Regulator Of Platelet Production Research Articles

Bone Marrow CD8 + Tissue-Resident Memory T Cell Promote B Cell Differentiation and Inhibit Platelet Production in Patients with Immune Thrombocytopenia

Immune thrombocytopenia (ITP) is a prevalent autoimmune disease in which enhanced anti-platelet T cell activity and dysfunction of CD8 + T cells are involved(Audia et al., 2013; Semple & Freedman, 1991). Persistent secretion of antibodies by long-lived plasma cells and memory B cells in the bone marrow contributes to ITP pathogenesis (Roeser, Lazarus, & Mahevas, 2023). However, the role of memory T cells, especially tissue-resident memory T cells (TRM), in ITP remains underexplored. TRM develop in specific tissues and play crucial role in local immunity. TRM exert their effects through mechanisms including the induction of local humoral responses, contributing to autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis (Chang et al., 2021; Gu, Song, Roh, Jung, & Kim, 2021). Bone marrow contains one of the largest pools of memory CD8 + T cells. However, the involvement of bone marrow TRM in ITP pathogenesis remains poorly understood. In this study, we observed a significantly higher proportion of CD8 + TRM (CD8 +CD45RA -CD45RO +CD69 +) in the bone marrow of ITP patients compared to healthy controls. However, no significant differences were observed in CD4 + TRM, CD8 + TCM and CD8 + TEM between the two groups. CD8 + TRM from ITP patients exhibited increased levels of IFN-γ, NLRP3, IL-1β, TNF-α, and decreased levels of IL-10 compared to those from healthy controls. Furthermore, CD8 + TRM proportions were significantly higher in glucocorticoid responders than non-responders, suggesting a potential role of CD8 + TRMs in mediating glucocorticoid therapeutic effects. Transcriptome sequencing revealed upregulated genes, including IGLC2, ASAP2, HIST2H2AA4, IGLV2-11, C1QA, ATP5MC1, IL5RA, and CYC1, and downregulated genes, including CCR9, HSPE1P18, RGCC, and ITGAE, in TRM cells from ITP patients compared to controls. GO/KEGG analysis of the top 500 upregulated genes highlighted pathways associated with B cell-associated immunity. To explore the effect of TRM on B cells, bone marrow CD19 + B cells were co-cultured with autologous TRM or memory T cells without TRM (TM) in the presence of IgM, and the proportion of memory B cells (CD19 +CD27 +CD38 -) and plasma cells (CD19 -CD24 -CD38 +) were measured by flow cytometry. The supernatants were collected, and cytokines and IgG level were tested by ELISA. Results showed that TRMs from ITP patients promoted the differentiation of memory B cells and IgG antibody production compared with those from healthy controls. However, no significant difference was observed in the proportion of naive B cells and plasma cells between the two groups. TRM, but not TM augmented B cell differentiation into memory B cells or plasma cells both in ITP patients and healthy controls. Additionally, TRM-primed B cells showed a stronger effect on antibody production and resulted in a higher level of IgG in supernatant compared with TM primed B cells. In line with these findings, the levels of B cell activating factor (BAFF), a proliferation inducing ligand (APRIL), and sCD40L, which were critical cytokines for B cell survival and differentiation, were significantly higher in supernatants from ITP patients than those from healthy controls. Moreover, the levels of BAFF, APRIL and CD40L in co-culture system with TRM were significantly increased compared to the system with TM, providing insights into the impact of TRM on B cell differentiation and function. To further investigate the role of TRM in regulating platelet production in ITP patients, we cocultured TRM with CD34 +cells in the presence of thrombopoietin (TPO), stem cell factor (SCF) and interleukin (IL)-3. Results showed that TRM did not suppress the production of CD41 + megakaryocytes from CD34 + cells, but reduced megakaryocyte apoptosis and increased the percentage of polyploidy (≥4N) megakaryocytes compared with TM. Additionally, TRM inhibited reticulated platelet formation and platelet production. Notably, dexamethasone (DXM) modulation alleviated certain suppressive effects on platelet production. In summary, our findings reveal the involvement of impaired bone marrow CD8 + TRM in the pathogenesis of ITP through the promotion of B cell differentiation and inhibition of platelet production. These results shed new light on the potential of immune therapy targeting TRM in the management of ITP.

