A circle of life: platelet and megakaryocyte cytoskeleton dynamics in health and disease.

Platelets are blood cells derived from megakaryocytes that play a central role in regulating haemostasis and vascular integrity. The microtubule cytoskeleton of megakaryocytes undergoes a critical dynamic reorganization during cycles of endomitosis and platelet biogenesis. Quiescent platelets have a discoid shape maintained by a marginal band composed of microtubule bundles, which undergoes remarkable remodelling during platelet activation, driving shape change and platelet function. Disrupting or enhancing this process can cause platelet dysfunction such as bleeding disorders or thrombosis. However, little is known about the molecular mechanisms underlying the reorganization of the cytoskeleton in the platelet lineage. Recent studies indicate that the emergence of a unique platelet tubulin code and specific pathogenic tubulin mutations cause platelet defects and bleeding disorders. Frequently, these mutations exhibit dominant negative effects, offering valuable insights into both platelet disease mechanisms and the functioning of tubulins. This review will highlight our current understanding of the role of the microtubule cytoskeleton in the life and death of platelets, along with its relevance to platelet disorders.

Chromatin modifier Hmga2 promotes adult hematopoietic stem cell function and blood regeneration in stress conditions.

The molecular mechanisms governing the response of hematopoietic stem cells (HSCs) to stress insults remain poorly defined. Here, we investigated effects of conditional knock-out or overexpression of Hmga2 (High mobility group AT-hook 2), a transcriptional activator of stem cell genes in fetal HSCs. While Hmga2 overexpression did not affect adult hematopoiesis under homeostasis, it accelerated HSC expansion in response to injection with 5-fluorouracil (5-FU) or in vitro treatment with TNF-α. In contrast, HSC and megakaryocyte progenitor cell numbers were decreased in Hmga2 KO animals. Transcription of inflammatory genes was repressed in Hmga2-overexpressing mice injected with 5-FU, and Hmga2 bound to distinct regions and chromatin accessibility was decreased in HSCs upon stress. Mechanistically, we found that casein kinase 2 (CK2) phosphorylates the Hmga2 acidic domain, promoting its access and binding to chromatin, transcription of anti-inflammatory target genes, and the expansion of HSCs under stress conditions. Notably, the identified stress-regulated Hmga2 gene signature is activated in hematopoietic stem progenitor cells of human myelodysplastic syndrome patients. In sum, these results reveal a TNF-α/CK2/phospho-Hmga2 axis controlling adult stress hematopoiesis.

An age-progressive platelet differentiation path from hematopoietic stem cells causes exacerbated thrombosis.

Platelet dysregulation is drastically increased with advanced age and contributes to making cardiovascular disorders the leading cause of death of elderly humans. Here, we reveal a direct differentiation pathway from hematopoietic stem cells into platelets that is progressively propagated upon aging. Remarkably, the aging-enriched platelet path is decoupled from all other hematopoietic lineages, including erythropoiesis, and operates as an additional layer in parallel with canonical platelet production. This results in two molecularly and functionally distinct populations of megakaryocyte progenitors. The age-induced megakaryocyte progenitors have a profoundly enhanced capacity to engraft, expand, restore, and reconstitute platelets in situ and upon transplantation and produce an additional platelet population in old mice. The two pools of co-existing platelets cause age-related thrombocytosis and dramatically increased thrombosis in vivo. Strikingly, aging-enriched platelets are functionally hyper-reactive compared with the canonical platelet populations. These findings reveal stem cell-based aging as a mechanism for platelet dysregulation and age-induced thrombosis.

Megakaryocytic IGF1 coordinates activation and ferroptosis to safeguard hematopoietic stem cell regeneration after radiation injury.

