Promotes Platelet Production Research Articles

Therapeutic Potential of Carica papaya Linn.: A Comprehensive Review of Its Pharmacological Properties and Health Benefits, with Emphasis on Dengue Fever Healing

For thousands of years, people have used various plant products, foods, spices, and herbs to treat and prevent illnesses. Sri Lankans have been using traditional and folk medicine to satisfy their basic health needs from the earliest days. Carica papaya Linn. (Caricaceae) is a commercially grown exotic plant in Sri Lanka. The entire plant, including the fruits both ripened and green, leaves, roots, peel, seed, and pulp, was used as medicine in traditional medicinal systems. Plant parts of Carica papaya Linn. were frequently used by ancient Sri Lankans to treat diabetes mellitus, hypertension, cancer, dengue fever, skin infections, renal disorders, and infections caused by bacteria, fungi, parasites, and viruses. Dengue fever is the most prevalent and significant arthropod-borne viral disease in humans, and it is extensively spread throughout Sri Lanka. Carica papaya Linn. extracts have been used to treat dengue patients to recover from its complications, which include thrombocytopenia, hemolysis, and leukopenia. Studies reveal that the papaya leaf extract can neutralize plasma with the dengue virus, reduce platelet aggregation, and increase the production of the enzyme ALOX12, which promotes platelet production. Moreover, the extract also upregulates the platelet-activating factor receptor (PTAFR) gene and inhibits serine proteases NS2B and NS3 involved in dengue virus replication. In this review, we have discussed extensively the ethnomedicinal and pharmacological properties of Carica papaya while highlighting its remarkable potency in healing dengue.

Avatrombopag for the salvage treatment of platelet transfusion refractoriness.

Platelet transfusion refractoriness (PTR) is a life-threatening and intractable condition in hematological patients. Thrombopoietin receptor agonists such as avatrombopag promote platelet production and modulate immune intolerance. However, its application in PTR has not been extensively studied. We aimed to compare the platelet response (PR) as well as bleeding events and mortality rate between the best available therapies (BATs) and avatrombopag (Ava) treatments in refractory PTR patients. A total of 71 refractory PTR patients were enrolled at Nanfang Hospital. Intravenous immunoglobulin, steroids, and human leucocyte antigen-matched platelet transfusions were administered to 30 patients in the BATs group. The Ava group included 41 patients. Data of refractory PTR patients were retrospectively collected. The primary endpoint was PR (defined as an increase of platelet count to ⩾50 × 109/L without platelet transfusion support for 7 consecutive days). Secondary endpoints included platelet-transfusion independence rate, cumulative platelet transfusion units, World Health Organization bleeding grades, adverse events, overall survival (OS), and bleeding event-free survival (EFS). There were 75.6% and 13.3% refractory PTR patients who reached PR within 3 months in Ava and BATs groups. The median platelet counts were significantly higher in Ava group from day 7. Platelet-transfusion independence rate in Ava was higher than BATs group. The median cumulative platelet transfusion unit in Ava was lower than that of BATs group. The OS and bleeding events-free EFS rate of Ava group improved within 3 months as compared to BATs group. Cox proportional hazards regression analysis revealed that Ava therapy was a protective factor for the OS and EFS. No primary disease progression or termination of avatrombopag was observed due to intolerability. Our study suggests that avatrombopag is an effective and safe treatment option for refractory PTR patients.

Early Efficacy of IST in Combination with Hetrombopag and Rhtpo As First-Line Treatment for Naïve Severe Aplastic Anemia

BACKGROUND: Immunosuppressive therapy (IST) (anti-thymocyte immunoglobulin (ATG) and cyclosporine (CSA)) is the treatment of choice for patients with acquired severe aplastic anemia (SAA) who are ineligible for hematopoietic stem cell transplantation (HSCT), which results in a hematological response (HR) in about 60-70% of SAA patients. TPO is a hepatic-secreted hematopoietic growth factor that specifically regulates platelet production, induces hematopoietic stem cell differentiation to megakaryocytes by binding to the TPO receptor (MPL), stimulates megakaryocyte proliferation and differentiation, and promotes platelet production and release. Several previous studies have shown that IST combined with rhTPO or hetrombopag can improve the early hematologic response of SAA patients, especially the proportion of good hematologic response (good HR) in the early stage. RhTPO and TPORA have different binding sites with thrombopoietin receptor (c-MPL) and do not have competitive binding, which may produce synergistic effect. A retrospective study showed that eltrombopag combined with rhTPO increased the IST HR and improved the quality of hematologic remission in SAA with mild adverse effects. Therefore the combination of TPORA and rhTPO in SAA patients undergoing IST treatment may result in faster and higher HR rates. Clinical studies have confirmed that hetrombopag, act similarly to eltrombopag in SAA, but there are no clinical studies of hetrombopag in combination with rhTPO in SAA. OBJECTIVE: To evaluate the early-efficacy and safety profile of IST in combination with hetrombopag, and rhTPO in patients with primary, severe aplastic anemia. METHODS: This study was designed for patients with primary aplastic anemia major aged 14-70 years after January 2023 from a single center. This group were planned to be get treat of IST combination with rhTPO 15000U/d for 8 weeks from the first day of ATG and hetrombopag 15mg/d for at least 6 months. The primary endpoint was to observe the good response rate, blood product transfusion and safety profile of the study regimen for the treatment of primary SAA for 3 months. Efficacy Criteria：Complete response (CR): HGB>100 g/L, PLT> 100×10 9/L and ANC>1.5×10 9/L; ② Good partial response (GPR): HGB>80g/L, PLT> 50×10 9/L and ANC>1.0×10 9/L; ③ Partial response (PR): withdrawal from blood product transfusion dependence (HGB>70g/L, PLT> 20×10 9/L), hematologic tests improved and no longer met the SAA criteria, but blood count did not meet the GPR criteria. ④No response to treatment (NR): Continuous transfusion dependency was classified as no response. and/or blood count still met SAA criteria. We defined CR+GPR+PR as HR and CR+GPR as good HR. RESULTS: The trial group proposes to enroll 39 patients and currently includes 8 patients who can be evaluated for 3-month efficacy of IST treatment. And the control group includes two groups of IST combined rhTPO group and IST combined HPAG group in our previous center, and the cohort study of the results of the three groups. In this study, the HR rate of 8 patients can be assessed at present as 75% (6/8), and the rate of good HR was 50% (4/8). The HR of 33 patients in the control IST+rhTPO group was 66.67% (22/33), and the rate of good HR was 36.4% (12/33); the HR rate of 32 patients in the control IST+HPAG group was 46.9% (15/32), and the rate of good HR was 21.9%. The HR rate and good HR rate of the trial group were higher than those of the IST+rhTPO group and the IST+HPAG group (75%v66.67%v46.9%; 50%v36.4%v21.9%). None of the 8 patients enrolled in the trial group had any transformation of myelofibrosis, myelodysplastic syndrome and acute myeloid leukemia during the follow-up period. CONCLUSION: HR was obtained at 3 months in 6 of the 8 patients with severe remodeling IST+rhTPO+ hetrombopag in this study, with a good HR rate of 50%, which is the highest good efficacy response we know of in a prospective study of Chinese SAA patients to date. This confirms that rhTPO combined with hetrombopag can synergize the recovery of bone marrow hematopoietic function in SAA patients treated with IST, and its clinical effect is superior to that of various TPO-Ras or rhTPO that have been widely used. It is expected that more cases will be included in the later stages and that better results will be obtained. 【Keywords】severe aplastic anemia; recombinant human thrombopoietin; hetrombopag ; immunosuppression treatment.

