Thrombocytopenia Independently Leads to Changes in Monocyte Immune Function.

BACKGROUND: While platelets have well-studied hemostatic functions, platelets are immune cells that circulate at the interface between the vascular wall and white blood cells. The physiological implications of these constant transient interactions are poorly understood. Activated platelets induce and amplify immune responses, but platelets may also maintain immune homeostasis in healthy conditions, including maintaining vascular integrity and T helper cell differentiation, meaning that platelets are central to both immune responses and immune quiescence. Clinical data have shown an association between low platelet counts (thrombocytopenia) and immune dysfunction in patients with sepsis and extracorporeal membrane oxygenation, further implicating platelets as more holistic immune regulators, but studies of platelet immune functions in nondisease contexts have had limited study. METHODS: We used in vivo models of thrombocytopenia and in vitro models of platelet and monocyte interactions, as well as RNA-seq and ATAC-seq (assay for transposase-accessible chromatin with sequencing), to mechanistically determine how resting platelet and monocyte interactions immune program monocytes. RESULTS: Circulating platelets and monocytes interact in a CD47-dependent manner to regulate monocyte metabolism, histone methylation, and gene expression. Resting platelet-monocyte interactions limit TLR (toll-like receptor) signaling responses in healthy conditions in an innate immune training-like manner. In both human patients with sepsis and mouse sepsis models, thrombocytopenia exacerbated monocyte immune dysfunction, including increased cytokine production. CONCLUSIONS: Thrombocytopenia immune programs monocytes in a manner that may lead to immune dysfunction in the context of sepsis. This is the first demonstration that sterile, endogenous cell interactions between resting platelets and monocytes regulate monocyte metabolism and pathogen responses, demonstrating platelets to be immune rheostats in both health and disease.

Novel chemical-structure TPOR agonist, TMEA, promotes megakaryocytes differentiation and thrombopoiesis via mTOR and ERK signalings.

BACKGROUND: Non-peptide thrombopoietin receptor (TPOR) agonists are promising therapies for the mitigation and treatment of thrombocytopenia. However, only few agents are available as safe and effective for stimulating platelet production for thrombocytopenic patients in the clinic. PURPOSE: This study aimed to develop a novel small molecule TPOR agonist and investigate its underlying regulation of function in megakaryocytes (MKs) differentiation and thrombopoiesis. METHODS: A potential active compound that promotes MKs differentiation and thrombopoiesis was obtained by machine learning (ML). Meanwhile, the effect was verified in zebrafish model, HEL and Meg-01 cells. Next, the key regulatory target was identified by Drug Affinity Responsive Target Stabilization Assay (DARTS), Cellular Thermal Shift Assay (CETSA), and molecular simulation experiments. After that, RNA-sequencing (RNA-seq) was used to further confirm the associated pathways and evaluate the gene expression induced during MK differentiation. In vivo, irradiation (IR) mice, C57BL/6N-TPORem1cyagen (Tpor-/-) mice were constructed by CRISPR/Cas9 technology to examine the therapeutic effect of TMEA on thrombocytopenia. RESULTS: A natural chemical-structure small molecule TMEA was predicted to be a potential active compound based on ML. Obvious phenotypes of MKs differentiation were observed by TMEA induction in zebrafish model and TMEA could increase co-expression of CD41/CD42b, DNA content, and promote polyploidization and maturation of MKs in HEL and Meg-01 cells. Mechanically, TMEA could bind with TPOR protein and further regulate the PI3K/AKT/mTOR/P70S6K and MEK/ERK signal pathways. In vivo, TMEA evidently promoted platelet regeneration in mice with radiation-induced thrombocytopenia but had no effect on Tpor-/- and C57BL/6 (WT) mice. CONCLUSION: TMEA could serve as a novel TPOR agonist to promote MKs differentiation and thrombopoiesis via mTOR and ERK signaling and could potentially be created as a promising new drug to treat thrombocytopenia.

Platelet augmentation activity of mature leaf juice of Sri Lankan wild type cultivar of Carica papaya L: Insights into potential cellular mechanisms.

