Leukemia (2001) 15, 1572–1581  2001 Nature Publishing Group All rights reserved 0887-6924/01 $15.00 www.nature.com/leu Early increase in DcR2 expression and late activation of caspases in the platelet storage lesion S Plenchette 1 , M Moutet 1 , M Benguella 2 , JP N’Gondara 2 , F Guigner 2 , C Coffe 2 , L Corcos 1 , A Bettaieb 1 and E Solary 1,3 1 INSERM U517, Dijon; 2 Etablissement Franc ¸ais du Sang de Bourgogne-Franche-Comte ´, and 3 Clinical Hematology Unit, CHU Le Bocage, Dijon, France Platelet transfusion is widely used to prevent bleeding in patients with severe thrombocytopenia. The maximal storage duration of platelet concentrates is usually 5 days, due to the platelet storage lesion that impairs their functions when stored for longer times. Some of the morphological and biochemical changes that characterize this storage lesion are reminiscent of cell death by apoptosis. The present study analyzed whether proteins involved in nucleated cell apoptosis could play a role in the platelet storage lesion. Storage of leukocyte-depleted platelets obtained by apheresis is associated with a late and limited activation of caspases, mainly caspase-3. This event correlates with an increased expression of the pro-apoptotic BH3-only protein Bim in the particulate fraction and a slight and late release of the pro-apoptotic mitochondrial protein Diablo/Smac in the cytosol. Platelets do not express the death receptors Fas, DR4 and DR5 on their plasma membrane, while the expression of the decoy receptor DcR2 increases progress- ively during platelet storage. Addition of low concentrations of the cryoprotector dimethylsulfoxide accelerates platelet cas- pase activation during storage, an effect that is partially pre- vented by the caspase inhibitor z-VAD-fmk. Altogether, DcR2 expression on the plasma membrane is an early event while caspase activation is a late event during platelet storage. These observations suggest that caspases are unlikely to account for the platelet storage lesion. As a consequence, addition of cas- pase inhibitors may not improve the quality of platelet concen- trates stored in standard conditions. Leukemia (2001) 15, 1572–1581. Keywords: platelets; caspase; aging; storage; apoptosis Introduction Platelets are anucleated blood cells derived from bone mar- row megakaryocytes. These cells, whose life-span in the circu- lation is about 10 to 12 days, are critical for normal hemost- asis. Transfusion of platelet concentrates is widely used to prevent bleeding in patients with severe thrombocytopenia as a consequence of various diseases or cytotoxic treatments. The two main methods currently used to prepare platelet con- centrates from healthy donors are the buffy coat (multiple donors) and the apheresis (single donor) methods. 1 In some countries, leukocyte depletion of platelet concentrates is per- formed systematically by using a filtration method. In France, the number of residual leukocytes per bag must be lower than 1 × 10 6 per concentrate that otherwise contains more than 2 × 10 11 platelets. Then, platelet concentrates are suspended in plasma and stored in gas-permeable plastic containers with gentle and continuous agitation on a horizontal reciprocator at a stable temperature of 20–24°C. The maximal storage dur- ation after whole blood withdrawal is 5 days. 2 Platelet storage duration is limited by the platelet storage lesion that associates morphological and metabolic changes, Correspondence: E Solary, INSERM U517, Faculty of Medicine, 7 boulevard Jeanne d’Arc, 21000 Dijon, France; Fax: 33 3 80 39 34 34 Received 26 March 2001; accepted 29 May 2001 leading to impaired functions. 3 The conditions of harvesting, processing and storage influence these platelet changes that can be associated with a decrease in post-transfusion recovery in vivo. The platelet storage lesion can be identified by a var- iety of in vitro tests. The thin discoid shape of circulating platelets is gradually lost within storage to become spherical or dendritic while the size of platelets decreases as a conse- quence of fragmentation into microvesicles. 4 Visual obser- vation of the swirling phenomenon is the simplest way to identify these platelet shape changes. Altered metabolic activity, as indicated by pH changes, increased lactate pro- duction and decreased glucose consumption, as well as lac- tate dehydrogenase (LDH) release, also reflect the conditions of storage. 3 Many of the other changes which take place in platelets during their storage are similar to the activation of fresh plate- lets, although they occur much more slowly (several days compared to a few seconds) and are partially reversible. Both activated and stored platelets undergo -granule release, as demonstrated by expression of CD62P (also termed P- selection) on the plasma membrane and release of cleaved CD62P in the plasma. 5–7 They also demonstrate critical plasma membrane changes such as externalization of phos- phatidylserine that serves as the major surface procoagulant for assembly of the prothrombinase complex in the coagu- lation pathway. 8 Several of the events associated with platelet activation are reminiscent of cell death by apoptosis. 9 Two main pathways have been shown to lead to apoptosis. The intrinsic pathway involves the release of pro-apoptotic molecules such as cyto- chrome c (cyt c), 10,11 apoptosis-inducing factor (AIF), 12 and Diablo/Smac 13,14 from the mitochondrial intermembrane space under the control of pro-apoptotic and anti-apoptotic proteins of the Bcl-2 family. Once in the cytosol, cytochrome c, in the presence of ATP, induces oligomerization of the adaptor molecule Apaf-1 (apoptosis protein activating factor- 1) that, in turn, recruits and activates caspase-9 and the down- stream cascade of effector caspases such as caspase-3. 15 The extrinsic pathway involves plasma membrane death receptors that, upon binding of their specific ligand, recruit the adaptor molecule FADD (Fas associated death domain) and the initiator caspase designated caspase-8 in a death-inducing sig- naling complex. Activated caspase-8, either directly or through the release of mitochondrial proapoptotic molecules, activates the cascade of effector caspases. 16,17 Caspases are a family of cysteine aspartate proteases that are synthesized as proenzymes and must be cleaved at aspartate residues to be activated. Effector caspases cleave a limited number of intra- cellular proteins, leading to the dismantling of the cells that characterizes apoptotic cell death. 18,19 Several observations suggested a role for these apoptotic pathways in the platelet storage lesion: (1) cell shrinkage, release of microvesicules and phosphatidylserine redistri- bution on the cell surface are observed in both activated plate-