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.

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.

ABCC4 impacts megakaryopoiesis and protects megakaryocytes against 6-mercaptopurine induced cytotoxicity

The role of ABCC4, an ATP-binding cassette transporter, in the process of platelet formation, megakaryopoiesis, is unknown. Here, we show that ABCC4 is highly expressed in megakaryocytes (MKs). Mining of public genomic data (ATAC-seq and genome wide chromatin interactions, Hi-C) revealed that key megakaryopoiesis transcription factors (TFs) interacted with ABCC4 regulatory elements and likely accounted for high ABCC4 expression in MKs. Importantly these genomic interactions for ABCC4 ranked higher than for genes with known roles in megakaryopoiesis suggesting a role for ABCC4 in megakaryopoiesis. We then demonstrate that ABCC4 is required for optimal platelet formation as in vitro differentiation of fetal liver derived MKs from Abcc4-/- mice exhibited impaired proplatelet formation and polyploidization, features required for optimal megakaryopoiesis. Likewise, a human megakaryoblastic cell line, MEG-01 showed that acute ABCC4 inhibition markedly suppressed key processes in megakaryopoiesis and that these effects were related to reduced cAMP export and enhanced dissociation of a negative regulator of megakaryopoiesis, protein kinase A (PKA) from ABCC4. PKA activity concomitantly increased after ABCC4 inhibition which was coupled with significantly reduced GATA-1 expression, a TF needed for optimal megakaryopoiesis. Further, ABCC4 protected MKs from 6-mercaptopurine (6-MP) as Abcc4-/- mice show a profound reduction in MKs after 6-MP treatment. In total, our studies show that ABCC4 not only protects the MKs but is also required for maximal platelet production from MKs, suggesting modulation of ABCC4 function might be a potential therapeutic strategy to regulate platelet production.

Toll-like Receptors and Thrombopoiesis.

Platelets are the second most abundant blood component after red blood cells and can participate in a variety of physiological and pathological functions. Beyond its traditional role in hemostasis and thrombosis, it also plays an indispensable role in inflammatory diseases. However, thrombocytopenia is a common hematologic problem in the clinic, and it presents a proportional relationship with the fatality of many diseases. Therefore, the prevention and treatment of thrombocytopenia is of great importance. The expression of Toll-like receptors (TLRs) is one of the most relevant characteristics of thrombopoiesis and the platelet inflammatory function. We know that the TLR family is found on the surface or inside almost all cells, where they perform many immune functions. Of those, TLR2 and TLR4 are the main stress-inducing members and play an integral role in inflammatory diseases and platelet production and function. Therefore, the aim of this review is to present and discuss the relationship between platelets, inflammation and the TLR family and extend recent research on the influence of the TLR2 and TLR4 pathways and the regulation of platelet production and function. Reviewing the interaction between TLRs and platelets in inflammation may be a research direction or program for the treatment of thrombocytopenia-related and inflammatory-related diseases.

Actin Bundling Protein L-plastin Regulates Platelet Production, Spreading, and Thrombosis

Actin Bundling Protein L-plastin Regulates Platelet Production, Spreading, and Thrombosis

MAPK-interacting kinase 1 regulates platelet production, activation, and thrombosis