ETHNOPHARMACOLOGICAL RELEVANCE: Carica papaya L., a common fruit crop of the family Caricaceae and its leaf juice/extract is a traditionally commended preparation against dengue and other thrombocytopenic diseases by many Asian countries. AIM OF THE STUDY: The present study posits the potential cellular mechanisms of platelet augmentation activity of mature leaf juice of Sri Lankan wild-type Carica papaya. MATERIALS AND METHODS: C. papaya leaf juice prepared from different cultivar types, maturity of the leaf, agro-climatic region, and preparation methods were orally administered to hydroxyurea-induced thrombocytopenic rats at 0.72 ml/100 g BW dosage to investigate the most potent platelet increasing preparation. The papaya juice doses; low dose (LD-0.18 ml/100 g BW), human equivalent dose (HED-0.36 ml/100 g BW), and high dose (HD-0.72 ml/100 g BW), were administered to thrombocytopenic rats (N = 6/group) daily for three consecutive days and post-treatment plasma levels of interleukin 6 (IL-6), thrombopoietin (TPO), and platelet-activating factor (PAF) were quantified using specific rat ELISA kits. The mature leaf juice of C. papaya induced IL-6 secretion from bone marrow cell (BMC) cultures was quantified using ELISA. The ability of papaya juice to protect the platelet membrane, from the damage caused by the lytic agent was analyzed in vitro using the lactate dehydrogenase (LDH) assay. The effect of the mature leaf juice of C. papaya on secondary hemostasis was investigated using blood coagulation and clot hydrolyzing activity. RESULTS: The comparative analysis revealed that the platelet increasing activity of C. papaya leaf did not significantly differ among different types of cultivar, maturity of the leaf, agro-climatic regions and preparation methods (p > 0.05). Both TPO and PAF levels in thrombocytopenic rats diminished when treated with all three doses of the mature leaf juice of C. papaya (p < 0.05), yet IL-6 plasma level was unaltered (p > 0.05). Nevertheless, ex vivo treatment of the mature leaf juice of C. papaya had significantly enhanced IL-6 levels of rat BMC cultures (p < 0.05). Pre-treatment of platelets with the mature leaf juice of C. papaya at different concentrations significantly inhibited LDH leakage from platelets and may have reduced the membrane damage caused by the lytic agent (p < 0.05). Treatment of mature leaf juice of C. papaya also significantly reduced blood clotting time through the extrinsic pathway of the blood coagulation cascade (p < 0.05). Further, prolonged incubation of the plasma clot with different concentrations of the papaya leaf juice revealed dose-dependent hydrolysis of the blood clot, indicating fibrinolysis activity. CONCLUSIONS: The current study exceeded the traditional medicinal claims, and scientifically affirmed the platelet augmentation activity of mature leaf juice of C. papaya. The mechanistic rationale tested herein explicated that the platelet augmentation activity of the papaya leaf juice can be partially attributed to the stimulation of bone marrow megakaryocytes via modulating thrombopoietic cytokines TPO and IL-6, and by inhibiting the secretion of PAF, while reducing the peripheral platelet destruction by stabilizing the platelet membrane. Further, mature leaf juice of C. papaya imparted both pro-coagulation and fibrinolysis activity of secondary hemostasis endorsing its potential against thrombocytopenia.

Efficacy & safety of Carica papaya leaf extract (CPLE) in severe thrombocytopenia (≤30,000/µl) in adult dengue - Results of a pilot study.