BACKGROUND: Hematopoietic stem cell (HSC) regeneration underlies hematopoietic recovery from myelosuppression, which is a life-threatening side effect of cytotoxicity. HSC niche is profoundly disrupted after myelosuppressive injury, while if and how the niche is reshaped and regulates HSC regeneration are poorly understood. METHODS: A mouse model of radiation injury-induced myelosuppression was built by exposing mice to a sublethal dose of ionizing radiation. The dynamic changes in the number, distribution and functionality of HSCs and megakaryocytes were determined by flow cytometry, immunofluorescence, colony assay and bone marrow transplantation, in combination with transcriptomic analysis. The communication between HSCs and megakaryocytes was determined using a coculture system and adoptive transfer. The signaling mechanism was investigated both in vivo and in vitro, and was consolidated using megakaryocyte-specific knockout mice and transgenic mice. RESULTS: Megakaryocytes become a predominant component of HSC niche and localize closer to HSCs after radiation injury. Meanwhile, transient insulin-like growth factor 1 (IGF1) hypersecretion is predominantly provoked in megakaryocytes after radiation injury, whereas HSCs regenerate paralleling megakaryocytic IGF1 hypersecretion. Mechanistically, HSCs are particularly susceptible to megakaryocytic IGF1 hypersecretion, and mTOR downstream of IGF1 signaling not only promotes activation including proliferation and mitochondrial oxidative metabolism of HSCs, but also inhibits ferritinophagy to restrict HSC ferroptosis. Consequently, the delicate coordination between proliferation, mitochondrial oxidative metabolism and ferroptosis ensures functional HSC expansion after radiation injury. Importantly, punctual IGF1 administration simultaneously promotes HSC regeneration and hematopoietic recovery after radiation injury, representing a superior therapeutic approach for myelosuppression. CONCLUSIONS: Our study identifies megakaryocytes as a last line of defense against myelosuppressive injury and megakaryocytic IGF1 as a novel niche signal safeguarding HSC regeneration.

Daucosterol regulates JAK2-STAT3 signaling pathway to promote megakaryocyte differentiation.

Immune thrombocytopenia (ITP) is an autoimmune disease caused by the loss of immune tolerance to platelet autoantigens, resulting in reduced platelet production and increased platelet destruction. Impaired megakaryocyte differentiation and maturation is a key factor in the pathogenesis and treatment of ITP. Sarcandra glabra, a plant of the Chloranthaceae family, is commonly used in clinical practice to treat ITP, and daucosterol (Dau) is one of its active ingredients. However, whether Dau can treat ITP and the key mechanism of its effect are still unclear. In this study, we found that Dau could effectively promote the differentiation and maturation of megakaryocytes and the formation of polyploidy in the megakaryocyte differentiation disorder model constructed by co-culturing Dami and HS-5 cells. In vivo experiments showed that Dau could not only increase the number of polyploidized megakaryocytes in the ITP rat model, but also promote the recovery of platelet count. In addition, through network pharmacology analysis, we speculated that the JAK2-STAT3 signaling pathway might be involved in the process of Dau promoting megakaryocyte differentiation. Western blot results showed that Dau inhibited the expression of P-JAK2 and P-STAT3. In summary, these results provide a basis for further studying the pharmacological mechanism of Dau in treating ITP.

Novel insights into the ontogeny of basophils.

Basophils are the least common granulocytes, accounting for <1% of peripheral blood leukocytes. In the last 20 years, analytical tools for mouse basophils have been developed, and we now recognize that basophils play critical roles in various immune reactions, including the development of allergic inflammation and protective immunity against parasites. Moreover, the combined use of flow cytometric analyses and knockout mice has uncovered several progenitor cells committed to basophils in mice. Recently, advancements in single-cell RNA sequencing (scRNA-seq) technologies have challenged the classical view of the differentiation of various hematopoietic cell lineages. This is also true for basophil differentiation, and studies using scRNA-seq analysis have provided novel insights into basophil differentiation, including the association of basophil differentiation with that of erythrocyte/megakaryocyte and the discovery of novel basophil progenitor cells in the mouse bone marrow. In this review, we summarize the recent findings of basophil ontogeny in both mice and humans, mainly focusing on studies using scRNA-seq analyses.

Modifying platelets at their birth: anti-thrombotic therapy without haemorrhage.

Cardiovascular disease is a leading cause of death. The current approach to the prevention of arterial thrombosis in cardiovascular disease is dependent on the use of therapies which inhibit the activation of platelets. Predictably these are associated with an increased risk of haemorrhage which causes significant morbidity. The thrombotic potential of an activated platelet is modifiable; being determined before thrombopoiesis. Increased megakaryocyte ploidy is associated with larger and more active platelets carrying an increased risk of thrombosis. The reduction in the ploidy of megakaryocytes is therefore a novel area of therapeutic interest for reducing thrombosis. We propose a new therapeutic approach for the prevention and treatment of thrombosis by targeting the reduction in ploidy of megakaryocytes. We examine the role of a receptor mediated event causing megakaryocytes to increase ploidy, the potential for targeting the molecular mechanisms underpinning megakaryocyte endomitosis and the existence of two separate regulatory pathways to maintain haemostasis by altering the thrombotic potential of platelets as targets for novel therapeutic approaches producing haemostatically competent platelets which are not prothrombotic.

