodsA rat animal model was used by performing an end-to-side anastomosis of the external jugular vein (EJV) to the common carotid artery. The model AVM was treated with radiosurgery (20 Gy) using a Leksell Gamma Knife. At 3 weeks following GKS a dose of a conjugate of annexin V and thrombin was administered intravenously. Groups injected with saline or thrombin alone were used as controls. At 4 weeks, an angiogram was performed with tissue harvested for histology.ResultsThere was angiographic evidence of AVM occlusion in 69% of conjugate-treated animals and none of the control animals (p=0.002). AVM occlusion occurred in 63% of the GKS group treated with conjugate (p=0.03), and in 75% of the sham-GKS conjugate treated group (p=0.009). Histological evidence of thrombus was present within the EJV and nidus of 62.5% of animals in the GKS with conjugate group and <25% of the control groups (p=0.026).ConclusionThis study demonstrates that targeting of PS with an annexin V-thrombin conjugate is effective in causing thrombotic occlusion in a model AVM. These results are the first of their kind and indicate that ligand vascular targeting with radiation sensitisation may become a potential future treatment in brain AVMs.