nitrophenols and Thrombocytopenia

Chemotherapy-induced thrombocytopenia is a common bleeding risk in cancer patients and limits chemotherapy dose and frequency. Recent data from mouse and human platelets revealed that activation of protein kinase A/G (PKA/PKG) not only inhibited thrombin/convulxin-induced platelet activation but also prevented the platelet pro-coagulant state. Here we investigated whether or not PKA/PKG activation could attenuate caspase-dependent apoptosis induced by the anti-cancer drugs ABT-737 (the precursor of navitoclax) and thymoquinone (TQ), thereby potentially limiting chemotherapy-induced thrombocytopenia. This is particularly relevant as activation of cyclic nucleotide signalling in combination chemotherapy is an emerging strategy in cancer treatment. However, PKA/PKG-activation, as monitored by phosphorylation of Vasodilator-stimulated phosphoprotein (VASP), did not block caspase-3-dependent platelet apoptosis induced by the compounds. In contrast, both substances induced PKA activation themselves and PKA activation correlated with platelet inhibition and apoptosis. Surprisingly, ABT-737- and TQ-induced VASP-phosphorylation was independent of cAMP levels and neither cyclases nor phosphatases were affected by the drugs. In contrast, however, ABT-737- and TQ-induced PKA activation was blocked by caspase-3 inhibitors. In conclusion, we show that ABT-737 and TQ activate PKA in a caspase-3-dependent manner, which correlates with platelet inhibition and apoptosis and therefore potentially contributes to the bleeding risk in chemotherapy patients.

Topics: Animals; Apoptosis; Benzoquinones; Biphenyl Compounds; Blood Platelets; Caspase 3; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP-Dependent Protein Kinases; Humans; Mice; Neoplasms; Nitrophenols; Piperazines; Platelet Activation; Sulfonamides; Thrombocytopenia

The simultaneous delivery of multiple cancer drugs in combination therapies to achieve optimal therapeutic effects in patients can be challenging. This study investigated whether co-encapsulation of the BH3-mimetic ABT-737 and the topoisomerase I inhibitor camptothecin (CPT) in PEGylated polymeric nanoparticles (NPs) was a viable strategy for overcoming their clinical limitations and to deliver both compounds at optimal ratios. We found that thrombocytopenia induced by exposure to ABT-737 was diminished through its encapsulation in NPs. Similarly, CPT-associated leukopenia and gastrointestinal toxicity were reduced compared with the administration of free CPT. In addition to the reduction of dose-limiting side effects, the co-encapsulation of both anticancer compounds in a single NP produced synergistic induction of apoptosis in both in vitro and in vivo colorectal cancer models. This strategy may widen the therapeutic window of these and other drugs and may enhance the clinical efficacy of synergistic drug combinations.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Biphenyl Compounds; Camptothecin; Cell Line, Tumor; Colorectal Neoplasms; Drug Compounding; Drug Synergism; Humans; Male; Mice; Mice, Inbred C57BL; Nanoparticles; Nitrophenols; Piperazines; Sulfonamides; Thrombocytopenia

Apoptotic caspases, including caspase-9, are thought to facilitate platelet shedding by megakaryocytes. They are known to be activated during platelet apoptosis, and have also been implicated in platelet hemostatic responses. However, the precise requirement for, and the regulation of, apoptotic caspases have never been defined in either megakaryocytes or platelets. To establish the role of caspases in platelet production and function, we generated mice lacking caspase-9 in their hematopoietic system. We demonstrate that both megakaryocytes and platelets possess a functional apoptotic caspase cascade downstream of Bcl-2 family-mediated mitochondrial damage. Caspase-9 is the initiator caspase, and its loss blocks effector caspase activation. Surprisingly, steady-state thrombopoiesis is unperturbed in the absence of caspase-9, indicating that the apoptotic caspase cascade is not required for platelet production. In platelets, loss of caspase-9 confers resistance to the BH3 mimetic ABT-737, blocking phosphatidylserine (PS) exposure and delaying ABT-737-induced thrombocytopenia in vivo. Despite this, steady-state platelet lifespan is normal. Casp9(-/-) platelets are fully capable of physiologic hemostatic responses and functional regulation of adhesive integrins in response to agonist. These studies demonstrate that the apoptotic caspase cascade is required for the efficient death of megakaryocytes and platelets, but is dispensable for their generation and function.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Biphenyl Compounds; Blood Platelets; Caspase 9; Cell Lineage; Hemostasis; Hirudins; Liver; Liver Transplantation; Megakaryocytes; Mice; Mice, Inbred C57BL; Mice, Knockout; Nitrophenols; Piperazines; Platelet Activation; Radiation Chimera; Sulfonamides; Thrombocytopenia; Thrombopoiesis