AbstractThe MAPK-interacting kinase (Mnk) family includes Mnk1 and Mnk2, which are phosphorylated and activated in response to extracellular stimuli. Mnk1 contributes to cellular responses by regulating messenger RNA (mRNA) translation, and mRNA translation influences platelet production and function. However, the role of Mnk1 in megakaryocytes and platelets has not previously been studied. The present study investigated Mnk1 in megakaryocytes and platelets using both pharmacological and genetic approaches. We demonstrate that Mnk1, but not Mnk2, is expressed and active in human and murine megakaryocytes and platelets. Stimulating human and murine megakaryocytes and platelets induced Mnk1 activation and phosphorylation of eIF4E, a downstream target of activated Mnk1 that triggers mRNA translation. Mnk1 inhibition or deletion significantly diminished protein synthesis in megakaryocytes as measured by polysome profiling and [35S]-methionine incorporation assays. Depletion of Mnk1 also reduced megakaryocyte ploidy and proplatelet forming megakaryocytes in vitro and resulted in thrombocytopenia. However, Mnk1 deletion did not affect the half-life of circulating platelets. Platelets from Mnk1 knockout mice exhibited reduced platelet aggregation, α granule secretion, and integrin αIIbβ3 activation. Ribosomal footprint sequencing indicated that Mnk1 regulates the translation of Pla2g4a mRNA (which encodes cPLA2) in megakaryocytes. Consistent with this, Mnk1 ablation reduced cPLA2 activity and thromboxane generation in platelets and megakaryocytes. In vivo, Mnk1 ablation protected against platelet-dependent thromboembolism. These results provide previously unrecognized evidence that Mnk1 regulates mRNA translation and cellular activation in platelets and megakaryocytes, endomitosis and thrombopoiesis, and thrombosis.

A novel MPIG6B gene mutation in an adolescent girl with congenital thrombocytopenia and myelofibrosis

The MPIG6B gene, which encodes G6b-B, regulates platelet production, aggregation, and activation. Loss-of-function of G6b-B can cause thrombocytopenia, myelofibrosis, and anemia in both humans and mice. Several pathogenic MPIG6B mutations have been reported, such as c.324C>A (p.C108*), c.61_61+1dup (p.Ala21GlyfsX159), c.149dup (p.Ala52GlyfsX128), G6b c.469G>A (p.Gly157Arg) c.392delC (p.P134Lfs*10), and c523C>T(p.Arg175Ter). We have added to this database by reporting a new homozygous nonsense mutation (c.420T>A(p.Tyr140Ter)) of MPIG6B in a 14-year-old girl who presented with pallor, scattered cutaneous petechia of the limb, thrombocytopenia, anemia and myelofibrosis. This novel MPIG6B gene mutation encodes a shorter mutated G6b-B that does contain the transmembrane region immunoreceptor tyrosine-based inhibitory motif. The patient was effectively treated with allogeneic hematopoietic stem cell transplantation with peripheral stem cells from a matched unrelated donor. Her symptoms and the MPIG6B mutation disappeared after treatment, and she was healthy and had returned to school at the last follow-up.

3208 – PROLONGED THROMBOCYTOPENIA IN BONE MARROW TRANSPLANT PATIENTS IS CAUSED BY IMPAIRED SUPPORT OF PLATELET PRECURSOR CELLS BY ALTERED BONE MARROW NICHES