Activating SRC/MAPK signaling via 5-HT1A receptor contributes to the effect of vilazodone on improving thrombocytopenia.

Thrombocytopenia caused by long-term radiotherapy and chemotherapy exists in cancer treatment. Previous research demonstrates that 5-Hydroxtrayptamine (5-HT) and its receptors induce the formation of megakaryocytes (MKs) and platelets. However, the relationships between 5-HT1A receptor (5-HTR1A) and MKs is unclear so far. We screened and investigated the mechanism of vilazodone as a 5-HTR1A partial agonist in promoting MK differentiation and evaluated its therapeutic effect in thrombocytopenia. We employed a drug screening model based on machine learning (ML) to screen the megakaryocytopoiesis activity of Vilazodone (VLZ). The effects of VLZ on megakaryocytopoiesis were verified in HEL and Meg-01 cells. Tg (itga2b: eGFP) zebrafish was performed to analyze the alterations in thrombopoiesis. Moreover, we established a thrombocytopenia mice model to investigate how VLZ administration accelerates platelet recovery and function. We carried out network pharmacology, Western blot, and immunofluorescence to demonstrate the potential targets and pathway of VLZ. VLZ has been predicted to have a potential biological action. Meanwhile, VLZ administration promotes MK differentiation and thrombopoiesis in cells and zebrafish models. Progressive experiments showed that VLZ has a potential therapeutic effect on radiation-induced thrombocytopenia in vivo. The network pharmacology and associated mechanism study indicated that SRC and MAPK signaling are both involved in the processes of megakaryopoiesis facilitated by VLZ. Furthermore, the expression of 5-HTR1A during megakaryocyte differentiation is closely related to the activation of SRC and MAPK. Our findings demonstrated that the expression of 5-HTR1A on MK, VLZ could bind to the 5-HTR1A receptor and further regulate the SRC/MAPK signaling pathway to facilitate megakaryocyte differentiation and platelet production, which provides new insights into the alternative therapeutic options for thrombocytopenia.

Alnustone promotes megakaryocyte differentiation and platelet production via the interleukin-17A/interleukin-17A receptor/Src/RAC1/MEK/ERK signaling pathway.

OBJECTIVES: Thrombocytopenia is a disease in which the number of platelets in the peripheral blood decreases. It can be caused by multiple genetic factors, and numerous challenges are associated with its treatment. In this study, the effects of alnustone on megakaryocytes and platelets were investigated, with the aim of developing a new therapeutic approach for thrombocytopenia. METHODS: Random forest algorithm was used to establish a drug screening model, and alnustone was identified as a natural active compound that could promote megakaryocyte differentiation. The effect of alnustone on megakaryocyte activity was determined using cell counting kit-8. The effect of alnustone on megakaryocyte differentiation was determined using flow cytometry, Giemsa staining, and phalloidin staining. A mouse model of thrombocytopenia was established by exposing mice to X-rays at 4 Gy and was used to test the bioactivity of alnustone in vivo. The effect of alnustone on platelet production was determined using zebrafish. Network pharmacology was used to predict targets and signaling pathways. Western blotting and immunofluorescence staining determined the expression levels of proteins. RESULTS: Alnustone promoted the differentiation and maturation of megakaryocytes in vitro and restored platelet production in thrombocytopenic mice and zebrafish. Network pharmacology and western blotting showed that alnustone promoted the expression of interleukin-17A and enhanced its interaction with its receptor, and thereby regulated downstream MEK/ERK signaling and promoted megakaryocyte differentiation. CONCLUSIONS: Alnustone can promote megakaryocyte differentiation and platelet production via the interleukin-17A/interleukin-17A receptor/Src/RAC1/MEK/ERK signaling pathway and thus provides a new therapeutic strategy for the treatment of thrombocytopenia.

From Hematopoietic Stem Cells to Platelets: Unifying Differentiation Pathways Identified by Lineage Tracing Mouse Models.

