Metabolic Arrest Suppresses Platelet Binding to and Phagocytosis by Macrophages.
Authors: Badlou Bahram Alamdary , Wu Yaping , Smid Martin W. , Akkerman Jan Willem N. 
Published: October 2019

Abstract
In previous work we have demonstrated that metabolic arrest prior to storage at 4°C better preserves expression of Glycoprotein Ib (GPIb) and GPIb-mediated platelet functions compared with control suspensions kept at room temperature without metabolic interference. However, cooling/rewarming of platelets is known to compromise post-transfusion recovery since it causes changes in the platelet membrane (chilled-rewarmed lesions) that trigger their rapid removal by liver macrophages. Binding and phagocytosis is thought to be mediated via cold-induced clustering of platelet GPIb that is recognized by hepatic macrophage complement type 3(CR3/ Mac-1) receptors. Aim of the present study is to investigate whether metabolic suppression of platelet prior to cold storage (4°C) can prevent binding to and phagocytosis by macrophages. Washed platelets were incubated without glucose and with antimycin A for 40 min at 37°C to suppress energy production. Then, metabolic suppressed platelets (MSP) were stored at 4°C for 48 hrs. Samples were collected at the end of the suppression phase and after 48 hrs storage and glycolytic energy supply was restored by incubation with glucose (20 mM glucose; 1 hr, 37°C). Controls were platelets in glucose-containing buffer stored at 22°C (controls 22°C) or cooled platelets stored at 4°C (controls 0°C) which were incubated with glucose for 1 hr at 37°C (controls 22°C) or rewarmed for 1 hr at 37°C respectively. Then, platelets were labelled with mepacrine and incubated with PMA-matured THP-1 cells for 60 min, 37°C. Platelet binding to macrophages was determined by measuring double positive particles (CD42b+/CD14+) and expressed as % of total cell count; phagocytosis by measuring mepacrine-labelled THP-1 cells and expressed as % of total number of THP-1 cells, all based on FACS analysis. Fresh platelets showed < 5% binding. At the end of the metabolic suppression phase, binding of MSP increased to 25±5%, and after 48 hrs storage to 30±4%. Compared with MSP (100%), binding of controls 22°C after 40min and 48 hr was 133±28 and 130±23% at these time intervals and of controls 0°C 188±28 and 167±30% (n=6). Fresh platelets showed < 2% phagocytosis. At the end of the metabolic suppression phase, phagocytosis of MSP increased to 26±7 % and after 48 hrs storage to 32±9%. Compared with MSP(100%), phagocytosis of controls 22°C was 155±14 and 132±19% at these time intervals and of controls 0°C 181±22 and 174±31% (n=5). Analysis before and after incubation with glucose showed that metabolic recovery induced a 30–40% fall in phagocytosis, reflecting a role of metabolic energy. Clustering of GPIb is known to be blocked by beta-N-acetyl-hexosaminidase (GlcNAc). In controls 22°C GLCNac decreased phagocytosis by 50 % but in MSP there was no effect of GlcNac treatment.
Collectively, these data show that metabolic suppression prior to storage at low temperature followed by recovery by incubation with glucose attenuates platelet-macrophage interaction and may help to develop means to decrease platelet removal by macrophages following transfusion.