Emma C. Josefsson

Two distinct pathways regulate platelet phosphatidylserine exposure and procoagulant function

Procoagulant platelets exhibit hallmark features of apoptotic cells, including membrane blebbing, microvesiculation, and phosphatidylserine (PS) exposure. Although platelets possess many well-known apoptotic regulators, their role in regulating the procoagulant function of platelets is unclear. To clarify this, we investigated the consequence of removing the essential mediators of apoptosis, Bak and Bax, or directly inducing apoptosis with the BH3 mimetic compound ABT-737. Treatment of platelets with ABT-737 triggered PS exposure and a marked increase in thrombin generation in vitro. This increase in procoagulant function was Bak/Bax- and caspase-dependent, but it was unaffected by inhibitors of platelet activation or by chelating extracellular calcium. In contrast, agonist-induced platelet procoagulant function was unchanged in Bak(-/-)Bax(-/-) or caspase inhibitor-treated platelets, but it was completely eliminated by extracellular calcium chelators or inhibitors of platelet activation. These studies show the existence of 2 distinct pathways regulating the procoagulant function of platelets.

The Dendritic Cell Receptor Clec9A Binds Damaged Cells via Exposed Actin Filaments

The immune system must distinguish viable cells from cells damaged by physical and infective processes. The damaged cell-recognition molecule Clec9A is expressed on the surface of the mouse and human dendritic cell subsets specialized for the uptake and processing of material from dead cells. Clec9A recognizes a conserved component within nucleated and nonnucleated cells, exposed when cell membranes are damaged. We have identified this Clec9A ligand as a filamentous form of actin in association with particular actin-binding domains of cytoskeletal proteins. We have determined the crystal structure of the human CLEC9A C-type lectin domain and propose a functional dimeric structure with conserved tryptophans in the ligand recognition site. Mutation of these residues ablated CLEC9A binding to damaged cells and to the isolated ligand complexes. We propose that Clec9A provides targeted recruitment of the adaptive immune system during infection and can also be utilized to enhance immune responses generated by vaccines.

Dual roles for hepatic lectin receptors in the clearance of chilled platelets.

Rapid chilling causes glycoprotein-Ib (GPIb) receptors to cluster on blood platelets. Hepatic macrophage β2 integrin binding to β-N-acetylglucosamine (β-GlcNAc) residues in the clusters leads to rapid clearance of acutely chilled platelets after transfusion. Although capping the β-GlcNAc moieties by galactosylation prevents clearance of short-term–cooled platelets, this strategy is ineffective after prolonged refrigeration. We report here that prolonged refrigeration increased the density and concentration of exposed galactose residues on platelets such that hepatocytes, through Ashwell-Morell receptor binding, become increasingly involved in platelet removal. Macrophages rapidly removed a large fraction of transfused platelets independent of their storage conditions. With prolonged platelet chilling, hepatocyte-dependent clearance further diminishes platelet recovery and survival after transfusion. Inhibition of chilled platelet clearance by both β2 integrin and Ashwell-Morell receptors may afford a potentially simple method for storing platelets in the cold.

Bcl-xL–inhibitory BH3 mimetics can induce a transient thrombocytopathy that undermines the hemostatic function of platelets

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.

Megakaryocytes possess a functional intrinsic apoptosis pathway that must be restrained to survive and produce platelets

Deletion of Bak and Bax, the effectors of mitochondrial apoptosis, does not affect platelet production, however, loss of prosurvival Bcl-xL results in megakaryocyte apoptosis and failure of platelet shedding.

Deciphering the molecular and biologic processes that mediate histone deacetylase inhibitor–induced thrombocytopenia

Histone deacetylase inhibitor (HDACI)-induced thrombocytopenia (TCP) is a major dose-limiting toxicity of this new class of drugs. Using preclinical models to study the molecular and biologic events that underpin this effect of HDACI, we found that C57BL/6 mice treated with both the HDAC1/2-selective HDACI romidepsin and the pan-HDACI panobinostat developed significant TCP. HDACI-induced TCP was not due to myelosuppression or reduced platelet lifespan, but to decreased platelet release from megakaryocytes. Cultured primary murine megakaryocytes showed reductions in proplatelet extensions after HDACI exposure and a dose-dependent increase in the phosphorylation of myosin light chain 2 (MLC2). Phosphorylation of MLC to phospho-MLC (pMLC) and subsequent proplatelet formation in megakaryocytes is regulated by the Rho-GTPase proteins Rac1, CDC42, and RhoA. Primary mouse megakaryocytes and the human megakaryoblastic cell line Meg-01 showed reductions in Rac1, CDC42, and RhoA protein levels after treatment with HDACIs. We were able to overcome HDACI-induced TCP by administering the mouse-specific thrombopoietin (TPO) mimetic AMP-4, which improved platelet numbers to levels similar to untreated controls. Our report provides the first detailed account of the molecular and biologic processes involved in HDACI-mediated TCP. Moreover, our preclinical studies provide evidence that dose-limiting TCP induced by HDACIs may be circumvented using a TPO mimetic.