Prolonged thrombocytopenia affects one-third of patients who undergo a bone marrow transplant (BMT). It is a serious complication associated with significantly reduced survival. There is no alternative treatment aside from platelet transfusion to assist in platelet recovery post-BMT, which carries risks and complications. Platelet production occurs in the bone marrow in a multi-stage process, with each stage involving different niche cells. Little is known about the niches that regulate different types of platelet precursors, including megakaryocyte progenitors (MkPs), or how they respond to a BMT. Consistent with humans, mice have persistent reductions in platelets post-BMT. This was accompanied by reduced MkPs and megakaryocytes, but not hematopoietic stem cells. There were reduced perivascular niche cells and sinusoidal endothelial cells, and increased numbers of adipocytes post-BMT. Recent studies on human biopsies also show that patients with persistent thrombocytopenia have altered perivascular niche cells. To identify the niche cells that interact with the platelet precursors in situ, we used 7-colour Opal immunofluorescence. We used markers to phenotype megakaryocytes, MkPs and niche cells in mouse bone marrow sections and quantified their interactions using image analysis. The megakaryocytes and MkPs interacted closely with sinusoids and arterioles but negatively with trabecular bone. Post-BMT, MkPs were closer to the sinusoids and arterioles while the megakaryocytes are further away from arterioles. To identify novel proteins that regulate platelet production we are currently profiling bone marrow niche cells from mice post-BMT vs control using scRNA-seq. Collectively, our results show that endothelial and perivascular niche cells are important for platelet production and disruption in these niche cells may contribute to prolonged thrombocytopenia post-BMT. Prolonged thrombocytopenia affects one-third of patients who undergo a bone marrow transplant (BMT). It is a serious complication associated with significantly reduced survival. There is no alternative treatment aside from platelet transfusion to assist in platelet recovery post-BMT, which carries risks and complications. Platelet production occurs in the bone marrow in a multi-stage process, with each stage involving different niche cells. Little is known about the niches that regulate different types of platelet precursors, including megakaryocyte progenitors (MkPs), or how they respond to a BMT. Consistent with humans, mice have persistent reductions in platelets post-BMT. This was accompanied by reduced MkPs and megakaryocytes, but not hematopoietic stem cells. There were reduced perivascular niche cells and sinusoidal endothelial cells, and increased numbers of adipocytes post-BMT. Recent studies on human biopsies also show that patients with persistent thrombocytopenia have altered perivascular niche cells. To identify the niche cells that interact with the platelet precursors in situ, we used 7-colour Opal immunofluorescence. We used markers to phenotype megakaryocytes, MkPs and niche cells in mouse bone marrow sections and quantified their interactions using image analysis. The megakaryocytes and MkPs interacted closely with sinusoids and arterioles but negatively with trabecular bone. Post-BMT, MkPs were closer to the sinusoids and arterioles while the megakaryocytes are further away from arterioles. To identify novel proteins that regulate platelet production we are currently profiling bone marrow niche cells from mice post-BMT vs control using scRNA-seq. Collectively, our results show that endothelial and perivascular niche cells are important for platelet production and disruption in these niche cells may contribute to prolonged thrombocytopenia post-BMT.

Impact of Non-Alcoholic Fatty Liver Disease (NAFLD) on Platelet Count

BACKGROUNDIt is well established that alcohol use and chronic viral hepatitis negatively affect the peripheral platelet count. However, less is known about the effects of non-alcoholic fatty liver disease (NAFLD) on the peripheral platelet count.Platelets are anucleate cells made in the bone marrow that are responsible for the initiation of the hemostatic system and ultimately the repair of damaged endothelium. Platelet production is stimulated by thrombopoietin (TPO), a glycoprotein made by the liver that regulates platelet production. Binding of TPO to its receptor, c-MPL, on platelets and megakaryocytes prevents apoptosis of megakaryocytes and increases their number, size, and ploidy. In the setting of liver disease, such as NAFLD, TPO production may be reduced, which could result in lower peripheral platelet counts.NAFLD is a growing concern in the United States where the prevalence of NAFLD is estimated to be 25% and is expected to continue to rise over the next 10-15 years. NAFLD comprises a wide spectrum of disorders from simple steatosis to steatohepatitis. It is caused by excessive fat accumulation in the liver due to a dysregulation of fat synthesis and utilization resulting in oxidative stress. NAFLD is closely tied to insulin resistance. A subset of those with NAFLD develop progressive liver disease characterized by hepatocyte injury, inflammation, and ultimately cirrhosis. Hispanics have the highest prevalence and often have components of metabolic syndrome including obesity, systemic hypertension, dyslipidemia, and insulin resistance or diabetes. Studies show conflicting results as to the association of NAFLD and thrombocytopenia. Thus, the effect of NAFLD on the peripheral platelet count warrants further investigation. METHODSWe performed a retrospective chart review of all patients aged greater than 18 years who presented to Harbor-UCLA Medical Center between October 1, 2015 and March 1, 2021 with the diagnosis of NAFLD based on imaging. Diagnosis was established based on radiologic evidence of NAFLD on abdominal ultrasound or computed tomography scan. Patients were excluded if they had cirrhosis, chronic viral hepatitis, splenomegaly, excessive alcohol consumption (≥ 30 g/day in men or ≥ 20 g/day in women), malignancy, consumption of drugs commonly associated with thrombocytopenia, or known immune thrombocytopenia. Platelet count, body mass index (BMI), bilirubin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin, protein, cholesterol, triglycerides, prothrombin time (PT), partial thromboplastin time (PTT), and HbA1c were measured. Comorbid conditions including hypertension, obstructive sleep apnea (OSA), cardiovascular disease (CVD), and chronic kidney disease (CKD), were recorded. RESULTSThere were a total of 587 patients with the diagnosis of NAFLD who met the inclusion criteria. 41.7% were female and 58.3% were male. Of these, 51.4% were Hispanic, 8.9% were White (European), 7.0% were African American, 2.7% were Asian, and 2.7% were Southeast Asian, and 27.3% were unknown. The average age was 49 years. The mean platelet count was 249.5 K/cumm. The mean BMI was 33.4. The mean AST was 60.23 (normal 15-41 U/L) and ALT was 20.23 U/L (normal 7-35 U/L). The mean bilirubin, albumin, protein, PT, and PTT were all within normal limits. The mean HbA1c was 6.89%. The mean total cholesterol was 178.81 mg/dL (normal 125-199mg/dL), HDL 44.07 mg/dL (normal >40mg/dL), and LDL 101.65 mg/dL (normal <99 mg/dL). Coexisting hypertension, CKD, CVD, and OSA occurred in 39.2%, 11.1%, 7.8%, and 4.4% respectively. CONCLUSIONSIn this study, we found that NAFLD was not associated with thrombocytopenia. Further studies may be done to elucidate the impact of NAFLD on TPO levels and resulting peripheral platelet levels. DisclosuresTomassetti: Parexel: Research Funding; Novartis: Research Funding; Natera: Research Funding; Beigene: Research Funding; Rigel: Research Funding; Seagene: Research Funding.

