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Bianka Sobolewska, Focke Ziemssen, Cornelia Grimmel, Jadwiga Kwiatkowska, Martin Spitzer, Meinrad Gawaz, Tilo Biedermann, Konstantinos Stellos; Influence of bevacizumab on Platelet activation profile in vitro. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4126. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Inhibitors of the vascular endothelial growth factor (VEGF) are increasingly used in the therapy of multiple retinal diseases. While the risk of thrombotic events or bleeding has been discussed, we wanted to evaluate the effect of the off-label drug bevacizumab on platelet activation.
Blood was drawn from the antecubital vein of healthy volunteers, who did not take any drugs during the previous 10 days. Platelet-rich plasma (PRP) was prepared by centrifuging blood at 1000 rpm for 10 min at room temperature. The number of platelets in the PRP was counted by using fully automated hematology analyzers and adjusted to 200.000 platelets/μl with platelet-poor plasma (PPP). Suspension of platelets was treated with bevacizumab (2.5 mg/ml) or bevacizumab solvent for 10 min and 30 min before addition to the platelets of TRAP (25 μM) or thrombin (0.02 unit/ml). As a control, vehicle, TRAP or thrombin, bevacizumab or its solvent were used. In order to measure the ability of the incubated platelets to get activated, the expression of PAC-1 (activated form of GPIIb/IIIa) and P-Selectin (CD62p) was determined on resting (non-activated) and activated platelets. The expression of platelet-bound SDF-1 (stromal-cell-derived factor-1) was also evaluated. CD42b-PE served as control antibody to identify the platelet population. Mouse IgG1-FITC and IgG1-PE were used as monoclonal immunoglobuline isotype control antibodies. Platelet-associated fluorescence was quantified (10.000 events) with FACS-Calibur Flow Cytometer. The specific monoclonal antibody binding was expressed as stimulation index (SI) of surface platelet protein expression.
No statistically significant differences were observed in the expression of P-Selectin and SDF-1 on both resting and TRAP-activated platelets, subsequent to the exposure of bevacizumab. However, the fibrinogen receptor (GPIIb/IIIa) showed significant changes after the exposure to bevacizumab. PAC-1, CD62p and SDF-1 were also significantly down-regulated on platelets if stimulated with thrombin after incubation with bevacizumab (mean ± SD of PAC-1: 0.5 ± 0.1, P=0.002 & P=0.001; CD62p: 0.08 ± 0.2, P=0.0002 & 1.1 ± 0.2, P=0.008; SDF-1: 0.9 ± 0.2, P=0.01 & 1.1 ± 0.1, P=0.002).
Different patterns of platelet activation after exposure to bevacizumab were observed and might be associated with an increased risk of bleeding.
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