Abstract
Purpose Growing evidence suggests different systemic exposure of anti-vascular endothelial growth factor (anti-VEGF) agents with repeated intravitreal application. Since the penetration of anti-VEGF agents through vascular barrier was reported, the interaction of anti-VEGF with nonresident platelets has become a topic of interest. The purpose of this study was to evaluate, with the help of visualization techniques, whether platelets take up the anti-VEGF agents ranibizumab, aflibercept, and bevacizumab. Methods The uptake of anti-VEGF agents with or without VEGF treatment was investigated using immunofluorescence and immunogold staining in human platelets. The role of actin filaments and clathrin-coated vesicles in the transport of ranibizumab, aflibercept, and bevacizumab was evaluated by two pharmacologic inhibitors: staurosporine (protein kinase C inhibitor) and cytochalasin D. Results All three anti-VEGF agents were taken up by platelets and colocalized with VEGF. Ranibizumab and aflibercept were mainly detected in alpha-granules; however, bevacizumab was equally localized in alpha-granules and in platelet vesicles. Both staurosporine and cytochalasin D completely inhibited the uptake of aflibercept into platelets. Both pharmacological inhibitors also decreased the transport of ranibizumab and bevacizumab into platelets. Bevacizumab was significantly more frequently colocalized within clathrin-coated vesicles than ranibizumab and aflibercept. Conclusion All three anti-VEGF agents are taken up by platelets and internalized in alpha-granules, which may result in a higher local exposure of anti-VEGF after the activation of platelets, potentially contributing to arterial thromboembolic events. Clathrin-coated vesicles seem to be more prominent in the transport of bevacizumab than ranibizumab and aflibercept. Nevertheless, whether the different localization and transport of bevacizumab are truly related to specific differences of receptor-mediated endocytosis has to be revealed by further research.
Highlights
Anti-vascular endothelial growth factor agents have a broad field of application due to their impact on tumor growth and metastasis in oncology or in sealing, and antiangiogenic effect in the treatment of neovascular agerelated macular degeneration or other retinal diseases. e inhibitors have a certain range of molecular properties: Ranibizumab is a recombinant humanized 48 kDa antibody fragment (Fab), designed to penetrate the retina with low serum concentrations [1,2,3]
We have previously shown that increased levels of FITC-labeled ranibizumab, aflibercept, and bevacizumab were found using fluorescenceactivated cell sorting (FACS) analysis after the activation of platelets with either thrombin receptor-activating peptide-6 (TRAP), proteinase-activated receptor 4-activating peptide (PAR-4-AP), or thrombin [21, 22]. erefore, the question arises as to whether all anti-vascular endothelial growth factors (VEGFs) agents are taken up by platelets and which mechanisms are involved in their endocytosis by platelets (Figure 1)
Anti-VEGF agents are widely used in treatment of cancer patients and are the first-line therapy of neovascular AMD and macular edema secondary to diabetes and retinal vein occlusion. erefore, the comparison between compounds is warranted, and the safety profile has gained interest since vascular events were observed in a recently approved drug [40, 41]. e adverse events are primarily explained by the blocking of VEGF signaling and the different suppression of serum proteins by anti-VEGF agents [3, 42, 43]
Summary
Anti-vascular endothelial growth factor (anti-VEGF) agents have a broad field of application due to their impact on tumor growth and metastasis in oncology or in sealing, and antiangiogenic effect in the treatment of neovascular agerelated macular degeneration (nAMD) or other retinal diseases. e inhibitors have a certain range of molecular properties: Ranibizumab is a recombinant humanized 48 kDa antibody fragment (Fab), designed to penetrate the retina with low serum concentrations [1,2,3]. Anti-vascular endothelial growth factor (anti-VEGF) agents have a broad field of application due to their impact on tumor growth and metastasis in oncology or in sealing, and antiangiogenic effect in the treatment of neovascular agerelated macular degeneration (nAMD) or other retinal diseases. E inhibitors have a certain range of molecular properties: Ranibizumab is a recombinant humanized 48 kDa antibody fragment (Fab), designed to penetrate the retina with low serum concentrations [1,2,3]. It has been reported several times that monoclonal antibodies are sequestered in platelets, similar to most other serum proteins and growth factors such as platelet-derived growth factors (PDGFs), transforming growth factors. Journal of Ophthalmology (TGFs), vascular endothelial growth factors (VEGFs), and epithelial growth factors (EGFs) [6,7,8]. Platelet activating factors contribute to the further recruitment and aggregation of platelets, as well as other cells including monocytes and fibroblasts, leading to stabilization of the hemostatic plug [10]
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