Ligand-induced cell death “ The death receptors” Ligand-induced trimerization FasL Trail TNF Death Domains Death Effectors Induced proximity of Caspase 8 Activation of Caspase 8
CASPASES CAN BE INHIBITED BY VIRUSES ... CrmA ... Baculovirüs p35 ... Ebstein Barr Virüs BHRFI proteini ... Ebstein Barr V irüs LMP-1 proteini
3 mechanisms of caspase activation a. Proteolytic cleavage e.g. pro-caspase 3 b. Induced proximity, e.g. pro-caspase 8 c. Oligomerization, e.g. cyt c, Apaf-1 & caspase 9 Back
Bcl-X L Bad Bcl-X L Bax Bcl-2 Bax Bax Bax Bcl-2 Bad CELL SURVIVAL CELL DEATH
Controlling the cell-proliferation and death machinery P53 is able to activate p21 P21 binds to the CDK-cyclin complex and inhibits its protein kinase enzymatic activity <ul><li>- CDK’s target proteins are not phosphorylated </li></ul><ul><li>- Cell cycle is unable to progress </li></ul><ul><li>When the DNA mismatches have been repaired, </li></ul><ul><li>the drop of p53 levels & a cessation of inhibition </li></ul>G1-to-S checkpoint block The cell cycle: negative intracellular controls Intracellular signals <ul><li>Fail-safe systems (checkpoints) ensure that the cell cycle does not progress </li></ul><ul><li>until the cell is competent. </li></ul>
The bcl-2 family Receptor domain phosphorylation Raf-1 calcineurin Pore formation Membrane anchor Ligand domain BH4 BH3 BH1 BH2 TM N C Group I Group II Group III Bcl-2 bax Bad bid bik
Bcl-2 Protein (with BH3 Peptide) Bcl-2 Protein
<ul><li>65000 papers </li></ul><ul><li>Apoptsis in anucleate platelets wa first reported in 1997 by Vanags et al. </li></ul><ul><li>It was demonstrated that apoptosis within megakaryocytes & megakaryoblastic is causal for platelet production (NO , TNF α , BCL 2 ) </li></ul>
<ul><li>Models of platelet apoptosis </li></ul><ul><li>apoptosis of platelets was induced by the calcium ionophores ionomycin , A23187 which induce apoptosis in nucleate cells. </li></ul><ul><li>apoptosis was provoked by platelet storage in culture where washed platelets were aged by incubation for 18–24 h at 37 C in a culture medium or plasma in capped tubes </li></ul><ul><li>apoptosis was induced by platelet aging in vitro during storage of leukodepleted platelet concentrates (PCs) under standard blood banking conditions at 22 C </li></ul><ul><li>apoptosis was associated with platelet aging in vivo in dogs with thrombopoiesis suppressed by estradiol injection . </li></ul><ul><li>platelet apoptosis was reported in mice with thrombocytopenia caused by malaria infection and induced by injection of TNF or anti-platelet antibodies </li></ul>
<ul><li>Apoptotic changes in platelet morphology </li></ul><ul><li>These morphologic changes included: </li></ul><ul><li>platelet shrinkage </li></ul><ul><li>cytoplasm condensation </li></ul><ul><li>plasma membrane blebbing </li></ul><ul><li>extension of filopodia. </li></ul>Originaly , these changes were described as “ platelet activation ” and only since 1997 did some investigetors begin to consider these morphologic chenges as apoptotic.
Li et al, found that platelets express mRNA for death ligand TRAIL , death receptors TNFR1, DR3 , DR4 and DR5 , and adapter proteins TRADD and RIP . In contrast , Fas receptor and Fas ligand were not detected in platelets as determined by mRNA and immunoblot and anti-Fas antibodies had no eﬀect on platelets.
ᴪ m In normal undamaged nucleate cells, mitochondria have a high ᴪ m; breakdown of ᴪ m is characteristic of early apoptosis . ᴪ m in platelets can be measured by the cell-permeable lipophilic cationic dyes JC-1 and DiOC6 . Using JC-1 , we have demonstrated depolarization of ᴪ m in PCs starting from days 13_14 of storage.
Cytochrome c, Diablo/Smac and Apaf-1. Cytochrome C and Apaf-1 have been found by immunoblot in whole lysates of fresh nonactivated platelets.