The fate of mitochondria during platelet activation.

Blood platelets undergo several successive motor-driven reorganizations of the cytoskeleton when they are recruited to an injured part of a vessel. These reorganizations take place during the platelet activation phase, the spreading process on the injured vessel or between fibrin fibers of the forming clot, and during clot retraction. All these steps require a lot of energy, especially the retraction of the clot when platelets develop strong forces similar to those of muscle cells. Platelets can produce energy through glycolysis and mitochondrial respiration. However, although resting platelets have only 5 to 8 individual mitochondria, they produce adenosine triphosphate predominantly via oxidative phosphorylation. Activated, spread platelets show an increase in size compared with resting platelets, and the question arises as to where the few mitochondria are located in these larger platelets. Using expansion microscopy, we show that the number of mitochondria per platelet is increased in spread platelets. Live imaging and focused ion beam-scanning electron microscopy suggest that a mitochondrial fission event takes place during platelet activation. Fission is Drp1 dependent because Drp1-deficient platelets have fused mitochondria. In nucleated cells, mitochondrial fission is associated with a shift to a glycolytic phenotype, and using clot retraction assays, we show that platelets have a more glycolytic energy production during clot retraction and that Drp1-deficient platelets show a defect in clot retraction.

Germline ETV6 mutation promotes inflammation and disrupts lymphoid development of early hematopoietic progenitors.

Germline mutations in ETV6 are associated with a syndrome of thrombocytopenia and leukemia predisposition, and ETV6 is among the most commonly mutated genes in leukemias, especially childhood B-cell acute lymphoblastic leukemia. However, the mechanisms underlying disease caused by ETV6 dysfunction are poorly understood. To address these gaps in knowledge, using CRISPR/Cas9, we developed a mouse model of the most common recurrent, disease-causing germline mutation in ETV6. We found defects in hematopoiesis related primarily to abnormalities of the multipotent progenitor population 4 (MPP4) subset of hematopoietic progenitor cells and evidence of sterile inflammation. Expression of ETV6 in Ba/F3 cells altered the expression of several cytokines, some of which were also detected at higher levels in the bone marrow of the mice with Etv6 mutation. Among these, interleukin-18 and interleukin-13 abrogated B-cell development of sorted MPP4 cells, but not common lymphoid progenitors, suggesting that inflammation contributes to abnormal hematopoiesis by impairing lymphoid development. These data, along with those from humans, support a model in which ETV6 dysfunction promotes inflammation, which adversely affects thrombopoiesis and promotes leukemogenesis.

Platelet microRNAs inhibit primary tumor growth via broad modulation of tumor cell mRNA expression in ectopic pancreatic cancer in mice.

We investigated the contributions of platelet microRNAs (miRNAs) to the rate of growth and regulation of gene expression in primary ectopic tumors using mouse models. We previously identified an inhibitory role for platelets in solid tumor growth, mediated by tumor infiltration of platelet microvesicles (microparticles) which are enriched in platelet-derived miRNAs. To investigate the specific roles of platelet miRNAs in tumor growth models, we implanted pancreatic ductal adenocarcinoma cells as a bolus into mice with megakaryocyte-/platelet-specific depletion of mature miRNAs. We observed an ~50% increase in the rate of growth of ectopic primary tumors in these mice compared to controls including at early stages, associated with reduced apoptosis in the tumors, in particular in tumor cells associated with platelet microvesicles-which were depleted of platelet-enriched miRNAs-demonstrating a specific role for platelet miRNAs in modulation of primary tumor growth. Differential expression RNA sequencing of tumor cells isolated from advanced primary tumors revealed a broad cohort of mRNAs modulated in the tumor cells as a function of host platelet miRNAs. Altered genes comprised 548 up-regulated transcripts and 43 down-regulated transcripts, mostly mRNAs altogether spanning a variety of growth signaling pathways-notably pathways related to epithelial-mesenchymal transition-in tumor cells from platelet miRNA-deleted mice compared with those from control mice. Tumors in platelet miRNA-depleted mice showed more sarcomatoid growth and more advanced tumor grade, indicating roles for host platelet miRNAs in tumor plasticity. We further validated increased protein expression of selected genes associated with increased cognate mRNAs in the tumors due to platelet miRNA depletion in the host animals, providing proof of principle of widespread effects of platelet miRNAs on tumor cell functional gene expression in primary tumors in vivo. Together, these data demonstrate that platelet-derived miRNAs modulate solid tumor growth in vivo by broad-spectrum restructuring of the tumor cell transcriptome.