Severe thrombocytopenia in dengue often prompts platelet transfusion primarily to reduce bleeding risk. In India, about 11-43% of dengue patients report receiving platelet transfusions which is considered scarce and expensive especially in resource limited settings. Herein, we evaluated the efficacy and safety of Carica papaya leaf extract (CPLE) in the management of severe thrombocytopenia (≤30,000/µL) in dengue infection. 51 laboratory confirmed adult dengue patients with platelet counts ≤30,000/µL were randomly assigned to either treatment (n = 26) or placebo (n = 24) group. By day 3, CPLE treated patients reported significantly (p = 0.007) increased platelet counts (482%± 284) compared to placebo (331%±370) group. In the treatment group, fewer patients received platelet transfusions (1/26 v/s 2/24) and their median time for platelets to recover to ≥ 50,000/µL was 2 days (IQR 2-3) compared to 3 days (IQR 2-4) in placebo. Overall, CPLE was safe and well tolerated with no significant decrease in mean hospitalization days. Plasma cytokine profiling revealed that by day 3, mean percent increase in TNFα and IFNγ levels in treatment group was less compared to that observed in placebos; (TNFα: 58.6% v/s 127.5%; p = 0.25 and IFNγ: 1.93% v/s 62.6% for; p = 0.12). While a mean percent increase in IL-6 levels occurred in placebos (15.92%±29.93%) by day 3, a decrease was noted in CPLE group (12.95%±21.75%; p = 0.0232). Inversely, CPLE treated patients reported a mean percent increase compared to placebo by day 3 (143% ±115.7% v/s 12.03%± 48.4%; p = 0.006). Further, by day 3, a faster clearance kinetics of viral NS1 antigenemia occurred-mean NS1 titers in treatment group decreased to 97.3% compared to 88% in placebos (p = 0.023). This study demonstrates safety and efficacy of CPLE in increasing platelet counts in severe thrombocytopenia in dengue infections. A possible immunomodulatory and antiviral activity may be attributed to CPLE treatment. These findings merit validation in larger prospective studies. Trial registration Name of the registry: Clinical Trials Registry-India (CTRI) Registration No.: CTRI-REF/2017/02/013314.

Effects of Shen Cao Granules on Chemotherapy-Induced Thrombocytopenia in Gastrointestinal Cancer Patients: A Randomized Controlled Trial.

BACKGROUND: To observe clinical effects of Shen Cao granules on thrombocytopenia in patients with gastrointestinal cancer undergoing chemotherapy. PATIENTS AND METHODS: Patients under a FOLFIRI chemotherapy regimen (n = 92) were randomly divided into study and control groups (n = 46 for each group) and were given 10 g of Shen Cao granules and a placebo, respectively, once daily on chemotherapy treatment days. Platelet counts were measured every other day and any adverse reaction recorded during the study and at follow-up. RESULTS: The incidence of thrombocytopenia (grades II-IV) in the study group was significantly decreased, and the length of hospitalization significantly reduced compared with the control group (11.21 ± 2.46 vs 15.34 ± 3.68 days, P < .05). The minimum numbers of post-chemotherapy platelets and the values of platelet counts 21 days after chemotherapy were significantly increased ([100.65 ± 63.16] × 109/L vs [60.21 ±37.22] × 109/L, P < .05; [267.81 ± 81.32] × 109/L vs [146.42 ± 70.54] × 109/L, P < .001), and the duration of thrombocytopenia and treatment with recombinant human interleukin-11 was significantly decreased in the Shen Cao treatment compared with the control group. No serious adverse events were observed. CONCLUSIONS: Shen Cao granules were effective in decreasing chemotherapy-induced thrombocytopenia, shortened the duration of thrombocytopenia, and reduced the length of hospital stay and costs.

Evaluation of anti-dengue activity of Carica papaya aqueous leaf extract and its role in platelet augmentation.

Dengue disease is characterized by a marked decrease in platelet count, which is life threatening. In the present study, we investigated the antiviral activity of an aqueous extract of Carica papaya leaves (PLE) against dengue virus (DENV) and its effect on platelet augmentation. The anti-dengue activity of PLE in DENV-infected THP-1 cells was examined by immunoblotting and flow cytometry. The effect of PLE on erythrocyte damage was investigated using hemolytic and anti-hemolytic assays. Virus-infected THP-1 cells were assayed for IFN-α secretion. The effect of PLE on platelet augmentation in rats with cyclophosphamide-induced thrombocytopenia was also investigated. The platelet count of blood from the retro-orbital plexus of rats was determined on the 1st, 4th, 7th, 11th and 14th day of study. On the 14th day, the rats were sacrificed for histopathological examination of the liver, kidney and spleen. Plasma of thrombocytopenic rats was tested for thrombopoietin (TPO) and IL-6 secretion. The data suggest that PLE significantly decreases the expression of the envelope and NS1 proteins in DENV-infected THP-1 cells. A marked decrease in intracellular viral load upon PLE treatment confirmed its antiviral activity. This also resulted in a significant decrease in erythrocyte damage and hydrogen-peroxide-induced lipid peroxidation. A significant increase in the number of platelets was found in thrombocytopenic rats treated with PLE, along with an increase in IL-6 and TPO levels. These findings suggest that PLE can potentially be used as an antiviral agent, as it helps in platelet augmentation and exhibits antiviral activity against DENV.

