Analysis of Platelet Degenerative Processes in a Sterile Closed System

HA Leaver, V Muzola, DB McPhail, G Cook. 1University of Edinburgh, Edinburgh, United Kingdom, 2Department of Pharmacy, University of Rwanda, Rwanda, Rwanda, 3Antoxis Limited, Aberdeen, United Kingdom, 4SNBTS Edinburgh, Edinburgh, United Kingdom.

Platelets are essential for haemostasis and are among the most labile of transfused blood products, undergoing degenerative changes when stored under blood bank conditions. However, the signalling processes in platelet ageing are incompletely understood, and agents improving platelet viability and function are the focus of current research. The aim of this study was to investigate the relation between mitochondrial cell death pathways and stress signals in ageing platelets in vitro, using calpain inhibitors and A0-1-530, a lipophilic antioxidant developed from a flavonoid scaffold, which is rapidly incorporated into cells and targets the mitochondrial compartment.

Platelets for transfusion are prepared and stored under blood bank conditions with continuous agitation at 20-24°C in gas permeable pack, and these conditions were used in this study. Platelets (40-50ml) from irradiated pooled platelet preparations from 44 healthy donors were transferred under aseptic conditions to 6-8 400ml aphesis packs (Fresenius Kabi). Stress signals associated with vascular disorders, including peroxide, and potential antagonists of these (antioxidant flavonoids, calpain inhibitors) or vehicle control were added under aseptic conditions to platelets 2 days after collection and stored for 2-20 days under blood bank conditions, and mitochondrial function (mitochondrial membrane potential, δψm) and associated caspase-9 activity (Promega), were analysed in 2ml samples, withdrawn at 2,5,7,9 and 12 days after collection (Leaver et al 2006).

The sterile closed system permitted analysis of platelet degenerative processes under blood bank conditions for up to 8 agonists, antagonists or doses (and approximately twice this number of determinations, if paediatric packs were used). Using the intrinsic cell death marker δψm, to monitor platelet ageing, a high correlation (0.983) was obtained in 10 samples obtained from this system, compared with 10 conventional single packs, stored under blood bank conditions. Over the 12-day incubation period, there was a decline in platelet count, hypotonic shock response (HSR), pH, δψm, and an associated increase in caspase-9 activity in platelets. Incubation with the oxidative stress signal tert-butyl-hydroperoxide (0.1-100μM) was associated with a dose dependent decrease in platelet count, HSR, pH, and δψm. Incubation with the calpain inhibitors calpeptin (50-500μM), MG-132 (5-50μM), ALLN (5-15μM), and the flavonoid A-01-530 (0.5-1.5μM), was associated with dose-dependent protection against age-associated decreases in platelet cell death signalling, implicating oxidative signalling and intracellular calcium in the degenerative pathways of platelet cell death.

Leaver HA et al (2006) Platelets 17, 368-77.