Desialylation accelerates platelet clearance after refrigeration and initiates GPIbα metalloproteinase-mediated cleavage in mice

When refrigerated platelets are rewarmed, they secrete active sialidases, including the lysosomal sialidase Neu1, and express surface Neu3 that remove sialic acid from platelet von Willebrand factor receptor (VWFR), specifically the GPIbα subunit. The recovery and circulation of refrigerated platelets is greatly improved by storage in the presence of inhibitors of sialidases. Desialylated VWFR is also a target for metalloproteinases (MPs), because GPIbα and GPV are cleaved from the surface of refrigerated platelets. Receptor shedding is inhibited by the MP inhibitor GM6001 and does not occur in Adam17(ΔZn/ΔZn) platelets expressing inactive ADAM17. Critically, desialylation in the absence of MP-mediated receptor shedding is sufficient to cause the rapid clearance of platelets from circulation. Desialylation of platelet VWFR therefore triggers platelet clearance and primes GPIbα and GPV for MP-dependent cleavage.

Dual requirement for the ETS transcription factors Fli-1 and Erg in hematopoietic stem cells and the megakaryocyte lineage

Fli-1 and Erg are closely related members of the Ets family of transcription factors. Both genes are translocated in human cancers, including Ewings sarcoma, leukemia, and in the case of Erg, more than half of all prostate cancers. Although evidence from mice and humans suggests that Fli-1 is required for megakaryopoiesis, and that Erg is required for normal adult hematopoietic stem cell (HSC) regulation, their precise physiological roles remain to be defined. To elucidate the relationship between Fli-1 and Erg in hematopoiesis, we conducted an analysis of mice carrying mutations in both genes. Our results demonstrate that there is a profound genetic interaction between Fli-1 and Erg. Double heterozygotes displayed phenotypes more dramatic than single heterozygotes: severe thrombocytopenia, with a significant deficit in megakaryocyte numbers and evidence of megakaryocyte dysmorphogenesis, and loss of HSCs accompanied by a reduction in the number of committed hematopoietic progenitor cells. These results illustrate an indispensable requirement for both Fli-1 and Erg in normal HSC and megakaryocyte homeostasis, and suggest these transcription factors may coregulate common target genes.

Mpl expression on megakaryocytes and platelets is dispensable for thrombopoiesis but essential to prevent myeloproliferation

Significance Blood platelets, the small circulating cells that coordinate hemostasis, are produced by specialized bone marrow cells called megakaryocytes. The cytokine thrombopoietin (TPO) is a key regulator of platelet production acting via its specific cell receptor, Mpl. Via genetic modification of the Mpl allele in mice, we precisely define the bone marrow cells that express Mpl and, by genetically removing Mpl from megakaryocytes and platelets, we show TPO signaling via Mpl is not required in megakaryocytes for their expansion, maturation, or platelet production. Rather, Mpl expression on megakaryocytes is essential for regulating TPO availability in the bone marrow microenvironment to prevent myeloproliferation, a model we suggest is important for human disease. Thrombopoietin (TPO) acting via its receptor, the cellular homologue of the myeloproliferative leukemia virus oncogene (Mpl), is the major cytokine regulator of platelet number. To precisely define the role of specific hematopoietic cells in TPO-dependent hematopoiesis, we generated mice that express the Mpl receptor normally on stem/progenitor cells but lack expression on megakaryocytes and platelets (MplPF4cre/PF4cre). MplPF4cre/PF4cre mice displayed profound megakaryocytosis and thrombocytosis with a remarkable expansion of megakaryocyte-committed and multipotential progenitor cells, the latter displaying biological responses and a gene expression signature indicative of chronic TPO overstimulation as the underlying causative mechanism, despite a normal circulating TPO level. Thus, TPO signaling in megakaryocytes is dispensable for platelet production; its key role in control of platelet number is via generation and stimulation of the bipotential megakaryocyte precursors. Nevertheless, Mpl expression on megakaryocytes and platelets is essential to prevent megakaryocytosis and myeloproliferation by restricting the amount of TPO available to stimulate the production of megakaryocytes from the progenitor cell pool.

Caspase-9 mediates the apoptotic death of megakaryocytes and platelets, but is dispensable for their generation and function

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.