latrunculin-a and benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone

Megakaryocytes assemble and release platelets through the extension of proplatelet processes, which are cytoplasmic extensions that extrude from the megakaryocyte and form platelets at their tips. Proplatelet formation and platelet release are complex processes that require a combination of structural rearrangements. While the signals that trigger the initiation of proplatelet formation process are not completely understood, it has been shown that inhibition of cytoskeletal signaling in mature megakaryocytes induces proplatelet formation. Megakaryocyte apoptosis may also be involved in initiation of proplatelet extension, although this is controversial. This study inquires whether the proplatelet production induced by cytoskeletal signaling inhibition is dependent on activation of apoptosis.. Megakaryocytes derived from human umbilical cord blood CD34+ cells were treated with the actin polymerization inhibitor latrunculin and their ploidy and proplatelet formation were quantitated. Apoptosis activation was analyzed by flow cytometry and luminescence assays. Caspase activity was inhibited by two compounds, ZVAD and QVD. Expression levels of pro-survival and pro-apoptosis genes were measured by quantitative RT-PCR. Protein levels of Bcl-XL, Bax and Bak were measured by western blot. Cell ultrastructure was analyzed by electron microscopy.. Actin inhibition resulted in increased ploidy and increased proplatelet formation in cultured umbilical cord blood-derived megakaryocytes. Actin inhibition activated apoptosis in the cultured cells. The effects of actin inhibition on proplatelet formation were blocked by caspase inhibition. Increased expression of both pro-apoptotic and pro-survival genes was observed. Pro-survival protein (Bcl-xL) levels were increased compared to levels of pro-apoptotic proteins Bak and Bax. Despite apoptosis being activated, the megakaryocytes underwent minimal ultrastructural changes during actin inhibition.. We report a correlation between increased proplatelet formation and activation of apoptosis, and that the increase in proplatelet formation in response to actin inhibition is caspase dependent. These findings support a role for apoptosis in proplatelet formation in this model.

Topics: Actins; Amino Acid Chloromethyl Ketones; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; bcl-X Protein; Blood Platelets; Bridged Bicyclo Compounds, Heterocyclic; Caspases; Cells, Cultured; Fetal Blood; Humans; Megakaryocytes; Microscopy, Electron, Transmission; Microscopy, Fluorescence; Polyploidy; Quinolines; Real-Time Polymerase Chain Reaction; Thiazolidines

Binding of either ligand or agonistic antibodies to the death receptor CD95 (APO-1/Fas) induces the formation of the death-inducing signaling complex (DISC). We now show that signal initiation of CD95 in type I cells can be further separated into at least four distinct steps. (i) The first step is ligand-induced formation of CD95 microaggregates at the cell surface. (ii) The second step is recruitment of FADD to form a DISC. This step is dependent on actin filaments. (iii) The third step involves formation of large CD95 surface clusters. This event is positively regulated by DISC-generated caspase 8. (iv) The fourth step is internalization of activated CD95 through an endosomal pathway. The latter step is again dependent on the presence of actin filaments. The data indicate that the signal initiation by CD95 is a complex process actively regulated at various levels, providing a number of new drug targets to specifically modulate CD95 signaling.

Topics: Actin Cytoskeleton; Adaptor Proteins, Signal Transducing; Amino Acid Chloromethyl Ketones; Animals; Anti-Bacterial Agents; Apoptosis; beta-Cyclodextrins; Bridged Bicyclo Compounds, Heterocyclic; Carrier Proteins; Caspase 8; Caspase 9; Caspase Inhibitors; Caspases; Cell Line; Cell Membrane; Cyclodextrins; Endocytosis; fas Receptor; Fas-Associated Death Domain Protein; Filipin; Fluorescent Dyes; Humans; Ionophores; Ligands; Lymphocytes; Membrane Microdomains; Membrane Potentials; Microscopy, Fluorescence; Mitochondria; Models, Biological; Nystatin; Receptor Aggregation; Signal Transduction; Thiazoles; Thiazolidines