To investigate the compatibility of recombinant tissue plasminogen activator (rtPA) and bevacizumab in vitro because during surgery, rtPA or rtPA-induced plasmin may cleave and inactivate bevacizumab.
To simulate the intraoperative range of mixing ratios of rtPA, bevacizumab, and subretinal blood, we calculated the volumes of 12 submacular hemorrhages (SHs) with a spherical cap formula using measurements derived from fundus photographs and spectral-domain optical coherence tomographic images. Bevacizumab was incubated with rtPA or plasmin before gel electrophoresis with Coomassie blue and silver staining. The anti-angiogenetic activity of bevacizumab in the presence of rtPA with or without clotted human blood or of plasmin was quantified by vascular endothelial growth factor–enzyme-linked immunosorbent assay after incubation with the supernatant of porcine retinal pigment epithelium cell cultures.
The mean (SD) volume of SH was 28.6 (24.7) mm3 (range, 6.2-94.6 mm3). In sodium dodecyl sulfate–polyacrylamid electrophoresis with Coomassie blue or silver staining, bevacizumab displayed characteristic patterns of protein bands. No additional fragments were detected in co-application of bevacizumab with either rtPA or plasmin. The anti-angiogenetic activity of bevacizumab remained unchanged in co-application with rtPA with or without blood or plasmin.
We demonstrated the absence of cleavage or functional inactivation of bevacizumab by rtPA in an in-vitro model of their intraoperative co-application as a treatment of SH.
In clinical practice, rtPA and bevacizumab can be co-applied as a treatment for neovascular age-related macular degeneration with SH to simultaneously clear SH and reduce choroidal new vessel activity.
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Figure 1. Protein bands as seen in representative sodium dodecyl sulfate–polyacrylamide gel electrophoresis after co-incubation of bevacizumab and recombinant tissue plasminogen activator (rtPA) or bevacizumab and plasmin, respectively, in Coomassie and silver staining. Mixtures were applied in 2 dilutions for better visual analysis. For molecular weight (MW) analysis of bands, only the 1:10 dilution was considered (see tables for MW). Protein band patterns in mixtures correspond to untreated proteins; loss of single bands is caused by dilution. Bev indicates bevacizumab untreated; M, marker; and plas, plasmin untreated. Mix = 250 μg of bevacizumab and 2 μg of rtPA incubated for 2 hours at 37° C; mix1 = 125 μg of bevacizumab incubated with 0.2 μg of plasmin; and mix2 = 125 μg bevacizumab incubated with 2 μg of plasmin.
Figure 2. Bevacizumab functionality. Ability of bevacizumab to impair the recognition of vascular endothelial growth factor (VEGF) by VEGF–enzyme-linked immunosorbent assay in the presence of recombinant tissue plasminogen activator (rtPA) (A), plasmin (B), and human blood clot dissolved by rtPA in simulated intraoperative conditions (C). Vascular endothelial growth factor content is depicted as a percentage of the control experiment. The ability of bevacizumab to inhibit VEGF is not altered in either experimental setting. Error bars depict standard deviation.
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