Deficiency of ARHGAP21 alters megakaryocytic cell lineage responses and enhances platelet hemostatic function

BackgroundMicrotubules, actin and Rho GTPase proteins are essential players in the megakaryocyte biology for platelet formation and function. ObjectivesTo investigate the role of ARHGAP21, a RhoGAP protein, in megakaryocytic differentiation and platelet function. MethodsCytoskeletal proteins were investigated in HEL cells silenced for ARHGAP21 and submitted to megakaryocyte differentiation. The role of Arhgap21 in platelet function was accessed using haploinsufficient (Arhgap21+/−) mice. Arhgap21+/− platelet aggregation and p-selectin exposure were evaluated in response to thrombin. Vessel occlusion time and thrombus formation were detected after injury of the carotid artery. Platelet morphology was accessed by electronic microscopy. ResultsARHGAP21 was upregulated during megakaryocytic differentiation of the cell line and primary mouse cells. In the HEL cell model, ARHGAP21 was detected in the cytoplasmic protrusions, colocalized and associated with α-tubulin and was mostly detected in the protein cell fraction containing the polymerized tubulin. Silencing of ARHGAP21 decreased the expression of Glu-tubulin, suggesting microtubule instability, and enhanced cell spreading. Platelets from Arhgap21+/− mice presented enhanced thrombin-induced aggregation and p-selectin exposure associated with increased size of α-granules. Arhgap21+/− mice also showed increased CDC42 and RHOA activities, shorter tail-bleeding and accelerated thrombus formation. ConclusionsOur results indicate that ARHGAP21 may be a critical protein in the regulation of platelet production and function through the control of cytoskeletal rearrangement.

Reduction of ARHGAP21 Alters Platelet Biogenesis in Vitro and Accelerates Hemostatic Response In Vivo