Water-Pipe Smoke Exposure-Induced Circulatory Disturbances in Mice, and the Influence of Betaine Supplementation Thereon.

BACKGROUND/AIMS: It has been shown, both experimentally and clinically, that water-pipe smoke (WPS) exposure adversely affects the cardiovascular system (CVS) through the generation of oxidative stress and inflammation. Betaine, a naturally occurring compound in common foods, has antioxidant and anti-inflammatory actions. However, its potential to mitigate the adverse effect of WPS on the CVS has never been reported before. This is the subject of this study in mice. METHODS: Mice were exposed daily for 30 min to either normal air (control), or to WPS for two consecutive weeks. Betaine was administered daily by gavage at a dose of 10mg/kg, 1h before either air or WPS exposure. RESULTS: Betaine mitigated the in vivo prothrombotic effect of WPS in pial arterioles and venules. Moreover, it reversed the WPS-induced decrease in circulating platelets. Likewise, betaine alleviated platelet aggregation in vitro, and the shortening of activated partial thromboplastin time and prothrombin time induced by WPS. Betaine reduced the increase of plasminogen activator inhibitor-1 and fibrinogen concentrations in plasma induced by WPS. Betaine also diminished the WPS-induced increase of plasma concentrations of interleukin 6 and tumor necrosis factor α, and attenuated the increase of lipid peroxidation and superoxide dismutase. Immunohistochemical analysis of the heart revealed an increase in the expression of inducible nitric oxide synthase and cytochrome C by cardiomyocytes of the WPS-exposed mice. These effects were averted by betaine. CONCLUSION: Our findings suggest that betaine treatment significantly mitigated WPS-induced hypercoagulability, and inflammation, as well as systemic and cardiac oxidative stress.

The contribution of mouse models to the understanding of constitutional thrombocytopenia.

Constitutional thrombocytopenias result from platelet production abnormalities of hereditary origin. Long misdiagnosed and poorly studied, knowledge about these rare diseases has increased considerably over the last twenty years due to improved technology for the identification of mutations, as well as an improvement in obtaining megakaryocyte culture from patient hematopoietic stem cells. Simultaneously, the manipulation of mouse genes (transgenesis, total or conditional inactivation, introduction of point mutations, random chemical mutagenesis) have helped to generate disease models that have contributed greatly to deciphering patient clinical and laboratory features. Most of the thrombocytopenias for which the mutated genes have been identified now have a murine model counterpart. This review focuses on the contribution that these mouse models have brought to the understanding of hereditary thrombocytopenias with respect to what was known in humans. Animal models have either i) provided novel information on the molecular and cellular pathways that were missing from the patient studies; ii) improved our understanding of the mechanisms of thrombocytopoiesis; iii) been instrumental in structure-function studies of the mutated gene products; and iv) been an invaluable tool as preclinical models to test new drugs or develop gene therapies. At present, the genetic determinants of thrombocytopenia remain unknown in almost half of all cases. Currently available high-speed sequencing techniques will identify new candidate genes, which will in turn allow the generation of murine models to confirm and further study the abnormal phenotype. In a complementary manner, programs of random mutagenesis in mice should also identify new candidate genes involved in thrombocytopenia.

Defects in TRPM7 channel function deregulate thrombopoiesis through altered cellular Mg(2+) homeostasis and cytoskeletal architecture.