Megakaryocyte-specific knockout of the Mir-99b/let7e/125a cluster lowers platelet count without altering platelet function.

The purpose of this research was to assess the effects of a microRNA (miRNA) cluster on platelet production. Human chromosome 19q13.41 harbors an evolutionarily conserved cluster of three miRNA genes (MIR99B, MIRLET7E, MIR125A) within 727 base-pairs. We now report that levels of miR-99b-5p, miR-let7e-5p and miR-125a-5p are strongly correlated in human platelets, and all are positively associated with platelet count, but not white blood count or hemoglobin level. Although the cluster regulates hematopoietic stem cell proliferation, the function of this genomic locus in megakaryocyte (MK) differentiation and platelet production is unknown. Furthermore, studies of individual miRNAs do not represent broader effects in the context of a cluster. To address this possibility, MK/platelet lineage-specific Mir-99b/let7e/125a knockout mice were generated. Compared to wild type littermates, cluster knockout mice had significantly lower platelet counts and reduced MK proplatelet formation, but no differences in MK numbers, ploidy, maturation or ultra-structural morphology, and no differences in platelet function. Compared to wild type littermates, knockout mice showed similar survival after pulmonary embolism. The major conclusions are that the effect of the Mir-99b/let7e/125a cluster is confined to a late stage of thrombopoiesis, and this effect on platelet number is uncoupled from platelet function.

CRISPR-edited megakaryocytes for rapid screening of platelet gene functions.

Human anucleate platelets cannot be directly modified using traditional genetic approaches. Instead, studies of platelet gene function depend on alternative models. Megakaryocytes (the nucleated precursor to platelets) are the nearest cell to platelets in origin, structure, and function. However, achieving consistent genetic modifications in primary megakaryocytes has been challenging, and the functional effects of induced gene deletions on human megakaryocytes for even well-characterized platelet genes (eg, ITGA2B) are unknown. Here we present a rapid and systematic approach to screen genes for platelet functions in CD34+ cell-derived megakaryocytes called CRIMSON (CRISPR-edited megakaryocytes for rapid screening of platelet gene functions). By using CRISPR/Cas9, we achieved efficient nonviral gene editing of a panel of platelet genes in megakaryocytes without compromising megakaryopoiesis. Gene editing induced loss of protein in up to 95% of cells for platelet function genes GP6, RASGRP2, and ITGA2B; for the immune receptor component B2M; and for COMMD7, which was previously associated with cardiovascular disease and platelet function. Gene deletions affected several select responses to platelet agonists in megakaryocytes in a manner largely consistent with those expected for platelets. Deletion of B2M did not significantly affect platelet-like responses, whereas deletion of ITGA2B abolished agonist-induced integrin activation and spreading on fibrinogen without affecting the translocation of P-selectin. Deletion of GP6 abrogated responses to collagen receptor agonists but not thrombin. Deletion of RASGRP2 impaired functional responses to adenosine 5'-diphosphate (ADP), thrombin, and collagen receptor agonists. Deletion of COMMD7 significantly impaired multiple responses to platelet agonists. Together, our data recommend CRIMSON for rapid evaluation of platelet gene phenotype associations.

Anti-apoptotic BCL2L2 increases megakaryocyte proplatelet formation in cultures of human cord blood.