Megakaryocyte differentiation and platelet biogenesis require profound cytoskeleton rearrangement regulated by the Rho family of GTPases. ARHGAP21 is a RhoGAP protein that has been shown to negatively regulate the activity of RhoA, RhoC and Cdc42. We have previously demonstrated that ARHGAP21 knockdown in human common myeloid progenitors and in bipotent megakaryocyte-erythrocyte progenitors may bias the fate decision toward the megakaryocyte lineage. In addition, a mouse model with reduction of Arhgap21 expression (Arhgap21+/-) present a slight reduction in platelet number and increased platelet volume. However, the participation of ARHGAP21 in platelet biogenesis and hemostatic response has never been investigated. We studied the role of ARHGAP21 on cytoskeletal changes during megakaryocyte differentiation in HEL cell line. Differentiation was induced with 20ηM phorbol-13 myristate-12 acetate (PMA) for four days. ARHGAP21 protein expression was increased during the differentiation and was mostly detected in the protein cell fraction containing polymerized tubulin, in comparison with cell extracts containing soluble tubulin. ARHGAP21 co-localized (R ≥ 0.86 in all days of differentiation) and interacted with α-tubulin on day 2 of megakaryocyte differentiation, when ARHGAP21 expression was the highest. Silencing of ARHGAP21 with siRNA decreased the expression of Glu-tubulin and enhanced CDC42 activity on days 2 and 3 of differentiation. Increased cell size and spreading and alteration of the adhesion proteins p-p130Cas, vinculin, p-zyxin and p-FAK925 were also observed upon ARHGAP21 inhibition. There was no change in the acquisition of CD61, CD41 and CD42 megakaryocytic markers, neither in the polyploidy of HEL cells during differentiation. We further investigated the effects of ARHGAP21 inhibition on platelet morphology and on the hemostatic response in vivo, using the C57BL/6 Arhgap21 heterozygous mouse model (Arhgap21+/-). The study was approved by the Ethical Committee of our Institution. No differences were observed in CD61+CD41+ nor in CD41+CD42+ bone marrow cells from Arhgap21+/- and wild type (WT) mice. However, transmission electron microscopy revealed that Arhgap21+/- platelets presented increased alpha-granule size when compared to wild-type (WT). Tail bleeding time of Arhgap21+/- mice was decreased compared to WT (P= 0.0008). Intravital microscopy of carotid artery injured by FeCl3 showed increased adhesion of platelet and white blood cells on the vessel wall of Arhgap21+/-, which reflected in accelerated occlusion time (twice as fast) compared to WT (P= 0.0150). In conclusion, ARHGAP21 silencing may alter cell morphology and lead to increased microtubule dynamic instability during megakaryocyte differentiation in vitro, without compromising the acquisition of differentiation markers. In vivo, deficiency of Arhgap21 increases platelet granule size and accelerates hemostatic response. Together, these results indicate that ARHGAP21 may be a critical protein in the regulation of platelet production and function through the control of cytoskeletal rearrangement. This study was supported by São Paulo Research Foundation (FAPESP) National Council for Scientific and Technological Development (CNPq) and Coordination for the Improvement of Higher Education Personnel (CAPES). Disclosures No relevant conflicts of interest to declare.

Immune Thrombocytopenia Plasma-Derived Exosomes Impaired Megakaryocyte and Platelet Production through an Apoptosis Pathway.

Reduced megakaryocyte (MK) apoptosis and insufficient platelet production play important roles in the pathogenesis of immune thrombocytopenia (ITP). The contribution of plasma-derived exosomes to the decreased platelet count in ITP has not been entirely understood. Here, we found the percentage of apoptotic MKs in patients with ITP was significantly lower than those in healthy volunteers. In the presence of ITP plasma-derived exosomes (ITP-Exo), the apoptosis of MKs was reduced during the process of MK differentiation in vitro, which contributed to the reduced platelet production by Bcl-xL/caspase signaling. Furthermore, in vivo study demonstrated that ITP-Exo administration led to significantly delayed platelet recovery in mice after 3.5 Gy of irradiation. All these findings indicated that ITP-Exo, as a regulator of platelet production, impaired MK apoptosis and platelet production through Bcl-xL/caspase signaling, unveiling new mechanisms for reduced platelet count in ITP.

Regulation of Platelet Production and Life Span: Role of Bcl-xL and Potential Implications for Human Platelet Diseases.

