Effects and Mechanisms of Vitamin C Post-Conditioning on Platelet Activation after Hypoxia/Reoxygenation

Demin Liu; Dongguo Pei; Haijuan Hu; Guoqiang Gu; Wei Cui

doi:10.1159/000500492

Effects and Mechanisms of Vitamin C Post-Conditioning on Platelet Activation after Hypoxia/Reoxygenation

Transfus Med Hemother. 2020 Apr;47(2):110-118. doi: 10.1159/000500492. Epub 2019 May 28.

Authors

Demin Liu¹, Dongguo Pei¹, Haijuan Hu¹, Guoqiang Gu¹, Wei Cui¹

Affiliation

¹ Department of Cardiology, Second Hospital of Hebei Medical University, Shijiazhuang, China.

Abstract

Background: Platelet activation occurs upon ischemia/reperfusion and is related to the generation of reactive oxygen species (ROS) during this process. Vitamin C (VC) is a powerful antioxidant. VC scavenges ROS, reduces platelet activation, and attenuates reperfusion injury. However, the effects of VC on platelets undergoing hypoxia/reoxygenation (H/R) remain unclear.

Objectives: Herein, we evaluated the effects of VC on platelets in vitro following H/R and the related mechanisms.

Method: Fresh platelets were collected from 67 volunteers at the Blood Center of Hebei Province. Platelets were diluted with saline to a concentration of 2.00 × 10¹¹/L. Aggregation and the curve slope were evaluated within 4 h with a whole-blood impedance analyzer. To determine the optimal experimental time, platelets were treated with hypoxia or reoxygenation for different times, and impedance aggregometry was carried out by measuring changes in electrical impedance induced by arachidonic acid (0.5 mM) and adenosine diphosphate (10 µM), thereby establishing the H/R model. Three antioxidants (VC, melatonin, and probucol) were used to treat platelets after H/R, and impedance aggregometry was used to determine their effects on platelet aggregation. The influence of VC on apoptosis-related indicators was detected. ROS and the mitochondrial membrane potential were observed by inverted fluorescence microscopy and flow cytometry, respectively. Related protein levels were detected by Western blotting.

Results: ROS scavengers inhibited platelet activation and aggregation in a concentration-dependent manner. VC post-conditioning scavenged ROS, downregulated cytochrome C, Bax, and caspase-9 proteins, and upregulated Bcl-2 protein. These effects collectively blocked platelet apoptosis and inhibited platelet aggregation.

Conclusions: VC inhibited platelet aggregation by blocking apoptosis. Thus, VC may have applications in the treatment of platelet-related diseases.

Keywords: Apoptosis; Platelet aggregation; Platelets; Reactive oxygen species; Vitamin C.