IST Combined with Full-Dose Rhtpo in the Treatment of Severe Aplastic Anemia -a Prospective Single-Center Clinical Study

BACKGROUND: Immunosuppressive therapy (IST) (anti-thymocyte immunoglobulin (ATG) and cyclosporine (CSA)) is the treatment of choice for patients with acquired severe aplastic anemia (SAA) who are ineligible for hematopoietic stem cell transplantation (HSCT), which results in a hematological response (HR) in about 60-70% of SAA patients, while 1/3 of patients fail to achieve a hematological response. TPO is a hepatic-secreted hematopoietic growth factor that specifically regulates platelet production, induces hematopoietic stem cell differentiation to megakaryocytes by binding to the TPO receptor (MPL), stimulates megakaryocyte proliferation and differentiation, and promotes platelet production and release. Previous retrospective studies have demonstrated that IST combined with rhTPO improves the early hematologic response in SAA patients, but efficacy reported by centers varied depending on the dose and duration of rhTPO. There are no prospective studies of rhTPO application in SAA to confirm its efficacy. So this study was designed to observe the early feeicacy and safety of the SAA patients who were treated with IST combination with full-dose rhTPO. METHODS: This study was a single-center, single-arm, prospective cohort clinical study, enrolling 33 naive SAA patients aged between 14-70 years who diagnosed after April 2021 in our study center. The patients treated with IST combined with full-dose rhTPO (300U/kg.d) for 8 weeks starting from the first day of ATG. The primary endpoint of the observational study protocol good response rate, blood product transfusion volume and safety profile at 3 months of treatment of initial SAA. The patients were also evaluated for total hematologic response rate and adverse effects of drug application, clonal shift rate, and recurrence rate at 3 and 6 months after treatment. Efficacy Criteria：Complete response (CR): HGB>100 g/L, PLT> 100×10 9/L and ANC>1.5×10 9/L; ② Good partial response (GPR): HGB>80g/L, PLT> 50×10 9/L and ANC>1.0×10 9/L; ③ Partial response (PR): withdrawal from blood product transfusion dependence (HGB>70g/L, PLT> 20×10 9/L), hematologic tests improved and no longer met the SAA criteria, but blood count did not meet the GPR criteria. ④No response to treatment (NR): Continuous transfusion dependency was classified as no response. and/or blood count still met SAA criteria. We defined CR+GPR+PR as HR and CR+GPR as good HR. RESULTS: The rhTPO group enrolled 33 patients, and the control group was 99 patients who applied the caliper value of 0.2 to match the previous unapplied TPO treatment of SAA. At 3 months after IST treatment efficacy, the HR rate of 33 patients in the rhTPO group was 66.67% (22 /33 ), and the rate of good HR was 36.4% (12/33). The HR rate in the control group was 32.32% (32 /99 ), and the rate of good HR was 12.12% (12/99). The HR rate and the good HR rate between the two group were significantly statistical differences (66.67% v 32.32%; 36.4% v 12.12%), (P value=0.001; 0.002). At 6 months after IST treatment, the HR rate and good HR rate in the rhTPO group had a significantly higher than those of the control group (66.67% v 32.32%; 36.4% v 12.12%), (P value=0.001; 0.002). No disease transformation such as myelofibrosis, myelodysplastic syndrome and acute myeloid leukemia occurred in all enrolled patients during the follow-up period. CONCLUSION: 22 of the 33 SAA patients in this study achieved HR at 3 months after IST treatment, including 36.4% cases achived good HR , which is the highest good efficacy response in a prospective study of SAA patients in China known to us to date. This confirms that full-dose rhTPO, as a macromolecular peptide, can also promote the recovery of bone marrow hematopoietic function, significantly improved the rapidity and strength of HR in SAA patients. And its clinical effect is no less than that of the various TPO-Ras that have been widely used so far, and may even be higher. 【Keywords】severe aplastic anemia; recombinant human thrombopoietin; immunosuppression treatment.