Mg(2+) plays a vital role in platelet function, but despite implications for life-threatening conditions such as stroke or myocardial infarction, the mechanisms controlling [Mg(2+)]i in megakaryocytes (MKs) and platelets are largely unknown. Transient receptor potential melastatin-like 7 channel (TRPM7) is a ubiquitous, constitutively active cation channel with a cytosolic α-kinase domain that is critical for embryonic development and cell survival. Here we report that impaired channel function of TRPM7 in MKs causes macrothrombocytopenia in mice (Trpm7(fl/fl-Pf4Cre)) and likely in several members of a human pedigree that, in addition, suffer from atrial fibrillation. The defect in platelet biogenesis is mainly caused by cytoskeletal alterations resulting in impaired proplatelet formation by Trpm7(fl/fl-Pf4Cre) MKs, which is rescued by Mg(2+) supplementation or chemical inhibition of non-muscle myosin IIA heavy chain activity. Collectively, our findings reveal that TRPM7 dysfunction may cause macrothrombocytopenia in humans and mice.

Preclinical models of nicotinamide phosphoribosyltransferase inhibitor-mediated hematotoxicity and mitigation by co-treatment with nicotinic acid.

Nicotinamide adenine dinucleotide (NAD) is an essential co-factor in glycolysis and is a key molecule involved in maintaining cellular energy metabolism. Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the rate-limiting step of an important salvage pathway in which nicotinamide is recycled into NAD. NAMPT is up-regulated in many types of cancer and NAMPT inhibitors (NAMPTi) have potential therapeutic benefit in cancer by impairing tumor metabolism. Clinical trials with NAMPTi APO-866 and GMX-1778, however, failed to reach projected efficacious exposures due to dose-limiting thrombocytopenia. We evaluated preclinical models for thrombocytopenia that could be used in candidate drug selection and risk mitigation strategies for NAMPTi-related toxicity. Rats treated with a suite of structurally diverse and potent NAMPTi at maximum tolerated doses had decreased reticulocyte and lymphocyte counts, but no thrombocytopenia. We therefore evaluated and qualified a human colony forming unit-megakaryocyte (CFU-MK) as in vitro predictive model of NAMPTi-induced MK toxicity and thrombocytopenia. We further demonstrate that the MK toxicity is on-target based on the evidence that nicotinic acid (NA), which is converted to NAD via a NAMPT-independent pathway, can mitigate NAMPTi toxicity to human CFU-MK in vitro and was also protective for the hematotoxicity in rats in vivo. Finally, assessment of CFU-MK and human platelet bioenergetics and function show that NAMPTi was toxic to MK and not platelets, which is consistent with the clinically observed time-course of thrombocytopenia.

Royal jelly attenuates azathioprine induced toxicity in rats.

In the present study, we investigated the potential protective effects of royal jelly against azathioprine-induced toxicity in rat. Intraperitoneal administration of azathioprine (50 mg/kgB.W.) induced a significant decrease in RBCs count, Hb concentration, PCV%, WBCs count, differential count and platelet count, hepatic antioxidant enzymes (reduced glutathione and glutathione s-transferase) and increase of serum transaminases (alanine aminotransferase and aspartate aminotransferase enzymes) activities, alkaline phosphatase and malondialdehyde formation. Azathioprine induced hepatotoxicity was reflected by marked pathological changes in the liver. Oral administration of royal jelly (200 mg/kgB.W.) was efficient in counteracting azathioprine toxicity whereas it altered the anemic condition, leucopenia and thrombocytopenia induced by azathioprine. Furthermore, royal jelly exerted significant protection against liver damage induced by azathioprine through reduction of the elevated activities of serum hepatic enzymes. Moreover, royal jelly blocked azathioprine-induced lipid peroxidation through decreasing the malondialdehyde formation. In conclusion, royal jelly possesses a capability to attenuate azathioprine-induced toxicity.

Demonstration of early functional compromise of bone marrow derived hematopoietic progenitor cells during bovine neonatal pancytopenia through in vitro culture of bone marrow biopsies.