Platelets are the terminal progeny of megakaryocytes, primarily produced in the bone marrow, and play critical roles in blood homeostasis, clotting, and wound healing. Traditionally, megakaryocytes and platelets are thought to arise from multipotent hematopoietic stem cells (HSCs) via multiple discrete progenitor populations with successive, lineage-restricting differentiation steps. However, this view has recently been challenged by studies suggesting that (1) some HSC clones are biased and/or restricted to the platelet lineage, (2) not all platelet generation follows the "canonical" megakaryocytic differentiation path of hematopoiesis, and (3) platelet output is the default program of steady-state hematopoiesis. Here, we specifically investigate the evidence that in vivo lineage tracing studies provide for the route(s) of platelet generation and investigate the involvement of various intermediate progenitor cell populations. We further identify the challenges that need to be overcome that are required to determine the presence, role, and kinetics of these possible alternate pathways.

SET domain containing 2 promotes megakaryocyte polyploidization and platelet generation through methylation of α-tubulin.

BACKGROUND: Megakaryocytes (MKs) are polyploid cells responsible for producing ∼1011 platelets daily in humans. Unraveling the mechanisms regulating megakaryopoiesis holds the promise for the production of clinical-grade platelets from stem cells, overcoming significant current limitations in platelet transfusion medicine. Previous work identified that loss of the epigenetic regulator SET domain containing 2 (SETD2) was associated with an increased platelet count in mice. However, the role of SETD2 in megakaryopoiesis remains unknown. OBJECTIVES: Here, we examined how SETD2 regulated MK development and platelet production using complementary murine and human systems. METHODS: We manipulated the expression of SETD2 in multiple in vitro and ex vivo models to assess the ploidy of MKs and the function of platelets. RESULTS: The genetic ablation of Setd2 increased the number of high-ploidy bone marrow MKs. Peripheral platelet counts in Setd2 knockout mice were significantly increased ∼2-fold, and platelets exhibited normal size, morphology, and function. By knocking down and overexpressing SETD2 in ex vivo human cell systems, we demonstrated that SETD2 negatively regulated MK polyploidization by controlling methylation of α-tubulin, microtubule polymerization, and MK nuclear division. Small-molecule inactivation of SETD2 significantly increased the production of high-ploidy MKs and platelets from human-induced pluripotent stem cells and cord blood CD34+ cells. CONCLUSION: These findings identify a previously unrecognized role for SETD2 in regulating megakaryopoiesis and highlight the potential of targeting SETD2 to increase platelet production from human cells for transfusion practices.

Differentiation route determines the functional outputs of adult megakaryopoiesis.

Emerging evidence has revealed a direct differentiation route from hematopoietic stem cells to megakaryocytes (direct route), in addition to the classical differentiation route through a series of restricted hematopoietic progenitors (stepwise route). This raises the question of the importance of two alternative routes for megakaryopoiesis. Here, we developed fate-mapping systems to distinguish the two routes, comparing their quantitative and functional outputs. We found that megakaryocytes were produced through the two routes with comparable kinetics and quantity under homeostasis. Single-cell RNA sequencing of the fate-mapped megakaryocytes revealed that the direct and stepwise routes contributed to the niche-supporting and immune megakaryocytes, respectively, but contributed to the platelet-producing megakaryocytes together. Megakaryocytes derived from the two routes displayed different activities and were differentially regulated by chemotherapy and inflammation. Our work links differentiation route to the heterogeneity of megakaryocytes. Alternative differentiation routes result in variable combinations of functionally distinct megakaryocyte subpopulations poised for different physiological demands.

Bioprinting Soft 3D Models of Hematopoiesis using Natural Silk Fibroin-Based Bioink Efficiently Supports Platelet Differentiation.