Treatment with Hetrombopag for Poor Platelet Engraftment after Allo-HSCT-a Single Center Retrospective, Observational Study

Background: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a common and important treatment for most hematopoietic diseases. Some patients may experience poor platelet engraftment after allo-HSCT, which may lead to transplant failure and is also an important influencing factor for post-HSCT complications such as graft versus host disease (GVHD) and non-relapse mortality (NRM). Thus, exploring methods to improve platelet engraftment is very important. Hetrombopag is a small-molecule thrombopoietin-receptor agonist (TPO-RA) produced in China, stimulates the proliferation and differentiation of megakaryocytes and promotes platelet production through binding to thrombopoietin receptor to stimulate multiple intracellular signaling pathways. It has been reported that hetrombopag was effective in promoting platelet engraftment and decreasing platelet transfusion after allo-HSCT. Here, we studied the efficacy and safety of hetrombopag for poor platelet engraftment after allo-HSCT, of whom did not respond to rhTPO treatment. Aims: To evaluate the efficacy of hetrombopag in patients with poor platelet engraftment after allogeneic hematopoietic stem cell transplantation. Methods: In this retrospective study, data from patients with poor platelet engraftment after allo-HSCT who had been treated firstly with rhTPO and then with hetrombopag at Henan Cancer Hospital from July 1, 2021 to July 1, 2023 were collected. Patients should meet the following criteria to enroll in the study: experienced poor platelet engraftment who had been treated with rhTPO from day +4 post-HSCT. Here we defined poor platelet engraftment as platelet count (PLT) < 20 × 10 9/L beyond day +28 post-HSCT. We analyzed 57 patients mentioned above and divided them into two groups in a ratio of 1:2, 19 of them were given hetrombopag until their PLT exceeded 50 × 10 9/L and maintained for more than 7 days without platelet transfusion (hetrombopag group), another 38 patients were treated without hetrombopag (control group). The initial dose of hetrombopag was 5, or 7.5 mg/d according to the patient's conditions and characteristics (e.g., age, PLT, and bleeding risk). After allo-HSCT, patients administered rhTPO at a dose of 300U/kg·d from day +4. We defined rhTPO as ineffective when PLT was still below 20×10 9/L beyond +21 days post-HSCT after rhTPO administration. At this time, rhTPO was replaced with hetrombopag, or IL-11, decitabine, platelet transfusion, etc. Results: The median ages of the two groups of patients were 38 years (hetrombopag group) and 41 years (control group), respectively. The median baseline platelet count in both groups were 20 × 10 9/L. The proportion of patients with PLT recovery to 50×10 9/L was significantly higher in the hetrombopag group than in the control group (47.3% vs 7.9%, χ2=26.03, P<0.0001). The median time to PLT recovery to 50×10 9/L was 10 (7-48) days in the hetrombopag group and 33 (21-94) days in the control group ( P=0.004). The mean units of platelet transfusion in the hetrombopag group was significantly lower than that in the control group (2.7 vs 6.3 U, P=0.001), and no thrombotic events occurred. There was no statistical difference in the incidence of aGVHD (42.0% vs 44.7%, P=0.850) and NRM (5.2% vs 7.9%, P=0.714) between the two groups, suggesting that hetrombopag had no significant effect on aGVHD and NRM. Conclusion: For allo-HSCT patients who experienced poor platelet engraftment after rhTPO treatment, hetrombopag can rapidly promote PLT recovery, reduce the units of platelet transfusion, and have no risk of the increase of aGVHD and NRM. These results still need further validation in a larger sample prospective confirmatory study in the future.

The Role of Gpibα in Control of Megakaryocyte Reactivity to Thrombopoietin

Thrombopoietin (TPO) was first described in 1958 as a humoral factor that regulates platelet production. TPO binds to c-Mpl and stimulates the expansion and differentiation of megakaryocyte precursors and the maturation of megakaryocytes. Notably, TPO has no significant effect on the formation stage of megakaryocyte boundary membrane system and platelet release. Studies investigating GPIbα deficiency have demonstrated its substantial effects on megakaryocyte proliferation, ploidy maturation, and resultant platelet count reduction. Furthermore, our previous study demonstrated that GPIbα deficiency in mice and patients with Bernard-Soulier syndrome (BSS) results in reduced circulating TPO levels. Surprisingly, this decrease in TPO levels is not due to increased TPO clearance by platelets lacking GPIbα but rather a decrease in TPO production, which is regulated by GPIbα through the interaction with liver cells. Based on these findings, we hypothesize that augmenting exogenous TPO levels may effectively promote platelet production in GPIbα deficiency. We first generated C57/BL6 background GPIbα deficiency mice and measured their platelet levels using an automatic blood routine counter, confirming previous findings of a lower platelet count in GPIbα deficiency mice. Subsequently, both GPIbα deficiency and wild-type mice were intraperitoneally injected with recombinant mouse TPO (rmTPO) simultaneously. Surprisingly, we observed that the GPIbα deficiency mice exhibited reduced reactivity to TPO, resulting in a significant decrease in platelet production compared to wild-type mice. To elucidate it mechanism, we conducted comprehensive studies in vitro, isolating bone marrow-derived lin- cells from both GPIbα-deficient and wild-type mice using magnetic beads. These cells were then cultured in the presence of rmTPO, and the results revealed that GPIbα deficiency led to the inhibition of megakaryocyte cell ploidy maturation and platelet production compared to wild-type mice. Furthermore, we designed RNA small interference sequences targeting GPIbα. By conducting in vitro interference experiment using wild-type mice bone marrow-derived lin- cells with TPO, the knockdown of GPIbα resulted in inhibited megakaryocyte cell ploidy maturation and platelet production, consistent with the findings from the deficiency mice. In addition to our prior experiments investigating the GPIbα mediated TPO generation, we have also explored the impact of GPIbα antibodies on TPO production, revealing a significant reduction in TPO levels among GPIbα-positive immune thrombocytopenia (ITP) patients. These data raise our curiosity regarding the responsiveness to TPO in ITP, particularly among GPIbα-positive ITP patients. To verify our hypothesis, a retrospective analysis was performed to assess the response to TPO-related treatments in ITP patients, including eltrombopag, romiplostim, avatrombopag and recombinant human thrombopoietin (rhTPO). The outcomes observed in ITP patients positive for GPIbα antibodies revealed noteworthy patterns. The retrospective analysis revealed that ITP patients exhibiting GPIbα positivity displayed a worse response to thrombopoietin receptor agonists (TPO-RA) and rhTPO treatment. To gain deeper insights into the aforementioned findings, we conducted in vitro investigations on antibody-bound megakaryocytes. Specifically, we cultured wild-type mice bone marrow-derived lin- cells in the presence of TPO, with or without anti-GPIbα antibody intervention. The antibody group exhibited inhibition of megakaryocyte ploidy maturation and platelet production compared to the negative control group. In summary, our findings suggest that GPIbα plays a critical role in control of TPO reactivity, and its absence or interference can lead to impaired megakaryocyte ploidy maturation and platelet production. Moreover, ITP patients with anti-GPIbα antibody is associated with decreased TPO responsiveness, probably contributing to the observed poor response to TPO-based treatments. These findings have significant clinical implications, shedding light on platelet-related disorders and potential therapeutic strategies. Specifically, understanding the influence of GPIbα on megakaryocyte TPO reactivity will offer avenues for novel therapeutic interventions targeting platelet-related disorders.