Apoptosis is a recognized limitation to generating large numbers of megakaryocytes in culture. The genes responsible have been rigorously studied in vivo in mice, but are poorly characterized in human culture systems. As CD34-positive (+) cells isolated from human umbilical vein cord blood were differentiated into megakaryocytes in culture, two distinct cell populations were identified by flow cytometric forward and side scatter: larger size, lower granularity (LLG), and smaller size, higher granularity (SHG). The LLG cells were CD41aHigh CD42aHigh phosphatidylserineLow, had an electron microscopic morphology similar to mature bone marrow megakaryocytes, developed proplatelets, and displayed a signaling response to platelet agonists. The SHG cells were CD41aLowCD42aLowphosphatidylserineHigh, had a distinctly apoptotic morphology, were unable to develop proplatelets, and showed no signaling response. Screens of differentiating megakaryocytes for expression of 24 apoptosis genes identified BCL2L2 as a novel candidate megakaryocyte apoptosis regulator. Lentiviral BCL2L2 overexpression decreased megakaryocyte apoptosis, increased CD41a+ LLG cells, and increased proplatelet formation by 58%. An association study in 154 healthy donors identified a significant positive correlation between platelet number and platelet BCL2L2 mRNA levels. This finding was consistent with the observed increase in platelet-like particles derived from cultured megakaryocytes over-expressing BCL2L2 BCL2L2 also induced small, but significant increases in thrombin-induced platelet-like particle αIIbß3 activation and P-selectin expression. Thus, BCL2L2 restrains apoptosis in cultured megakaryocytes, promotes proplatelet formation, and is associated with platelet number. BCL2L2 is a novel target for improving megakaryocyte and platelet yields in in vitro culture systems.

Human megakaryocytes possess intrinsic antiviral immunity through regulated induction of IFITM3.

Evolving evidence indicates that platelets and megakaryocytes (MKs) have unexpected activities in inflammation and infection; whether viral infections upregulate biologically active, antiviral immune genes in platelets and MKs is unknown, however. We examined antiviral immune genes in these cells in dengue and influenza infections, viruses that are global public health threats. Using complementary biochemical, pharmacological, and genetic approaches, we examined the regulation and function of interferon-induced transmembrane protein 3 (IFITM3), an antiviral immune effector gene not previously studied in human platelets and MKs. IFITM3 was markedly upregulated in platelets isolated from patients during clinical influenza and dengue virus (DENV) infections. Lower IFITM3 expression in platelets correlated with increased illness severity and mortality in patients. Administering a live, attenuated DENV vaccine to healthy subjects significantly increased platelet IFITM3 expression. Infecting human MKs with DENV selectively increased type I interferons and IFITM3. Overexpression of IFITM3 in MKs was sufficient to prevent DENV infection. In naturally occurring, genetic loss-of-function studies, MKs from healthy subjects harboring a homozygous mutation in IFITM3 (rs12252-C, a common single-nucleotide polymorphism in areas of the world where DENV is endemic) were significantly more susceptible to DENV infection. DENV-induced MK secretion of interferons prevented infection of bystander MKs and hematopoietic stem cells. Thus, viral infections upregulate IFITM3 in human platelets and MKs, and IFITM3 expression is associated with adverse clinical outcomes. These observations establish, for the first time, that human MKs possess antiviral functions, preventing DENV infection of MKs and hematopoietic stem cells after local immune signaling.

Platelet abnormalities in Huntington's disease.

Huntington's disease (HD) is a hereditary disorder that typically manifests in adulthood with a combination of motor, cognitive and psychiatric problems. The pathology is caused by a mutation in the huntingtin gene which results in the production of an abnormal protein, mutant huntingtin (mHtt). This protein is ubiquitously expressed and known to confer toxicity to multiple cell types. We have recently reported that HD brains are also characterised by vascular abnormalities, which include changes in blood vessel density/diameter as well as increased blood-brain barrier (BBB) leakage. OBJECTIVES: Seeking to elucidate the origin of these vascular and BBB abnormalities, we studied platelets that are known to play a role in maintaining the integrity of the vasculature and thrombotic pathways linked to this, given they surprisingly contain the highest concentration of mHtt of all blood cells. METHODS: We assessed the functional status of platelets by performing ELISA, western blot and RNA sequencing in a cohort of 71 patients and 68 age- and sex-matched healthy control subjects. We further performed haemostasis and platelet depletion tests in the R6/2 HD mouse model. RESULTS: Our findings indicate that the platelets in HD are dysfunctional with respect to the release of angiogenic factors and functions including thrombosis, angiogenesis and vascular haemostasis. CONCLUSION: Taken together, our results provide a better understanding for the impact of mHtt on platelet function.