BACKGROUND: Bovine neonatal pancytopenia (BNP) is a syndrome characterised by thrombocytopenia associated with marked bone marrow destruction in calves, widely reported since 2007 in several European countries and since 2011 in New Zealand. The disease is epidemiologically associated with the use of an inactivated bovine virus diarrhoea (BVD) vaccine and is currently considered to be caused by absorption of colostral antibody produced by some vaccinated cows ("BNP dams"). Alloantibodies capable of binding to the leukocyte surface have been detected in BNP dams and antibodies recognising bovine MHC class I and ß-2-microglobulin have been detected in vaccinated cattle. In this study, calves were challenged with pooled colostrum collected from BNP dams or from non-BNP dams and their bone marrow hematopoietic progenitor cells (HPC) cultured in vitro from sternal biopsies taken at 24 hours and 6 days post-challenge. RESULTS: Clonogenic assay demonstrated that CFU-GEMM (colony forming unit-granulocyte/erythroid/macrophage/megakaryocyte; pluripotential progenitor cell) colony development was compromised from HPCs harvested as early as 24 hour post-challenge. By 6 days post challenge, HPCs harvested from challenged calves failed to develop CFU-E (erythroid) colonies and the development of both CFU-GEMM and CFU-GM (granulocyte/macrophage) was markedly reduced. CONCLUSION: This study suggests that the bone marrow pathology and clinical signs associated with BNP are related to an insult which compromises the pluripotential progenitor cell within the first 24 hours of life but that this does not initially include all cell types.

Antithrombotic activity of garlic: its inhibition of the synthesis of thromboxane-B2 during infusion of arachidonic acid and collagen in rabbits.

Rabbits were given collagen and arachidonic acid intravenously. Blood pressure, platelet counts, plasma thromboxane-B2 (TXB2) and plasma 6-keto-prostaglandin F1 alpha, (6-keto-PGF1 alpha) were determined. Both thrombogenic agents, upon infusion of a lethal dose, caused thrombocytopenia, indicative of in vivo platelet aggregation and hypotension. These changes were associated with an increase in plasma levels of TXB2 and 6-keto-PGF1 alpha measured by radioimmunoassay (RIA). Pretreatment of rabbits with an aqueous extract of garlic (500 mgkg) provided protection from thrombocytopenia and hypotension. Thromboxane-B2 synthesis was significantly reduced in animals pretreated with garlic and then injected with a lethal dose of either collagen or arachidonic acid. The amount of TXB2 synthesized in these animals was not sufficient to induce thrombocytopenia or hypotension. All animals pretreated with garlic were well protected against the effects of collagen or arachidonate infusion, and no apparent symptoms were observed in these animals. These observations indicate that garlic may be beneficial in the prevention of thrombosis.

Detection of thrombopoietic activity in plasma by stimulation of suppressed thrombopoiesis.

Rabbits in which thrombocytosis had been produced by five daily transfusions of platelet concentrates had suppressed endogenous thrombopoiesis, as reflected by decreased incorporation of selenomethionine-(75)Se ((75)SeM) into the circulating platelet mass. Rabbits in which endogenous thrombopoiesis had been suppressed by transfusion-induced thrombocytosis were used to detect thrombopoietic activity in rabbit plasma. Thrombopoietic activity was demonstrated in the plasma of both normal and thrombocytopenic donor rabbits. A dose response relationship was observed between the incorporation of (75)SeM into platelets and the dose of plasma administered. Infusion of 20-150 ml of plasma from thrombocytopenic donors increased the incorporation of (75)SeM into platelets from 52 to 107% above control values. A dose response effect also was seen after infusion of normal plasma, but normal plasma produced less effect than comparable doses of plasma from thrombocytopenic donors. Rabbits with transfusion-induced thrombocytosis appear to be more sensitive assay animals for the detection of thrombopoietic activity than animals with normal platelet counts. Changes in the rate of appearance and levels of (75)SeM may primarily indicate changes in platelet protein or platelet size and are apparently more sensitive indicators of the state of thrombopoiesis than are alterations in the numbers of circulating platelets. The results strongly support the concept of a humoral agent, i.e. thrombopoietin, that acts on megakaryocytes to regulate platelet production.