Xanthotoxin, a novel inducer of platelet formation, promotes thrombocytopoiesis via IL-1R1 and MEK/ERK signaling

BackgroundThrombocytopenia is a common hematological disease caused by many factors. It usually complicates critical diseases and increases morbidity and mortality. The treatment of thrombocytopenia remains a great challenge in clinical practice, however, its treatment options are limited. In this study, the active monomer xanthotoxin (XAT) was screened out to explore its medicinal value and provide novel therapeutic strategies for the clinical treatment of thrombocytopenia. MethodsThe effects of XAT on megakaryocyte differentiation and maturation were detected by flow cytometry, Giemsa and phalloidin staining. RNA-seq identified differentially expressed genes and enriched pathways. The signaling pathway and transcription factors were verified through WB and immunofluorescence staining. Tg (cd41: eGFP) transgenic zebrafish and mice with thrombocytopenia were used to evaluate the biological activity of XAT on platelet formation and the related hematopoietic organ index in vivo. ResultsXAT promoted the differentiation and maturation of Meg-01 cells in vitro. Meanwhile, XAT could stimulate platelet formation in transgenic zebrafish and recover platelet production and function in irradiation-induced thrombocytopenia mice. Further RNA-seq prediction and WB verification revealed that XAT activates the IL-1R1 target and MEK/ERK signaling pathway, and upregulates the expression of transcription factors related to the hematopoietic lineage to promote megakaryocyte differentiation and platelet formation. ConclusionXAT accelerates megakaryocyte differentiation and maturation to promote platelet production and recovery through triggering IL-1R1 and activating the MEK/ERK signaling pathway, providing a new pharmacotherapy strategy for thrombocytopenia.

Application and investigation of thrombopoiesis-stimulating agents in the treatment of thrombocytopenia.

Platelets, derived from a certain subpopulation of megakaryocytes, are closely related to hemostasis, coagulation, metastasis, inflammation, and cancer progression. Thrombopoiesis is a dynamic process regulated by various signaling pathways in which thrombopoietin (THPO)-MPL is dominant. Thrombopoiesis-stimulating agents could promote platelet production, showing therapeutic effects in different kinds of thrombocytopenia. Some thrombopoiesis-stimulating agents are currently used in clinical practices to treat thrombocytopenia. The others are not in clinical investigations to deal with thrombocytopenia but have potential in thrombopoiesis. Their potential values in thrombocytopenia treatment should be highly regarded. Novel drug screening models and drug repurposing research have found many new agents and yielded promising outcomes in preclinical or clinical studies. This review will briefly introduce thrombopoiesis-stimulating agents currently or potentially valuable in thrombocytopenia treatment and summarize the possible mechanisms and therapeutic effects, which may enrich the pharmacological armamentarium for the medical treatment of thrombocytopenia.

Combination of Thrombopoietin Receptor Agonist and Recombinant Human Thrombopoietin for Treating Cancer Therapy Induced Thrombopenia

Background Cancer therapy induced thrombopenia (CTIT) refer to thrombopenia caused by various anti-cancer therapy which include chemotherapy, radiotherapy, targeted therapy and immunotherapy. Chemotherapy-induced thrombopenia (CIT) could increase the risk of bleeding, modification of treatment, cost of treatment and even influence overall survival. Recombinant human thrombopoietin (rhTPO) was approved for CIT by national medical products administration (NMPA). However, certain patients still could not benefit from these agents. Hetrombopag, a new orally active small molecule thrombopoietin receptor agonist (TPO-RA), acts through binding to thrombopoietin receptor to stimulate multiple intracellular signaling pathways, including JAK/STAT, PI3K/AKT and ERK1/2. This will stimulate the proliferation and differentiation of megakaryocytes and promote platelet production. Combination of TPO-RA and rhTPO is anticipated to improve the outcome compared with rhTPO alone for treating CTIT in cancer patients. Methods Between January 2021 and June 2022, we conducted a retrospective analysis of patients (18 - 80 years) with cancer therapy induced thrombopenia (platelet count < 50 x 109/L) treated with rhTPO (subcutaneous injection, 300 U/kg/d) alone or combination of rhTPO and hetrombopag (oral, 5 mg/d) at Beijing Luhe Hospital, Capital Medical University. Treatment will discontinue when platelet count increased more than 50 x 109/L or platelet count doubled than baseline. The maximum treatment was 14 days. Results In total, 58 patients were included for analysis (28 patients received rhTPO plus hetrombopag and 30 patients received rhTPO alone). The baseline characteristics were generally similar between two groups. Patients with baseline platelet count less than 30 x109/L were 28.6% vs 10% in rhTPO plus hetrombopag group and rhTPO alone group, respectively. There were 21 patients (75.0%) vs 9 patients (30.0%) achieved response (platelet count increased more than 50 x 109/L or doubled than baseline or reached more than 100 x 109/L without platelet transfusion) within 7-day treatment in rhTPO plus hetrombopag group and rhTPO alone group, respectively. Furthermore, 11 patients (39.3%) and 3 patients (10.0%) reached platelet count ≥100 x 109/L in rhTPO plus hetrombopag group and rhTPO alone group (Table 1). The median platelet count at baseline in two groups were similar. The platelet count of post treatment in two groups were both increased. While the platelet count increase in rhTPO plus hetrombopag group were higher than rhTPO group in day 3, day 5, day 7 and day 9 after treatment (Figure 1). Median time of treatment was 6.5 days (range 4-13) for rhTPO plus hetromnopag group and 9.5 days (range 5-14) for rhTPO group (P < 0.0001). One patient (3.3%) in rhTPO group reported bleeding (WHO bleeding grade 2) while no bleeding events were reported in rhTPO plus hetrombopag group. Five patients (16.7%) and three patients (10.7%) in rhTPO group and rhTPO plus hetrombopag group received platelet transfusion. Cancer patients treated with rhTPO or rhTPO plus hetrombopag were well tolerated, the treatment related adverse events were mainly alanine aminotransferase/aspartate aminotransferase increase or total bilirubin increased (all grade 1/2). The incidence of such adverse event was not significantly different between two groups. Conclusion Compared with rhTPO, combined rhTPO with hetrombopag for treating cancer therapy induced thrombopenia showed faster and deeper response without raise the safety concern. Further study needed to consolidate this result. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

High-affinity autoreactive plasma cells disseminate through multiple organs in patients with immune thrombocytopenic purpura.