Hematopoietic stem and progenitor cells (HSPCs) continuously generate platelets throughout one's life. Inherited Platelet Disorders affect ≈ 3 million individuals worldwide and are characterized by defects in platelet formation or function. A critical challenge in the identification of these diseases lies in the absence of models that facilitate the study of hematopoiesis ex vivo. Here, a silk fibroin-based bioink is developed and designed for 3D bioprinting. This bioink replicates a soft and biomimetic environment, enabling the controlled differentiation of HSPCs into platelets. The formulation consisting of silk fibroin, gelatin, and alginate is fine-tuned to obtain a viscoelastic, shear-thinning, thixotropic bioink with the remarkable ability to rapidly recover after bioprinting and provide structural integrity and mechanical stability over long-term culture. Optical transparency allowed for high-resolution imaging of platelet generation, while the incorporation of enzymatic sensors allowed quantitative analysis of glycolytic metabolism during differentiation that is represented through measurable color changes. Bioprinting patient samples revealed a decrease in metabolic activity and platelet production in Inherited Platelet Disorders. These discoveries are instrumental in establishing reference ranges for classification and automating the assessment of treatment responses. This model has far-reaching implications for application in the research of blood-related diseases, prioritizing drug development strategies, and tailoring personalized therapies.

Transcription factor 3 is dysregulated in megakaryocytes in myelofibrosis.

Transcription factor 3 (TCF3) is a DNA transcription factor that modulates megakaryocyte development. Although abnormal TCF3 expression has been identified in a range of hematological malignancies, to date, it has not been investigated in myelofibrosis (MF). MF is a Philadelphia-negative myeloproliferative neoplasm (MPN) that can arise de novo or progress from essential thrombocythemia [ET] and polycythemia vera [PV] and where dysfunctional megakaryocytes have a role in driving the fibrotic progression. We aimed to examine whether TCF3 is dysregulated in megakaryocytes in MPN, and specifically in MF. We first assessed TCF3 protein expression in megakaryocytes using an immunohistochemical approach analyses and showed that TCF3 was reduced in MF compared with ET and PV. Further, the TCF3-negative megakaryocytes were primarily located near trabecular bone and had the typical "MF-like" morphology as described by the WHO. Genomic analysis of isolated megakaryocytes showed three mutations, all predicted to result in a loss of function, in patients with MF; none were seen in megakaryocytes isolated from ET or PV marrow samples. We then progressed to transcriptomic sequencing of platelets which showed loss of TCF3 in MF. These proteomic, genomic and transcriptomic analyses appear to indicate that TCF3 is downregulated in megakaryocytes in MF. This infers aberrations in megakaryopoiesis occur in this progressive phase of MPN. Further exploration of this pathway could provide insights into TCF3 and the evolution of fibrosis and potentially lead to new preventative therapeutic targets.

What is the context? We investigated TCF3 (transcription factor 3), a gene that regulates megakaryocyte development, for genomic and proteomic changes in myelofibrosis.Myelofibrosis is the aggressive phase of a group of blood cancers called myeloproliferative neoplasms, and abnormalities in development and maturation of megakaryocytes is thought to drive the development of myelofibrosis.What is new? We report detection of three novel TCF3 mutations in megakaryocytes and decreases in TCF3 protein and gene expression in primary megakaryocytes and platelets from patients with myelofibrosis.This is the first association between loss of TCF3 in megakaryocytes from patients and myelofibrosis.What is the impact? TCF3 dysregulation may be a novel mechanism that is responsible for the development of myelofibrosis and better understanding of this pathway could identify new drug targets.

Deficiency of thioredoxin-interacting protein results in age-related thrombocytopenia due to megakaryocyte oxidative stress.

BACKGROUND: Platelets are generated from megakaryocytes (MKs), mainly located in the bone marrow (BM). Megakaryopoiesis can be affected by genetic disorders, metabolic diseases, and aging. The molecular mechanisms underlying platelet count regulation have not been fully elucidated. OBJECTIVES: In the present study, we investigated the role of thioredoxin-interacting protein (TXNIP), a protein that regulates cellular metabolism in megakaryopoiesis, using a Txnip-/- mouse model. METHODS: Wild-type (WT) and Txnip-/- mice (2-27-month-old) were studied. BM-derived MKs were analyzed to investigate the role of TXNIP in megakaryopoiesis with age. The global transcriptome of BM-derived CD41+ megakaryocyte precursors (MkPs) of WT and Txnip-/- mice were compared. The CD34+ hematopoietic stem cells isolated from human cord blood were differentiated into MKs. RESULTS: Txnip-/- mice developed thrombocytopenia at 4 to 5 months that worsened with age. During ex vivo megakaryopoiesis, Txnip-/- MkPs remained small, with decreased levels of MK-specific markers. Critically, Txnip-/- MkPs exhibited reduced mitochondrial reactive oxygen species, which was related to AKT activity. Txnip-/- MkPs also showed elevated glycolysis alongside increased glucose uptake for ATP production. Total RNA sequencing revealed enrichment for oxidative stress- and apoptosis-related genes in differentially expressed genes between Txnip-/- and WT MkPs. The effects of TXNIP on MKs were recapitulated during the differentiation of human cord blood-derived CD34+ hematopoietic stem cells. CONCLUSION: We provide evidence that the megakaryopoiesis pathway becomes exhausted with age in Txnip-/- mice with a decrease in terminal, mature MKs that response to thrombocytopenic challenge. Overall, this study demonstrates the role of TXNIP in megakaryopoiesis, regulating mitochondrial metabolism.

