Abstract
BackgroundApoptotic pathways in platelets are important for their survival and function. Platelet apoptosis may be involved in the pathogenesis of immune thrombocytopenia (ITP), an autoimmune-mediated disease. In contrast to the intrinsic apoptosis pathway, not much is known about the extrinsic pathway mechanisms in platelets.ObjectivesTo investigate the expression of proteins involved in the extrinsic apoptosis pathway, including the death receptors, adaptor and regulator proteins in human platelets. To determine a possible trigger of the extrinsic apoptosis pathway in platelets.MethodsTo investigate the expression of key markers of the extrinsic pathway we used targeted immunofluorescence and flow cytometry assays. To study their expression and interaction we performed Western blotting and co-immunoprecipitation. Treated platelets with different apoptosis triggers were subjected to flow cytometry.ResultsWe could identify the protein expression of the pro-apoptotic proteins TRADD (Tumor Necrosis Factor Receptor type 1- Associated DEATH Domain protein), TRAF2/5, (TNF Associated Factor) and DEDAF (Death Effector Domain- Associated Factor), FADD (Fas-Associated protein with death domain) as well as the anti-apoptotic proteins DJ-1 (Deglycase 1) and c-FLIP in human platelets. ABT-737 treatment induced a disruption in the co-localization of DJ-1 with FADD. Platelets treated with ABT-737 showed an activation in caspase-3 and -8. The exposure to TNF (Tumor Necrosis Factor), FasL (Fas ligand), and TWEAK or to plasma derived from ITP patients, did not lead to changes in caspase-3 and -8 activation in platelets.ConclusionsHuman platelets express some proteins of the extrinsic apoptosis pathway which can be modulated only by ABT-737 treatment. However so far, no other apoptosis trigger or interaction with an external receptor have been yet identified.
Highlights
Apoptosis is the major cellular mechanism, which regulates the cell life span and the removal of damaged or infected nucleated cells
Human platelets express some proteins of the extrinsic apoptosis pathway which can be modulated only by ABT-737 treatment
We were able to identify the expression of pro-apoptotic proteins FADD, TRADD, DEDAF and the anti-apoptotic proteins c-FLIP and DJ-1 in washed human platelets by both methods (Fig 1)
Summary
Apoptosis is the major cellular mechanism, which regulates the cell life span and the removal of damaged or infected nucleated cells. In nucleated cells two major apoptosis pathways are known: the intrinsic apoptosis pathway, regulated by pro- and anti-apoptotic members of the Bcl-2 family at the level of mitochondria, and the extrinsic pathway, that is initiated by the interaction between death ligands and death receptors of tumor-necrosis factor receptor (TNF-R) superfamily and is regulated by death inducing signaling complexes (DISC) [1]. Each of these pathways can be triggered by specific stimuli that leads to activation first of initiator caspases, caspase-8 and/or -10 (extrinsic pathway) and caspase-9 (intrinsic pathway) and of executor caspase-3, -6, and -7. In contrast to the intrinsic apoptosis pathway, not much is known about the extrinsic pathway mechanisms in platelets
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