The major therapeutic goal for immune thrombocytopenic purpura (ITP) is to restore normal platelet counts using drugs to promote platelet production or by interfering with mechanisms responsible for platelet destruction. Eighty percent of patients with ITP possess anti–integrin αIIbβ3 IgG autoantibodies that cause platelet opsonization and phagocytosis. The spleen is considered the primary site of autoantibody production by autoreactive B cells and platelet destruction. The immediate failure in approximately 50% of patients to recover a normal platelet count after anti-CD20 rituximab-mediated B cell depletion and splenectomy suggests that autoreactive, rituximab-resistant, IgG-secreting B cells (IgG-SCs) reside in other anatomical compartments. We analyzed more than 3,300 single IgG-SCs from spleen, bone marrow, and/or blood of 27 patients with ITP, revealing high interindividual variability in affinity for αIIbβ3, with variations over 3 logs. IgG-SC dissemination and range of affinities were, however, similar for each patient. Longitudinal analysis of autoreactive IgG-SCs upon treatment with the anti-CD38 mAb daratumumab demonstrated variable outcomes, from complete remission to failure with persistence of high-affinity anti–αIIbβ3 IgG-SCs in the bone marrow. This study demonstrates the existence and dissemination of high-affinity autoreactive plasma cells in multiple anatomical compartments of patients with ITP that may cause the failure of current therapies.

Thrombocytopenia and insufficient thrombopoietin production in human small-for-gestational-age infants

BackgroundSmall-for-gestational-age (SGA) infants are at increased risk for transient thrombocytopenia. The aim of this study was to determine whether thrombocytopenia in human SGA infants is due to insufficient thrombopoietin (TPO) production.MethodsA prospective study of 202 infants with gestational age less than 37 weeks was conducted; 30 of them were SGA infants, and 172 were non-SGA infants. Thrombocytopenia was seen in 17 of 30 SGA infants and 40 of 172 non-SGA infants.ResultsPlatelet counts were significantly lower in the SGA group than in the non-SGA group at the time of the lowest platelet count within 72 h of birth. The platelet count and immature platelet fraction (IPF) were negatively correlated in non-SGA infants, but not in SGA infants. In addition, the platelet count and TPO were negatively correlated in non-SGA infants. IPF and TPO were significantly lower in SGA than in non-SGA infants with thrombocytopenia.ConclusionIPF increased with thrombocytopenia to promote platelet production in non-SGA infants due to increasing TPO, but not in SGA infants. This study found an association between insufficient TPO production and thrombocytopenia in SGA infants. In addition, this study is important for understanding the etiology of thrombocytopenia in SGA infants.ImpactThe immature platelet fraction was low, and serum thrombopoietin was not increased in small-for-gestational-age (SGA) infants with thrombocytopenia.Thrombocytopenia in SGA infants is due to insufficient thrombopoietin production.This study is important for understanding the etiology of thrombocytopenia in SGA infants.

Correlation between thrombopoietin and inflammatory factors, platelet indices, and thrombosis in patients with sepsis: A retrospective study.

BACKGROUNDThrombopoietin (TPO) is a primary regulator of thrombopoiesis in physiological conditions. TPO, in combination with its specific cytokine receptor c-Mpl, drives platelet production by inducing the proliferation and differentiation of megakaryocytes. However, the role of TPO in sepsis is not well determined. The elevated levels of TPO are often accompanied by a decrease of platelet count (PLT) in systemic infected conditions, which is contrary to the view that TPO promotes platelet production under physiological conditions. In addition, whether TPO mediates organ damage in sepsis remains controversial.AIMTo explore the relationships between TPO and inflammatory factors, platelet indices, and thrombotic indicators in sepsis.METHODSA total of 90 patients with sepsis diagnosed and treated at the emergency medicine department of The First People’s Hospital of Foshan between January 2020 and March 2021 were enrolled in this study. In addition, 110 patients without sepsis who came to the emergency medicine department were included as controls. Clinical and laboratory parameters including age, gender, TPO, blood cell count in peripheral blood, platelet indices, inflammatory factors such as high-sensitivity C-reactive protein (hs-CRP), interleukin (IL)-21, and IL-6, organ damage indicators, and thrombotic indicators were collected and analyzed by using various statistical approaches.RESULTSThe results showed that the TPO levels were higher in the sepsis group than in controls [86.45 (30.55, 193.1) vs 12.45 (0.64, 46.09) pg/mL, P < 0.001], but PLT was lower (P < 0.001). Multivariable analysis showed that white blood cell count (WBC) [odds ratio (OR) = 1.32; 95% confidence interval (CI): 1.01-1.722; P = 0.044], TPO (OR = 1.02; 95%CI: 1.01-1.04; P = 0.009), IL-21 (OR = 1.02; 95%CI: 1.00-1.03; P = 0.019), troponin I (OR = 55.20; 95%CI: 5.69-535.90; P = 0.001), and prothrombin time (PT) (OR = 2.24; 95%CI: 1.10-4.55; P = 0.027) were independent risk factors associated with sepsis. TPO levels were positively correlated with IL-21, IL-6, hs-CRP, creatinine, D-dimer, PT, activated prothrombin time, international normalized ratio, fibrinogen, WBC count, and neutrophil count, and negatively correlated with PLT, thrombin time, red blood cell count, and hemoglobin concentration (P < 0.05). Receiver operating characteristic analysis showed that TPO had fair predictive value in distinguishing septic patients and non-septic patients (the area under the curve: 0.788; 95%CI: 0.723-0.852; P < 0.001). With an optimized cutoff value (28.51 pg/mL), TPO had the highest sensitivity (79%) and specificity (65%).CONCLUSIONTPO levels are independently associated with sepsis. High TPO levels and low PLT suggest that TPO might be an acute-phase response protein in patients with infection.

