Different Effects of Ranibizumab and Bevacizumab on Platelet Activation Profile

Abstract

Purpose: The aim of the study was to evaluate the potential influence of ranibizumab and bevacizumab on platelet activation and aggregation, which are critical processes in the pathogenesis of arterial thromboembolic events (ATEs). Methods: For the assessment of platelet function, flow cytometry and aggregometry were employed. Platelets were isolated from healthy volunteers and exposed to ranibizumab (1 mg/ml and 150 ng/ml) and bevacizumab (2.5 mg/ml and 3 μg/ml) or their solvents for 10 and 30 min prior to the addition of TRAP (25 μ<smlcap>M</smlcap>), PAR-4-AP (25 μ<smlcap>M</smlcap>) or thrombin (0.02 U/ml). The surface expression of activated GP IIb/IIIa, P-selectin (CD62P) and platelet-bound stromal cell-derived factor-1 (SDF-1) was measured on resting (nonactivated) and activated platelets by flow cytometry. The platelet aggregation capacity was examined using light transmission aggregometry. Results: The expression of surface activation markers did not differ significantly between nonstimulated and TRAP-, PAR-4-AP- or thrombin-activated platelets after incubating with ranibizumab. However, GP IIb/IIIa, CD62P and SDF-1 were significantly downregulated in PAR-4-AP- and thrombin-activated platelets after exposure to bevacizumab 2.5 mg/ml. In addition, ranibizumab- and bevacizumab-FITC were significantly increased in all activated platelets. No significant differences were observed in the aggregation of activated platelets after incubation with ranibizumab or bevacizumab. Conclusion: All ranibizumab concentrations as well as the bevacizumab concentration of 3 μg/ml had no influence on platelet activation and aggregation. Therefore, this in vitro study did not show any relationship between the exposition of activated platelets to ranibizumab or bevacizumab and the development of ATEs. However, the highest level of bevacizumab interfered with platelet activation, leading to downregulation of platelet activation markers. This observation might explain why the systemic treatment with high-dose bevacizumab could be associated with an increased risk of bleeding. Regarding the use of lower intravitreal dosages, further research should focus on the complex interactions between platelets and other cells, such as endothelial cells, which might stronger relate to a potentially increased risk of ATEs and depend on systemic vascular endothelial growth factor levels. Facing the different activation profiles, the diverse effects of the drugs on the cellular level have to be critically scrutinized for their clinical relevance.

Human Platelets Take up Anti-VEGF Agents

“…In the previous experiments, we observed that activation-dependent platelet function is more impaired with aflibercept and bevacizumab compared to ranibizumab, without any impact on platelet aggregation. Moreover, FITC-labeled aflibercept and bevacizumab, as well as ranibizumab, were significantly upregulated in activated platelets [21,22]. erefore, the anti-VEGF agents might be transported into platelets and then localized in one of three major granule types: α-granules, dense granules, and lysosomes; this can be demonstrated by immunofluorescence and electron microscopy [46][47][48][49].…”

“…Until now, the uptake and intracellular transport of anti-VEGF agents have been investigated in retinal endothelium and retinal pigment epithelium; however, little is known about these mechanisms in platelets [6,[19][20][21]. 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-VEGF agents are taken up by platelets and which mechanisms are involved in their endocytosis by platelets (Figure 1).…”

Effect of intravitreal injection of aflibercept on blood coagulation parameters in patients with age-related macular degeneration

“…Nevertheless, platelet exposure to higher bevacizumab concentrations resulted in inhibition of platelet activation. 10 Another study demonstrated different results. More precisely, platelet exposure to a mixture of bevacizumab and VEGF-A or to a mixture of aflibercept and VEGF-B or placental growth factor resulted in their activation, while ranibizumab had no effect on the platelets.…”

“…5 Several biomarkers have been evaluated to study coagulation alterations that result in hypercoagulability, and therefore, potentially increased thromboembolic risk in patients under systemic chemotherapy or under treatment with intravitreal injections of ranibizumab or bevacizumab. 6–11…”

The Provocative Roles of Platelets in Liver Disease and Cancer

“…In another study, oxaliplatin-based chemotherapy affected platelets in the liver; the number attached to liver cells positively correlated with sinusoidal-obstruction syndrome severity (221). Oxaliplatin-based damage to hepatic sinusoids can possibly attract and activate platelets (228)(229)(230). Once activated, platelets secrete growth factors such as plateletactivating factor and thromboxane A 2 , causing liver injury, vascular and sinusoidal occlusion, and collagen deposition (231,232).…”