A pivotal step in the mitochondrial pathway of apoptosis is activation of Bak and Bax, although the molecular mechanism remains controversial. To examine whether mitochondrial apoptosis can be induced by just a lack of antiapoptotic Bcl-2-like proteins or requires direct activators of the BH3-only proteins including Bid and Bim, we studied the molecular requisites for platelet apoptosis induced by Bcl-xL deficiency. Severe thrombocytopenia induced by thrombocyte-specific Bcl-xL knock-out was fully rescued in a Bak and Bax double knock-out background but not with single knock-out of either one. In sharp contrast, deficiency of either Bid, Bim, or both did not alleviate thrombocytopenia in Bcl-xL knock-out mice. An in vitro study revealed that ABT-737, a Bad mimetic, induced platelet apoptosis in association with a conformational change of the amino terminus, translocation from the cytosol to mitochondria, and homo-oligomerization of Bax. ABT-737-induced Bax activation and apoptosis were also observed in Bid/Bim-deficient platelets. Human platelets, upon storage, underwent spontaneous apoptosis with a gradual decline of Bcl-xL expression despite a decrease in Bid and Bim expression. Apoptosis was attenuated in Bak/Bax-deficient or Bcl-xL-overexpressing platelets but not in Bid/Bim-deficient platelets upon storage. In conclusion, platelet lifespan is regulated by a fine balance between anti- and proapoptotic multidomain Bcl-2 family proteins. Despite residing in platelets, BH3-only activator proteins Bid and Bim are dispensable for Bax activation and mitochondrial apoptosis.

Topics: Animals; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; bcl-X Protein; BH3 Interacting Domain Death Agonist Protein; Biphenyl Compounds; Blood Platelets; Cytosol; Humans; Mice; Mice, Knockout; Mitochondria; Nitrophenols; Piperazines; Protein Transport; Sulfonamides; Thrombocytopenia

Apoptosis in megakaryocytes results in the formation of platelets. The role of apoptotic pathways in platelet turnover and in the apoptotic-like changes seen after platelet activation is poorly understood. ABT-263 (Navitoclax), a specific inhibitor of antiapoptotic BCL2 proteins, which is currently being evaluated in clinical trials for the treatment of leukemia and other malignancies, induces a dose-limiting thrombocytopenia. In this study, the relationship between BCL2/BCL-X(L) inhibition, apoptosis, and platelet activation was investigated. Exposure to ABT-263 induced apoptosis but repressed platelet activation by physiologic agonists. Notably, ABT-263 induced an immediate calcium response in platelets and the depletion of intracellular calcium stores, indicating that on BCL2/BCL-X(L) inhibition platelet activation is abrogated because of a diminished calcium signaling. By comparing the effects of ABT-263 and its analog ABT-737 on platelets and leukemia cells from the same donor, we show, for the first time, that these BCL2/BCL-X(L) inhibitors do not offer any selective toxicity but induce apoptosis at similar concentrations in leukemia cells and platelets. However, reticulated platelets are less sensitive to apoptosis, supporting the hypothesis that treatment with ABT-263 induces a selective loss of older platelets and providing an explanation for the transient thrombocytopenia observed on ABT-263 treatment.

