Cytoskeletal reorganization after preparation of platelet concentrates, using the buffy coat method, and during their storage

Abstract

BACKGROUND AND OBJECTIVE: The use of platelet transfusions has risen considerably in the last years. Changes occur in platelet biochemical and membrane properties during storage. We have analyzed the effect of platelet preparation and storage of platelet function through the evaluation of platelet cytoskeletal reorganization. METHODS: A blood sample was obtained from the donor and platelets were separated as standard platelet-rich plasma (PRP) (120 g, 20 min) (PRE sample). Aliquots were also collected immediately after preparation using buffy coat procedure of platelet concentrates (day 0) and after 1, 3 and 5 days of storage. Cytoskeleton composition in both low- and high-speed cytoskeletal fractions of detergent-lysed platelets was analyzed by gradient SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Presence of each contractile protein was quantified by densitometry. RESULTS: The method used to prepare platelet concentrates induced actin polymerization (actin increased to 163.5 +/- 4.8%, mean +/- SEM, n = 8, p < 0.001, considering actin values in PRE sample as 100%) with a concurrent increase in the association of actin-binding protein (ABP), myosin and alpha-actinin to the low-speed cytoskeletal fraction. During the first 24 hours of storage, cytoskeletal assembly was partially reversed (134.8 +/- 2.6% of actin, p < 0.001) and actin polymerization increased gradually to 144.3 +/- 5.8% and 153.2 +/- 5.1% at days 3 and 5, respectively (p < 0.001 for both days). ABP, myosin and alpha-actinin showed similar tendencies to those referred for actin. Conversely, during platelet preparation and storage, the contractile proteins associated with the high-speed cytoskeletal fraction decreased, due to reorganization of the contractile proteins to the low speed fraction. INTERPRETATION AND CONCLUSIONS: The method used to prepare platelet concentrates (buffy coat procedure) induced cytoskeletal polymerization. This activating effect was partially reversed after 1 day of storage, although it increased progressively after 3 days of storage. The storage lesion may lead to defective cytoskeletal assembly in response to further stimulus. Analysis of cytoskeletal assembly is a sensitive method for detecting platelet activation caused by the concentrate preparation method and the storage conditions.