title: Thrombin is an extracellular signal that activates intracellular death protease pathways inducing apoptosis in model motor neurons.
abstract: Apoptosis, often also termed "programmed cell death", occurs in normal development in the brain and spinal cord. Important to concepts of disease and potential intervention is the exciting finding that apoptosis is also found after neurotrauma and in a number of neurodegenerative diseases. Although the precise mechanism of neuronal cell loss remains unknown, much emphasis has been placed recently on the activation of cell death protease cascades within the cell. How these cascades may be activated, especially from extracellular influences, is currently poorly understood. Thrombin, the multifunctional coagulation protease, is an early phase modulator at sites of tissue injury and has been shown to induce cell death in neurons by an apoptotic mechanism by activating its receptor, PAR-1. Using a model motor neuronal cell line, NSC19, which we have shown undergoes apoptosis after treatment with classic apoptosis inducers such as the topoisomerase inhibitors camptothecin and etoposide, we unambiguously found that nanomolar thrombin induced characteristic signs of apoptosis. Strikingly, endonucleolysis was accompanied by an increase in caspase-3-like activity in cellular extracts, which correlated with both detection of caspase-induced signature cleavage of the cortical cytoskeleton component nonerythroid spectrin (alpha-fodrin) and identification of increased accessibility of a caspase cleavage domain, using an antibody (Ab127) made against a synthetic peptide KGDEVD. Demonstrating that thrombin activation of death proteases was linked to cell death, we were able to inhibit thrombin-induced apoptosis by using a caspase family inhibitor, benzyloxycarbonyl-Asp-(oMe)-fluoromethyl ketone (Boc-D-FMK). These novel results demonstrate that thrombin serves as an extracellular "death signal" to activate intracellular protease pathways. These pathways lead to apoptotic cell death and can be modulated by inhibiting caspase activity downstream to PAR-1.
authors.affiliation: Neurobiology Research Laboratory (151R), Department of Veterans Affairs Medical Center, Kansas City, Missouri 64128, USA.
authors.forename: I V
authors.initials: IV
authors.lastname: Smirnova
authors.forename: S X
authors.initials: SX
authors.lastname: Zhang
authors.forename: B A
authors.initials: BA
authors.lastname: Citron
authors.forename: P M
authors.initials: PM
authors.lastname: Arnold
authors.forename: B W
authors.initials: BW
authors.lastname: Festoff
mesh_terms: D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D053938:DNA Fragmentation; D010450:Endopeptidases / Q000502:physiology*; D004789:Enzyme Activation / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005110:Extracellular Space / Q000502:physiology*; D007425:Intracellular Membranes / Q000201:enzymology*; D051379:Mice; D009046:Motor Neurons / Q000187:drug effects / Q000201:enzymology / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology*; D013917:Thrombin / Q000494:pharmacology / Q000502:physiology*; D059004:Topoisomerase I Inhibitors; D059005:Topoisomerase II Inhibitors
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D015853:Cysteine Proteinase Inhibitors; D004791:Enzyme Inhibitors; D059004:Topoisomerase I Inhibitors; D059005:Topoisomerase II Inhibitors; C103192:butyloxycarbonyl-O-methyl-aspartyl-fluoromethyl ketone; D010450:Endopeptidases; D013917:Thrombin; D003546:Cysteine Endopeptidases