Topics: Aniline Compounds; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; bcl-X Protein; Biphenyl Compounds; Blood Platelets; Calcium Signaling; Gene Expression; Homeostasis; Humans; Kinetics; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphocytes; Molecular Targeted Therapy; Nitrophenols; Piperazines; Platelet Activation; Platelet Aggregation Inhibitors; Proto-Oncogene Proteins c-bcl-2; Sulfonamides; Thrombocytopenia; Thrombopoiesis

BH3 mimetics are a new class of proapo-ptotic anticancer agents that have shown considerable promise in preclinical animal models and early-stage human trials. These agents act by inhibiting the pro-survival function of one or more Bcl-2-related proteins. Agents that inhibit Bcl-x(L) induce rapid platelet death that leads to thrombocytopenia; however, their impact on the function of residual circulating platelets remains unclear. In this study, we demonstrate that the BH3 mimetics, ABT-737 or ABT-263, induce a time- and dose-dependent decrease in platelet adhesive function that correlates with ectodomain shedding of the major platelet adhesion receptors, glycoprotein Ibα and glycoprotein VI, and functional down-regulation of integrin α(IIb)β(3). Analysis of platelets from mice treated with higher doses of BH3 mimetics revealed the presence of a subpopulation of circulating platelets undergoing cell death that have impaired activation responses to soluble agonists. Functional analysis of platelets by intravital microscopy revealed a time-dependent defect in platelet aggregation at sites of vascular injury that correlated with an increase in tail bleeding time. Overall, these studies demonstrate that Bcl-x(L)-inhibitory BH3 mimetics not only induce thrombocytopenia but also a transient thrombocytopathy that can undermine the hemostatic function of platelets.

Topics: Aniline Compounds; Animals; Apoptosis; bcl-X Protein; Biphenyl Compounds; Blood Platelets; Blotting, Western; Collagen; Dose-Response Relationship, Drug; Down-Regulation; Hemostasis; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Microscopy, Electron, Scanning; Nitrophenols; Phosphatidylserines; Piperazines; Platelet Adhesiveness; Platelet Aggregation; Platelet Count; Platelet Glycoprotein GPIb-IX Complex; Platelet Glycoprotein GPIIb-IIIa Complex; Platelet Membrane Glycoproteins; Sulfonamides; Thrombocytopenia; Time Factors

Platelets are anuclear cytoplasmic fragments essential for blood clotting and wound healing. Despite much speculation, the factors determining their life span in the circulation are unknown. We show here that an intrinsic program for apoptosis controls platelet survival and dictates their life span. Pro-survival Bcl-x(L) constrains the pro-apoptotic activity of Bak to maintain platelet survival, but as Bcl-x(L) degrades, aged platelets are primed for cell death. Genetic ablation or pharmacological inactivation of Bcl-x(L) reduces platelet half-life and causes thrombocytopenia in a dose-dependent manner. Deletion of Bak corrects these defects, and platelets from Bak-deficient mice live longer than normal. Thus, platelets are, by default, genetically programmed to die by apoptosis. The antagonistic balance between Bcl-x(L) and Bak constitutes a molecular clock that determines platelet life span: this represents an important paradigm for cellular homeostasis, and has profound implications for the diagnosis and treatment of disorders that affect platelet number and function.

Topics: Animals; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; bcl-X Protein; Biomimetics; Biphenyl Compounds; Blood Platelets; Caspases; Cell Nucleus; Crosses, Genetic; Ethylnitrosourea; Female; Male; Mice; Mice, Inbred BALB C; Models, Molecular; Mutagenesis; Nitrophenols; Piperazines; Protein Structure, Tertiary; Sulfonamides; Thrombocytopenia

Topics: Acid Phosphatase; Blood Cell Count; Blood Coagulation Tests; Blood Platelets; Carcinoma; Clot Retraction; Depression, Chemical; Heparin; Humans; Male; Nitrophenols; Prostatic Neoplasms; Temperature; Thrombocytopenia