The role of PKC in X-ray-induced megakaryocyte apoptosis and thrombocytopenia.

Thrombocytopenia is a critical complication after radiation therapy and exposure. Dysfunction of megakaryocyte development and platelet production are key pathophysiological stages in ionizing radiation (IR)-induced thrombocytopenia. Protein kinase C (PKC) plays an important role in regulating megakaryocyte development and platelet production. However, it remains unclear how PKC regulates IR-induced megakaryocyte apoptosis. In this study, we found that pretreatment of PKC pan-inhibitor Go6983 delayed IR-induced megakaryocyte apoptosis, and inhibited IR-induced mitochondrial membrane potential and ROS production in CMK cells. Moreover, suppressing PKC activation inhibited cleaved caspase3 expression and reduced p38 phosphorylation levels, and IR-induced PKC activation might be regulated by p53. In vivo experiments confirmed that Go6983 promoted platelet count recovery after 21 days of 3 Gy total body irradiation. Furthermore, Go6983 reduced megakaryocyte apoptosis, increased the number of megakaryocyte and polyploid formation in bone marrow, and improved the survival rate of 6 Gy total body irradiation. In conclusion, our results provided a potential therapeutic target for IR-induced thrombocytopenia.

Serum CRP interacts with SPARC and regulate immune response in severe cases of COVID-19 infection.

Serum C-reactive protein (CRP) has been found elevated during COVID-19 infection, and associated with systematic inflammation as well as a poor clinical outcome. However, how did CRP participated in the COVID-19 pathogenesis remains poorly understood. Here, we report that serum C-reactive protein (CRP) levels are correlated with megakaryocyte marker genes and could regulate immune response through interaction with megakaryocytes. Molecular dynamics simulation through ColabFold showed a reliable interaction between monomeric form of CRP (mCRP) and the secreted protein acidic and rich in cysteine (SPARC). The interaction does not affect the physiological activities of SPARC while would be disturbed by pentamerization of CRP. Interplay between SPARC and mCRP results in a more intense immune response which may led to poor prognosis. This study highlights the complex interplay between inflammatory markers, megakaryocytes, and immune regulation in COVID-19 and sheds light on potential therapeutic targets.

Reprogramming Megakaryocytes for Controlled Release of Platelet-like Particles Carrying a Single-Chain Thromboxane A2 Receptor-G-Protein Complex with Therapeutic Potential.

In this study, we reported that novel single-chain fusion proteins linking thromboxane A2 (TXA2) receptor (TP) to a selected G-protein α-subunit q (SC-TP-Gαq) or to α-subunit s (SC-TP-Gαs) could be stably expressed in megakaryocytes (MKs). We tested the MK-released platelet-linked particles (PLPs) to be used as a vehicle to deliver the overexpressed SC-TP-Gαq or the SC-TP-Gαs to regulate human platelet function. To understand how the single-chain TP-Gα fusion proteins could regulate opposite platelet activities by an identical ligand TXA2, we tested their dual functions-binding to ligands and directly linking to different signaling pathways within a single polypeptide chain-using a 3D structural model. The immature MKs were cultured and transfected with cDNAs constructed from structural models of the individual SC-TP-Gαq and SC-TP-Gαs, respectively. After transient expression was identified, the immature MKs stably expressing SC-TP-Gαq or SC-TP-Gαs (stable cell lines) were selected. The stable cell lines were induced into mature MKs which released PLPs. Western blot analysis confirmed that the released PLPs were carrying the recombinant SC-TP-Gαq or SC-TP-Gαs. Flow cytometry analysis showed that the PLPs carrying SC-TP-Gαq were able to perform the activity by promoting platelet aggregation. In contrast, PLPs carrying SC-TP-Gαs reversed Gq to Gs signaling to inhibit platelet aggregation. This is the first time demonstrating that SC-TP-Gαq and SC-TP-Gαs were successfully overexpressed in MK cells and released as PLPs with proper folding and programmed biological activities. This bio-engineering led to the formation of two sets of biologically active PLP forms mediating calcium and cAMP signaling, respectively. As a result, these PLPs are able to bind to identical endogenous TXA2 with opposite activities, inhibiting and promoting platelet aggregation as reprogrammed for therapeutic process. Results also demonstrated that the nucleus-free PLPs could be used to deliver recombinant membrane-bound GPCRs to regulate cellular activity in general.

