TY - JOUR

T1 - Influence of aflibercept on platelet activation profile

AU - Sobolewska, B

AU - Golenko, J

AU - Poeschel, S

AU - Grimmel, C

AU - Gatsiou, A

AU - Sopova, K

AU - Biedermann, T

AU - Schenke-Layland, K

AU - Stellos, K

AU - Ziemssen, F

N1 - Copyright © 2018 Elsevier Ltd. All rights reserved.

PY - 2018/10/31

Y1 - 2018/10/31

N2 - Aflibercept appears to accumulate in systemic circulation following intravitreal injections in therapy of neovascular age-related macular degeneration. This gives raise to the question of whether aflibercept affects platelets and their function such as activation and aggregation, which are substantial in the pathogenesis of an arterial thromboembolic event (ATE). In order to determine the effect of aflibercept in platelet activation, platelets from healthy volunteers were treated with aflibercept and its solvents at equal concentrations (0.04 μg/mL - 4 μg/mL - 40 μg/mL - 400 μg/mL - 4 mg/mL) for 10 and 30 min before addition of agonists. IgG1 antibody was used as a control. The surface expression of GPIIb/IIIa, P-selectin, and platelet-bound stromal-cell-derived factor-1, which are potential blood biomarkers for ATEs, was determined on resting and activated platelets by the multispectral imaging flow cytometry, combining the features of flow cytometry with fluorescence microscopy. Platelet aggregation was assessed with light transmission aggregometry. To determine whether aflibercept directly interacts with platelets, aflibercept was labeled with the fluorescence FITC. Co-treatment of platelets with thrombin or PAR-4-AP and aflibercept resulted in increased activation of the fibrinogen receptor GPIIb/IIIa in comparison to controls (P < 0.05). Interestingly, the expression of platelet-derived P-selectin and SDF-1 was not affected by aflibercept, except thrombin-activated CD62P with 0.04 μg/mL aflibercept (aflibercept vs. its solvent: MSI = 1.54, IC = 1.201-1.879 vs. MSI = 1.37, IC = 1.136-1.604 [P = 0.031]) and SDF-1 with 4 mg/mL aflibercept (aflibercept vs. its solvent: MSI = 1.971, IC = 1.206-2.737 vs. MSI = 1.200, IC = 0.738-1.662 [P = 0.041]). Although the levels of platelet-bound aflibercept-FITC were significantly increased in all activated platelets, no effect was observed in platelet aggregation. Albeit no impact of aflibercept was found on platelet aggregation under the studied experimental conditions, the increased activation of the fibrinogen receptor GPIIb/IIIa and the presence of a direct interaction between aflibercept and platelets may partially explain the risk of ATE in patients under aflibercept treatment due to FcγRIIa mediated αIIbβ3 outside-in integrin signaling and transport of aflibercept into platelets. Therefore, the Fc domain seems to be involved in interactions between aflibercept and platelets. Further research is needed to explain the role of Fc containing aflibercept in the pathogenesis of drug-associated vascular events involving platelets, coagulation cascade, extracellular matrix proteins and other cells.

AB - Aflibercept appears to accumulate in systemic circulation following intravitreal injections in therapy of neovascular age-related macular degeneration. This gives raise to the question of whether aflibercept affects platelets and their function such as activation and aggregation, which are substantial in the pathogenesis of an arterial thromboembolic event (ATE). In order to determine the effect of aflibercept in platelet activation, platelets from healthy volunteers were treated with aflibercept and its solvents at equal concentrations (0.04 μg/mL - 4 μg/mL - 40 μg/mL - 400 μg/mL - 4 mg/mL) for 10 and 30 min before addition of agonists. IgG1 antibody was used as a control. The surface expression of GPIIb/IIIa, P-selectin, and platelet-bound stromal-cell-derived factor-1, which are potential blood biomarkers for ATEs, was determined on resting and activated platelets by the multispectral imaging flow cytometry, combining the features of flow cytometry with fluorescence microscopy. Platelet aggregation was assessed with light transmission aggregometry. To determine whether aflibercept directly interacts with platelets, aflibercept was labeled with the fluorescence FITC. Co-treatment of platelets with thrombin or PAR-4-AP and aflibercept resulted in increased activation of the fibrinogen receptor GPIIb/IIIa in comparison to controls (P < 0.05). Interestingly, the expression of platelet-derived P-selectin and SDF-1 was not affected by aflibercept, except thrombin-activated CD62P with 0.04 μg/mL aflibercept (aflibercept vs. its solvent: MSI = 1.54, IC = 1.201-1.879 vs. MSI = 1.37, IC = 1.136-1.604 [P = 0.031]) and SDF-1 with 4 mg/mL aflibercept (aflibercept vs. its solvent: MSI = 1.971, IC = 1.206-2.737 vs. MSI = 1.200, IC = 0.738-1.662 [P = 0.041]). Although the levels of platelet-bound aflibercept-FITC were significantly increased in all activated platelets, no effect was observed in platelet aggregation. Albeit no impact of aflibercept was found on platelet aggregation under the studied experimental conditions, the increased activation of the fibrinogen receptor GPIIb/IIIa and the presence of a direct interaction between aflibercept and platelets may partially explain the risk of ATE in patients under aflibercept treatment due to FcγRIIa mediated αIIbβ3 outside-in integrin signaling and transport of aflibercept into platelets. Therefore, the Fc domain seems to be involved in interactions between aflibercept and platelets. Further research is needed to explain the role of Fc containing aflibercept in the pathogenesis of drug-associated vascular events involving platelets, coagulation cascade, extracellular matrix proteins and other cells.

KW - Angiogenesis Inhibitors/pharmacology

KW - Blood Platelets/drug effects

KW - Chemokine CXCL12/blood

KW - Flow Cytometry

KW - Humans

KW - Microscopy, Fluorescence

KW - P-Selectin/blood

KW - Platelet Activation/physiology

KW - Platelet Aggregation/physiology

KW - Platelet Glycoprotein GPIIb-IIIa Complex/metabolism

KW - Receptors, Vascular Endothelial Growth Factor

KW - Recombinant Fusion Proteins/pharmacology

KW - Retrospective Studies

KW - Vascular Endothelial Growth Factor A/antagonists & inhibitors

U2 - 10.1016/j.exer.2018.06.009

DO - 10.1016/j.exer.2018.06.009

M3 - Journal article

C2 - 29908884

VL - 175

SP - 166

EP - 172

JO - Experimental Eye Research

JF - Experimental Eye Research

SN - 0014-4835

ER -