Platelet microparticles infiltrating solid tumors transfer miRNAs that suppress tumor growth.

Platelet-derived microparticles (PMPs) are associated with enhancement of metastasis and poor cancer outcomes. Circulating PMPs transfer platelet microRNAs (miRNAs) to vascular cells. Solid tumor vasculature is highly permeable, allowing the possibility of PMP-tumor cell interaction. Here, we show that PMPs infiltrate solid tumors in humans and mice and transfer platelet-derived RNA, including miRNAs, to tumor cells in vivo and in vitro, resulting in tumor cell apoptosis. MiR-24 was a major species in this transfer. PMP transfusion inhibited growth of both lung and colon carcinoma ectopic tumors, whereas blockade of miR-24 in tumor cells accelerated tumor growth in vivo, and prevented tumor growth inhibition by PMPs. Conversely, Par4-deleted mice, which had reduced circulating microparticles (MPs), supported accelerated tumor growth which was halted by PMP transfusion. PMP targeting was associated with tumor cell apoptosis in vivo. We identified direct RNA targets of platelet-derived miR-24 in tumor cells, which included mitochondrial mt-Nd2, and Snora75, a noncoding small nucleolar RNA. These RNAs were suppressed in PMP-treated tumor cells, resulting in mitochondrial dysfunction and growth inhibition, in an miR-24-dependent manner. Thus, platelet-derived miRNAs transfer in vivo to tumor cells in solid tumors via infiltrating MPs, regulate tumor cell gene expression, and modulate tumor progression. These findings provide novel insight into mechanisms of horizontal RNA transfer and add multiple layers to the regulatory roles of miRNAs and PMPs in tumor progression. Plasma MP-mediated transfer of regulatory RNAs and modulation of gene expression may be a common feature with important outcomes in contexts of enhanced vascular permeability.

Neonatal NET-inhibitory factor and related peptides inhibit neutrophil extracellular trap formation.

Neutrophil granulocytes, also called polymorphonuclear leukocytes (PMNs), extrude molecular lattices of decondensed chromatin studded with histones, granule enzymes, and antimicrobial peptides that are referred to as neutrophil extracellular traps (NETs). NETs capture and contain bacteria, viruses, and other pathogens. Nevertheless, experimental evidence indicates that NETs also cause inflammatory vascular and tissue damage, suggesting that identifying pathways that inhibit NET formation may have therapeutic implications. Here, we determined that neonatal NET-inhibitory factor (nNIF) is an inhibitor of NET formation in umbilical cord blood. In human neonatal and adult neutrophils, nNIF inhibits key terminal events in NET formation, including peptidyl arginine deiminase 4 (PAD4) activity, neutrophil nuclear histone citrullination, and nuclear decondensation. We also identified additional nNIF-related peptides (NRPs) that inhibit NET formation. nNIFs and NRPs blocked NET formation induced by pathogens, microbial toxins, and pharmacologic agonists in vitro and in mouse models of infection and systemic inflammation, and they improved mortality in murine models of systemic inflammation, which are associated with NET-induced collateral tissue injury. The identification of NRPs as neutrophil modulators that selectively interrupt NET generation at critical steps suggests their potential as therapeutic agents. Furthermore, our results indicate that nNIF may be an important regulator of NET formation in fetal and neonatal inflammation.

Germline mutations in ETV6 are associated with thrombocytopenia, red cell macrocytosis and predisposition to lymphoblastic leukemia.