Thrombopoietin receptor agonists for refractory thrombocytopenia in patients after autologous hematopoietic stem cell transplantation.

OBJECTIVE: Autologous hematopoietic stem cell (ASC) transplantation (ASCT) is an effective treatment method for patients with hematological disorders and malignant diseases. The patient's ASCs are harvested prior to radiotherapy/chemotherapy, cryopreserved and then transfused back after the high-dose radiotherapy/chemotherapy conditioning treatment. Since some patients develop thrombocytopenia after receiving ASCT, it is difficult for them to bear simultaneously the management of their original disease and thrombocytopenia. The present study aimed to evaluate the efficacy and safety of thrombocytopenia therapy with thrombopoietin receptor agonists (TPORAs) after ASCT. METHODS: We retrospectively analyzed the clinical safety and efficacy of TPORA treatment for the enrolled 20 patients who developed thrombocytopenia after ASCT. The measured parameters were prolonged isolated thrombocytopenia (PIT), secondary failure of platelet recovery (SFPR) and other calculated response index. Patients with platelet count (PC) ≤ 50×109/L were treated with TPORA, namely with either eltrombopag (Elt), hetrombopag (Het), or avatrobopag (Ava). RESULTS: The group of 20 patients, who received TPORA administration for their thrombocytopenia after ASCT, had a median age of 50 years (ranging between 17 and 60 years). The median administration time of TPORA application was 48 days (ranging from 7 to 451 days); an overall response rate (ORR) was 85% with no response in 15% of patients, while with complete response (CR) in 70% of patients and partial response (PR) in 15% of patients. The median platelet count was 19 × 109/L before TPORA treatment and increased to 87×109)/L after the treatment. The TPORA treatment was safe as only 4 patients (20%) displayed a mild transaminase elevation. No other reported side effects occurred, such as thrombosis, joint pain, diarrhea, and myelofibrosis. It was demonstrated that the short response time to TPORA treatment correlated to the fast platelet recovery, when the number of megakaryocytes in the bone marrow smear exceeded 35/4.5 cm2 under a low magnification of 100 times (p = 0.015). CONCLUSION: TPORA therapy for thrombocytopenia occurring after the radiotherapy/ chemotherapy-conditioned ASCT was well tolerated and effective for platelets recovery.

Mycophenolate mofetil increases the risk of diarrhea in allogeneic hematopoietic stem cell transplantation recipients.

OBJECTIVES: Mycophenolate mofetil (MMF) was reported to be a main cause of diarrhea following organ transplantation. However, research on MMF-induced diarrhea following allogeneic hematopoietic stem cell transplantation (allo-HSCT) is currently insufficient. This retrospective study examined the incidence of MMF-induced diarrhea among allo-HSCT recipients. METHODS: Recipients were divided according to the receipt of MMF and the appearance of diarrhea. The differences in clinical information, MMF usage and trough concentrations, and the occurrence of diarrhea from the first day of conditioning treatment to 100 days after transplantation were analyzed. RESULTS: In total, 32.9% of the recipients reported diarrhea. The incidence rate of diarrhea was higher in the MMF group than in the non-MMF group (40.0% vs. 16.7%). MMF-induced diarrhea usually occurred within 9 days of MMF initiation and persisted for 7.27 ± 3.54 days. The average body weight, MMF daily dose, and MMF trough concentration were higher in patients with diarrhea. CONCLUSION: MMF increased the risk of diarrhea in allo-HSCT recipients, and the risk was related to the MMF dose and trough concentration. The difference in onset time could be a basis for identifying the cause of diarrhea in allo-HSCT recipients.