Abstract
Purpose :
The human’s front-line defense involves the innate and adaptive immune response in coordination with the complement system. Although platelet activity is primarily involved in coagulation, it is also involved in amplifying complement recruitment during phosphatidylserine exposure, a central event in the sequestration and elimination of dead cells, harmful bacteria, and damage-associated molecular pattern ligands. The purpose of this study is to measure platelet levels of complement C3b in patients with dry age-related macular degeneration (AMD).
Methods :
Whole blood was obtained from AMD (n=5) and control subjects (n=15) after informed consent. Platelets were isolated by centrifugation and challenged with thrombin and convulxin (T/C) or lipopolysaccharide (LPS). Platelet surface levels of C3b and the percentage of superactivated platelets (SAPs) were measured by flow cytometry (CytoFLEX S, Beckman Coulter) at baseline and after activation with T/C or LPS. C3b was identified using an anti-C3b monoclonal antibody (Biolegend). SAPs were identified using anti-PAC1 (Thermo-Fisher) and biotin-fibrinogen with labeled streptavidin (Thermo-Fisher). Significance was determined using paired t-tests for independent samples and unpaired t-tests for dependent samples. All procedures were approved by the University of Illinois at Chicago Institutional Review Board.
Results :
Baseline levels of platelet C3b were significantly higher in AMD (25.3±4.1%) compared with controls (16.9±4.0%, p=0.02). After challenge with T/C, C3b levels increased from baseline in both AMD (60.4±6.0%, p=0.0003) and controls (48.9±3.5%, p=0.01) and were higher in AMD (p=0.03). The percentage of SAPs was also higher in AMD (44.4±4.2% vs. 32.4±6.0%, p=0.0007). LPS increased C3b levels moderately from baseline in both AMD (39.0±4.8%, p=0.005) and controls (25.7±4.9%, p=0.01).
Conclusions :
Surface levels of complement C3b are increased in AMD in resting platelets and after activation with T/C or LPS. SAPs are also increased in AMD after activation with T/C. Normally, C3b is negatively regulated by complement factor H (CFH). Loss of function mutations in the CFH gene, however, may lead to increased levels of complement activation and platelet activity. Increased platelet C3b may play a role in vascular abnormalities of the choriocapillaris in AMD.
This is a 2020 ARVO Annual Meeting abstract.