Delving into the Therapeutic Potential of Carica papaya Leaf against Thrombocytopenia.

Thrombocytopenia is a clinical manifestation that refers to the low platelet count, i.e., <150 × 103/μL, of blood, resulting in imbalanced hemostasis, which leads to several fatal complications. The causative factors vary greatly, but, as a consequence, they interfere with platelet production and promote destruction, leading to death. Carica papaya leaf has unique therapeutic and medicinal characteristics against thrombocytopenia, and this is supported by scientific studies. Secondary metabolites and minerals in the leaf, such as carpaine and quercetin, promote platelet production, inhibit platelet destruction, and maintain platelet membrane through gene expression activity and the ceasing of viral proteases, respectively. This review explores the scientific studies that support the role of papaya leaf in the form of juice, extract, or powder against thrombocytopenia through animal modeling and clinical trials. Phytochemical profiles of C. papaya leaf revealed the presence of flavonoids, alkaloids, phenols, cardiac glycosides, tannins, terpenes, and saponins, which impart therapeutic potential to the leaf. The therapeutic benefits of the leaf include immunomodulatory, antiviral, antidiabetic, anticancer, antimalarial, antiangiogenic, antibacterial, and antioxidant activities. Several conducted scientific research studies have proved the efficacy of C. papaya leaf against thrombocytopenia, expanding the implication of natural sources to eradicate numerous ailments.

Inhibition of LDHA to induce eEF2 release enhances thrombocytopoiesis

AbstractTranslation is essential for megakaryocyte (MK) maturation and platelet production. However, how the translational pathways are regulated in this process remains unknown. In this study, we found that MK/platelet–specific lactate dehydrogenase A (LdhA) knockout mice exhibited an increased number of platelets with remarkably accelerated MK maturation and proplatelet formation. Interestingly, the role of LDHA in MK maturation and platelet formation did not depend on lactate content, which was the major product of LDHA. Mechanism studies revealed that LDHA interacted with eukaryotic elongation factor 2 (eEF2) in the cytoplasm, controlling the participation of eEF2 in translation at the ribosome. Furthermore, the interaction of LDHA and eEF2 was dependent on nicotinamide adenine dinucleotide (NADH), a coenzyme of LDHA. NADH-competitive inhibitors of LDHA could release eEF2 from the LDHA pool, upregulate translation, and enhance MK maturation in vitro. Among LDHA inhibitors, stiripentol significantly promoted the production of platelets in vivo under a physiological state and in the immune thrombocytopenia model. Moreover, stiripentol could promote platelet production from human cord blood mononuclear cell–derived MKs and also have a superposed effect with romiplostim. In short, this study shows a novel nonclassical function of LDHA in translation that may serve as a potential target for thrombocytopenia therapy.

Promotion of Platelet Production by Co-Transplantation of Mesenchymal Stem Cells in Bone Marrow Transplantation.

Therapeutic strategies that can promote platelet production are in demand to enhance clinical outcomes of bone marrow transplantation (BMT). Our research group has studied human tonsil-derived mesenchymal stem cells (T-MSCs) and their effectiveness in promoting bone marrow (BM) engraftment. Here, we analyzed the effects of T-MSCs on platelet production and hemostasis. Donor BM cells (BMCs) were isolated from C57BL/6 mice and transplanted with or without T-MSCs to BALB/c recipient mice. Mice were sacrificed and blood cells were counted using an Auto Hematology Analyzer. Femur sections were stained with CD41 antibody to analyze megakaryocytes in the BM. Growth factor secretion from MSCs was analyzed using the Quantibody Array. Effects of T-MSC conditioned medium (CM) on megakaryopoiesis were investigated using the MegaCult assay. In a mouse model of BMT, T-MSC CM was injected with or without anti-placental growth factor (α-PlGF) blocking antibody, and blood cell numbers and coagulation were analyzed. T-MSC co-transplantation increased percent survival of BMT mice. Platelet numbers were significantly lower in the BMC-only group, whereas T-MSC co-transplantation restored circulating platelets to levels similar to those of the control group. Significantly reduced numbers of CD41 + megakaryocytes in Bu-Cy and BMC groups were increased by T-MSC co-transplantation. PlGF secretion from T-MSCs were detected and enhanced megakaryopoiesis, platelet production, and coagulation by T-MCS CM were disrupted in the presence of the α-PlGF blocking antibody. We demonstrated the effectiveness of T-MSC co-transplantation in promoting platelet production and coagulation after BMT. These findings highlight the potential therapeutic relevance of T-MSCs for preventing thrombocytopenia after BMT.

Single-Cell Tracking By Time Lapse Imaging Confirms Thrombopoietin Promotes Megakaryocytic-Erythroid Progenitor Self Renewal, but Does Not Instruct Lineage Commitment