Some familial platelet disorders are associated with predisposition to leukemia, myelodysplastic syndrome (MDS) or dyserythropoietic anemia. We identified a family with autosomal dominant thrombocytopenia, high erythrocyte mean corpuscular volume (MCV) and two occurrences of B cell-precursor acute lymphoblastic leukemia (ALL). Whole-exome sequencing identified a heterozygous single-nucleotide change in ETV6 (ets variant 6), c.641C>T, encoding a p.Pro214Leu substitution in the central domain, segregating with thrombocytopenia and elevated MCV. A screen of 23 families with similar phenotypes identified 2 with ETV6 mutations. One family also had a mutation encoding p.Pro214Leu and one individual with ALL. The other family had a c.1252A>G transition producing a p.Arg418Gly substitution in the DNA-binding domain, with alternative splicing and exon skipping. Functional characterization of these mutations showed aberrant cellular localization of mutant and endogenous ETV6, decreased transcriptional repression and altered megakaryocyte maturation. Our findings underscore a key role for ETV6 in platelet formation and leukemia predisposition.

Proteasome function is required for platelet production.

The proteasome inhibiter bortezomib has been successfully used to treat patients with relapsed multiple myeloma; however, many of these patients become thrombocytopenic, and it is not clear how the proteasome influences platelet production. Here we determined that pharmacologic inhibition of proteasome activity blocks proplatelet formation in human and mouse megakaryocytes. We also found that megakaryocytes isolated from mice deficient for PSMC1, an essential subunit of the 26S proteasome, fail to produce proplatelets. Consistent with decreased proplatelet formation, mice lacking PSMC1 in platelets (Psmc1(fl/fl) Pf4-Cre mice) exhibited severe thrombocytopenia and died shortly after birth. The failure to produce proplatelets in proteasome-inhibited megakaryocytes was due to upregulation and hyperactivation of the small GTPase, RhoA, rather than NF-κB, as has been previously suggested. Inhibition of RhoA or its downstream target, Rho-associated protein kinase (ROCK), restored megakaryocyte proplatelet formation in the setting of proteasome inhibition in vitro. Similarly, fasudil, a ROCK inhibitor used clinically to treat cerebral vasospasm, restored platelet counts in adult mice that were made thrombocytopenic by tamoxifen-induced suppression of proteasome activity in megakaryocytes and platelets (Psmc1(fl/fl) Pdgf-Cre-ER mice). These results indicate that proteasome function is critical for thrombopoiesis, and suggest inhibition of RhoA signaling as a potential strategy to treat thrombocytopenia in bortezomib-treated multiple myeloma patients.

Hematopoietic and nonhematopoietic cell tissue factor activates the coagulation cascade in endotoxemic mice.

Tissue factor (TF) is the primary activator of the coagulation cascade. During endotoxemia, TF expression leads to disseminated intravascular coagulation. However, the relative contribution of TF expression by different cell types to the activation of coagulation has not been defined. In this study, we investigated the effect of either a selective inhibition of TF expression or cell type-specific deletion of the TF gene (F3) on activation of coagulation in a mouse model of endotoxemia. We found that inhibition of TF on either hematopoietic or nonhematopoietic cells reduced plasma thrombin-antithrombin (TAT) levels 8 hours after administration of bacterial lipopolysaccharide (LPS). In addition, plasma TAT levels were significantly reduced in endotoxemic mice lacking the TF gene in either myeloid cells (TF(flox/flox),LysM(Cre) mice) or in both endothelial cells (ECs) and hematopoietic cells (TF(flox/flox),Tie-2(Cre) mice). However, deletion of the TF gene in ECs alone had no effect on LPS-induced plasma TAT levels. Similar results were observed in mice lacking TF in vascular smooth muscle cells. Finally, we found that mouse platelets do not express TF pre-mRNA or mRNA. Our data demonstrate that in a mouse model of endotoxemia activation of the coagulation cascade is initiated by TF expressed by myeloid cells and an unidentified nonhematopoietic cell type(s).