Blood platelets have important roles in haemostasis, where they quickly stop bleeding in response to vascular damage. They have also recognised functions in thrombosis, immunity, antimicrobal defense, cancer growth and metastasis, tumour angiogenesis, lymphangiogenesis, inflammatory diseases, wound healing, liver regeneration and neurodegeneration. Their brief life span in circulation is strictly controlled by intrinsic apoptosis, where the prosurvival Bcl-2 family protein, Bcl-xL, has a major role. Blood platelets are produced by large polyploid precursor cells, megakaryocytes, residing mainly in the bone marrow. Together with Mcl-1, Bcl-xL regulates megakaryocyte survival. This review describes megakaryocyte maturation and survival, platelet production, platelet life span and diseases of abnormal platelet number with a focus on the role of Bcl-xL during these processes.

SUN-038 HOMOCYSTEINE EXACERBATES IRI-INDUCED ACUTE KIDNEY INJURY VIA PROMOTING MEGAKARYOCYTE MATURATION AND PROPLATELET FORMATION

Platelets are crucial regulatory factors of acute kidney injury (AKI), as they are present in high numbers in the circulation and could release massive bioactive factors, which participate in intra-renal inflammation and haemostasis. After ischaemia-reperfusion injury (IRI), unbalanced macrocirculation and microcirculation lead to hypoxaemic injury and inflammatory response and haemostatic processes (platelet sequestration and thrombin). Homocysteine (Hcy), the important intermediate product of methionine cycle, is elevated as related genetic enzymatic deficiencies or nutritional defects which interfere metabolism of methionine. Moderate hyperhomocysteinemia (HHcy) is defined as total plasma Hcy in the range of 16-30uM, which is emerged as prothrombotic factor, influencing coagulative status and endothelial function and modulating platelet aggregation. We therefore aimed to evaluate the effects of hcy levels on platelet in IRI-induced AKI. Experiments were performed using 6 to 8 weeks old male C57BL/6 mice. To generate HHcy mice, mice were fed with high methionine diet containing 19.56g/kg methionine for 3 weeks. IRI induced AKI were performed by bilateral renal pedicles clamped for 30min in 37℃ with or without oral clopidogrel. After 48h, mice were euthanized and blood and kidney tissues were collected. For FeCl3 carotid artery injury, filter paper soaked in FeCl3 solution (3.5%) was placed on the right carotid artery for 90 seconds. Flow was recorded by Doppler Image scanner. As to deep venous thrombosis, the inferior vena cava was ligated for 48h and the length and weight of thrombus were recorded. A preparation of MKs was obtained from femora and tibiae BM of C57BL/6. Fetal liver-derived MKs was from the liver of mouse fetuses aged 13-15 days. The MKs was incubated with Hcy or not. In the present research, we found hyperhomocysteinemia mice developed more severe renal injury after IRI shown as more severe renal tubular damage and higher serum creatinine. Furthermore, the excess injury would be counteracted by oral clopidogrel compared with normal plasma Hcy level mice. Intriguingly, we observed that platelet counts rise by 30% in methionine-induced HHcy. The time required to form an occlusive thrombus in mice subjected to FeCl3 carotid artery injury was sharply shorter and the length and weight of deep venous thrombosis was significantly increased in HHcy model. We hypothesized that Hcy could regulate platelet production by affecting megakaryocytes. MKs cultured with Hcy or obtained from HHcy model increased proplatelet production and had higher ploidy. However, the amount of MKs and precursor cells were not change differently. In conclusion, the present study demonstrates that HHcy increases platelet counts by promoting megakaryocyte maturation and proplatelet formation. The results further suggest that mechanism of HHcy exacerbates IRI-induced acute Kidney Injury.

Phosphatidylinositol 3 monophosphate metabolizing enzymes in blood platelet production and in thrombosis.