The molecular mechanisms underlying lineage commitment of stem and progenitor cells have implications for deriving specific cell types in vitro for regenerative medicine purposes. Current approaches to derive transfusable amounts of erythrocytes and platelets fall short of producing physiologically relevant amounts of each cell type. Thrombopoietin (TPO), commonly used in culture systems to increase platelet yield from cultured megakaryocytes, increases megakaryocyte (Mk) numbers and promotes platelet production. With the goal of generating adequate quantities of transfusion products in mind, establishing differentiation cultures that start with highly purified progenitors with Mk and E lineage potential as well as expansion potential is a very attractive option. However, a pervasive misconception exists that supplementing MEP cultures with TPO will increase the frequency with which the MEP produce megakaryocyte-committed daughter cells, thereby increasing the pool of lineage-restricted progenitor cells with high expansion capacity that ultimately yield higher numbers of transfusable platelets. We investigated whether TPO plays a role in instructing Megakaryocytic-Erythroid Progenitor (MEP) [1] commitment to the Mk lineage utilizing our previously presented time-lapse imaged colony forming unit assay and cell tracking approach [2]. This allowed us to calculate the frequency with which MEP give rise to lineage restricted progenitors in control conditions (with SCF, IL3, IL6, EPO, and TPO) compared to conditions lacking TPO to test the hypothesis that absence of TPO may diminish the frequency with which MEP give rise to Mk-restricted progenitors.We found that in the absence of TPO, MEP colony forming efficiency significantly (p<0.05) dropped by 22.4%. Importantly, the distribution of colony types was unaffected by the absence of TPO (Figure 1). By time-lapse imaging we observed the frequency with which MEP commit to the E lineage versus the Mk lineage to be approximately 2:1 in both control and -TPO conditions, demonstrating that TPO does not affect MEP lineage commitmentTime-lapse imaging also confirmed that MEP cultured without TPO underwent significantly (p<0.05) fewer symmetric self-renewal divisions resulting in less expansion of MEP, as well as less asymmetric self-renewal divisions resulting in more rapid exhaustion of MEP (Figure 2). 80% of control MEP divisions resulted in self renewal for an average of 8 generations and expansion divisions dropped below 10% after the 6 th generation, whereas MEP cultured without TPO lost the same propensity to self-renew within 3 generations. This confirms that TPO supports MEP self-renewal and expansion, similarly to how TPO supports HSC expansion [3].Cell death rates were at least 5-fold higher in the absence of TPO compared to control culture conditions containing TPO, and this effect was observed in both E and Mk-destined cells, demonstrating that TPO supports survival of both E and Mk-destined progenitors (p<0.05; Figure 3). Of note, CD41 mean fluorescence intensity was 2-fold dimmer in Mk progeny in the -TPO condition compared to control condition after 6 days in culture, which could indicate a delay in megakaryocytic maturation resulting in slower up-regulation of CD41.Our results confirm the hypothesis that TPO allows for greater self-renewal of MEP, as well as the survival of both the E and Mk-destined progenitors, however TPO is not instructive in MEP lineage commitment as evidenced by no change in the frequency of colony types. These findings also support the use of TPO in cultures of less committed progenitors to promote expansion leading to higher production of transfusable cell types. Further uses of this approach can include measuring kinetics of cellular processes such as division rate and motility, which may correlate with cell state and permit predictions of the probability of lineage choices downstream of specific progenitor populations.

Novel therapies for immune thrombocytopenia.

Current therapies for immune thrombocytopenia (ITP) are successful in providing a haemostatic platelet count in over two-thirds of patients. Still, some patients have an inadequate response and there is a need for other therapies. A number of novel therapies for ITP are currently being developed based upon the current pathophysiology of ITP. Many therapies are targetted at reducing platelet destruction by decreasing anti-platelet antibody production by immunosuppression with monoclonal antibodies targetted against CD40, CD38 and the immunoproteasome or physically reducing the anti-platelet antibody concentration by inhibition of the neonatal Fc receptor. Others target the phagocytic system by inhibiting FcγR function with staphylococcal protein A, hypersialylated IgG, polymeric Fc fragments, or Bruton kinase. With a recognition that platelet destruction is also mediated by complement, inhibitors of C1s are also being tested. Inhibition of platelet desialylation may also play a role. Other novel therapies promote platelet production with new oral thrombopoietin receptor agonists or the use of low-level laser light to improve mitochondrial activity and prevent megakaryocyte apoptosis. This review will focus on these novel mechanisms for treating ITP and assess the status of treatments currently under development. Successful new treatments for ITP might also provide a pathway to treat other autoimmune disorders.

Low-Dose Decitabine Restores Immune Tolerance in ITP By Modulating Regulatory T Cells

Immune thrombocytopenia (ITP) is an aquired autoimmune disease triggered by increased platelet destruction and impaired platelet production. Loss of immune tolerance is considered to be the pivotal mechanism of ITP. Decitabine, a hypomethylating agent promoting cell differentiation at low dose, has been applied in the treatment of myelodysplastic syndrome (MDS) with a considerable platelet response. Our previous and current studies demonstrated that low-dose decitabine could significantly increase the number of mature polyploidy megakaryocytes and had a long-term efficacy that could not be explained by its role on promoting platelet production in ITP patients. Recent reports have demonstrated that decitabine could decrease the production of interferon-γ (IFN-γ) and tumor necrosis factor-a (TNF-a), and increase immune suppressive regulatory T cells. Thus, low-dose decitabine might correct the loss of immune tolerance to induce a more durable response in patients with ITP.CD4+CD25+Foxp3+ regulatory T cells (Tregs), which play an important role in maintenance of peripheral tolerance, were reported to be decreased in number and function in ITP (Stasi R et. al. Blood 2008). In the in vitro study, peripheral blood mononuclear cells (PBMCs) were isolated and cultured with 100nM decitabine or PBS for 72h. We performed flow cytometry to determine the percentage of Tregs. It was shown that the percentage of Tregs in PBMCs and CD4+ T cells were significantly elevated after decitabine treatment. We also sorted the CD4+CD25+CD127- Tregs from ITP patients and co-cultured them with CFSE-labeled CD4+CD25-T cells to evaluate the suppressive activity of Tregs. The results indicated that the inhibitory function of Tregs after decitbine treatments was significantly elevated compared with controls.As compared to healthy individuals, patients with ITP also had increased number of TH1, TH17, TH22, Bregs and CD16+ monocytes. We subsequently investigated the effects of low-dose decitabine on those cells in patients with active ITP. Three cycles of decitabine were administered intravenously at a dose of at 3.5 mg/m2 for 3 days per cycle with a 4-week interval between cycles. After decitabine treatment, there was a significant increase in the relative number of CD25+Foxp3+ Tregs in the CD4+ T cell population. We also observed an increase in Tregs function after the decitabine treatment. In contrast, decitabine could significantly decrease the percentage of CD4+IFN-γ+TH1 in the CD4+T cell population and the percentage of CD16+monoyctes. However, we have not observed the effect of decitabine on the CD4+IFN-γ-CD17+TH17, CD4+IFN-γ-CD22+TH22, and CD19+CD24+CD38+Bregs by now.We further established the murine ITP model by transferring the splenocytes of C57BL/6 CD61 knockout mice, which were immunized against platelets from wild-type syngeneic C57BL/6 mice into severe combined immunodeficient (SCID) mice. We treated the ITP mice model with a low-dose range of decitabine (0.01, 0.03 or 0.10 mg/kg) intravenously three times a week. Our data showed a significant effect on PLT increase and the expansion of the Tregs population and the suppression of TH1 cells and CD16+ monocytes .In addition, programmed death-1 (PD-1) is a co-receptor that is expressed predominantly by T cells as well as B cells, monocytes and was found to be lower on the PBMCs and in serum of ITP patients (Zhong J et. al. Hematology. 2016). And research has reported that PD-1 signaling pathway is important in the pathogenesis of autoimmune diseases by inhibition of self-reactive T cells and increasing the development and functions of Tregs. Furthermore, it was reported that the expression of PD-1 is enhanced by decitabine in MDS (Yang H et. al. Leukemia 2014). In our current studies, decreased PD-1 expression on CD4+T cells of ITP patients was observed and decitabine could elevate the PD-1 expression on CD4+T cells after the decitabine treatment in ITP patients. Therefore, decitabine may correct the loss of immune tolerance by upregulating the Expression of PD-1 and be a promising therapeutic strategy for the management in ITP patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A Novel Megakaryocyte-Based Screening System Identifies Compounds That Promote Megakaryocyte Maturation and Platelet Production in Vitro

Platelets are anucleate cytoplasmic fragments derived from megakaryocytes (MKs) that play crucial roles in blood clotting system and innate immune response. The clinical needs of platelet transfusion are extensive and increasing, while current donor-derived platelet supply confronts challenges including limited donors, short shelf life of platelet products, the risk of bacterial and viral contamination and transfusion refractoriness. The construction of a non-donor-dependent system for efficient platelet generation to provide a consistent supply of platelets could address these concerns. Therefore, in vitro differentiation of human pluripotent stem cells (PSCs) have been studied to serve as a potential source of MKs and platelets to meet the demands of clinical transfusion. The immortalized megakaryocyte cell lines (imMKCLs) were previously established as cryopreservable master cells that can be robustly expanded and then produce platelets upon maturation, but the yield of platelets was far too short from that postulated in vivo (Nakamura, 2014). To overcome this issue, we sought to establish a reporter system that can monitor imMKCL maturation in vitro in high throughput manner to identify compounds that improve their maturation and platelet release.Herein we generated TUBB1-VENUS reporter imMKCLs with a VENUS transgene integrated into the β1 tubulin (TUBB1) locus using CRISPR/Cas9 system. The expression of the cytoskeleton molecule TUBB1 has been identified to be restricted in MK/platelet lineage, and it is indispensable for platelet formation and function. The maturation of MKs accompanies the increased expression of TUBB1 along with crucial transcription factors including GATA1, FOG1 and NF-E2.With this reporter line, the maturation of imMKCL is indicated by the fluorescence intensity of VENUS. Using this reporter imMKCL system for drug screening, we identified several compounds from a commercial drug library on facilitating platelet production efficiency. Interestingly, despite diverse categories (i.e. a Wnt inhibitor and a FLT3 inhibitor) of compounds were included, most drugs consistently induced significant suppression of the mRNA expression of CYP1B1, a typical downstream target of aryl-hydrocarbon receptor (AhR) pathway. Indeed, such molecules revealed an antagonizing action to aryl-hydrocarbon receptor (AhR) by pharmacological competitive assay using FICZ, an agonist to AhR. However, overexpression or knockdown of CYP1B1 did not directly affect platelet biogenesis in the presence of candidate drugs. These findings suggested that major MK maturation signaling is mediated by inhibition of AhR but independently of CYP1B1, a conventional AhR component.The imMKCL reporter system identified several drugs that could contribute to ex vivo production of platelets through inhibition of AHR signaling. The system could also be useful as an experimental tool for studying MK maturation machinery. DisclosuresNo relevant conflicts of interest to declare.

Thrombopoietin Receptor Agonists (TPO-RAs): Drug Class Considerations for Pharmacists.

The thrombopoietin receptor agonists (TPO-RAs) romiplostim, eltrombopag, avatrombopag, and lusutrombopag carry unique US Food and Drug Administration (US FDA)- and European Medicines Agency (EMA)-approved indications and may be used to increase platelet counts in a variety of conditions. Current indications for available TPO-RAs include treatment of chronic immune thrombocytopenia (ITP) in cases of insufficient response to prior treatment (avatrombopag, eltrombopag, romiplostim), management of thrombocytopenia in adult patients with chronic liver disease who are scheduled to undergo a procedure (avatrombopag, lusutrombopag), management of severe aplastic anemia (eltrombopag), and management of thrombocytopenia associated with interferon-based therapy for hepatitis C (eltrombopag). Across current indications, pharmacists can assist in stabilizing platelet counts and help to reduce large undulations commonly seen when starting, stopping, or transitioning between these agents. If therapy modifications may benefit the patient, pharmacists should discuss possible changes with the patient’s treatment team or treating physician. When used for ITP, romiplostim, eltrombopag, and avatrombopag stimulate TPO receptors on hematopoietic stem cells (also known as c-Mpl, or CD110) to promote platelet production; however, romiplostim is the only TPO-RA that binds at the same site as endogenous TPO. These subtle mechanistic differences may explain why switching TPO-RA may be clinically advantageous in some situations. As pharmacists are called to counsel patients on TPO-RA use, a deep understanding of potential adverse events and management strategies, as well as appropriate monitoring, will increase the likelihood that patients meet their goals of therapy in the shortest timeframe. Other uses of TPO-RAs are also discussed in this review, including use following hematopoietic stem cell transplant, use in myelodysplastic syndrome, and use in chemotherapy-induced thrombocytopenia.