Blood platelets, produced by the fragmentation of megakaryocytes, play a key role in hemostasis and thrombosis. Being implicated in atherothrombosis and other thromboembolic disorders, they represent a major therapeutic target for antithrombotic drug development. Several recent studies have highlighted an important role for the lipid phosphatidylinositol 3 monophosphate (PtdIns3P) in megakaryocytes and platelets. PtdIns3P, present in small amounts in mammalian cells, is involved in the control of endocytic trafficking and autophagy. Its metabolism is finely regulated by specific kinases and phosphatases. Class II (α, β and γ) and III (Vps34) phosphoinositide-3-kinases (PI3Ks), INPP4 and Fig4 are involved in the production of PtdIns3P whereas PIKFyve, myotubularins (MTMs) and type II PIPK metabolize PtdIns3P. By regulating the turnover of different pools of PtdIns3P, class II (PI3KC2α) and class III (Vps34) PI3Ks have been recently involved in the regulation of platelet production and functions. These pools of PtdIns3P appear to modulate membrane organization and intracellular trafficking. Moreover, PIKFyve and INPP4 have been recently implicated in arterial thrombosis. In this review, we will discuss the role of PtdIns3P metabolizing enzymes in platelet production and function. Potential new anti-thrombotic therapeutic perspectives based on inhibitors targeting specifically PtdIns3P metabolizing enzymes will also be commented.

Case report of a novel MPIG6B gene mutation in a Chinese boy with pancytopenia and splenomegaly

MPIG6B has orthologous physiological effects in human and mice, which regulates platelet production and function. For this reason, germline loss-of-function mutations in this gene cause congenital thrombocytopenia that is associated with bone marrow fibrosis, organomegaly and subsequent anemia. This was described in a consanguineous Arabian family with a novel truncation mutation (p.C108*) in chromosome 6, open reading frame 25 gene, also known as MPIG6B. In our case, we identified a homozygous frameshift variation (c.392delC,p.P134Lfs*10) in a ten-month-old boy presenting with signs of pallor, splenomegaly and resistant hemocytopenia. Interestingly, this is a new form of a MPIG6B variation, which could disrupt the effector protein for the key hematopoiesis regulators. This report adds to the growing number of mutations causing complex clinical manifestations associated with pancytopenia and splenomegaly in children.

Thrombopoietin Metabolically Primes Hematopoietic Stem Cells to Megakaryocyte-Lineage Differentiation

During acute myelosuppression or thrombocytopenia, bone marrow (BM) hematopoietic cells respond rapidly to replenish peripheral blood platelets. While the cytokine thrombopoietin (Thpo) both regulates platelet production and maintains HSC potential, whether Thpo controls megakaryocyte (Mk)-lineage differentiation of HSCs is unclear. Here, we show that Thpo rapidly upregulates mitochondrial activity in HSCs, an activity accompanied by differentiation to an Mk lineage. Moreover, in unperturbed hematopoiesis, HSCs with high mitochondrial activity exhibit Mk-lineage differentiation invitro and myeloid lineage-biased reconstitution invivo. Furthermore, Thpo skewed HSCs to express the tetraspanin CD9, a pattern correlated with mitochondrial activity. Mitochondria-active HSCs are resistant to apoptosis and oxidative stress upon Thpo stimulation. Thpo-regulated mitochondrial activity associated with mitochondrial translocation of STAT3 phosphorylated at serine 727. Overall, we report an important role for Thpo in regulating rapid Mk-lineage commitment. Thpo-dependent changes in mitochondrial metabolism prime HSCs to undergo direct differentiation to an Mk lineage.

A dual role for the class III PI3K, Vps34, in platelet production and thrombus growth

AbstractTo uncover the role of Vps34, the sole class III phosphoinositide 3-kinase (PI3K), in megakaryocytes (MKs) and platelets, we created a mouse model with Vps34 deletion in the MK/platelet lineage (Pf4-Cre/Vps34lox/lox). Deletion of Vps34 in MKs led to the loss of its regulator protein, Vps15, and was associated with microthrombocytopenia and platelet granule abnormalities. Although Vps34 deficiency did not affect MK polyploidisation or proplatelet formation, it dampened MK granule biogenesis and directional migration toward an SDF1α gradient, leading to ectopic platelet release within the bone marrow. In MKs, the level of phosphatidylinositol 3-monophosphate (PI3P) was significantly reduced by Vps34 deletion, resulting in endocytic/trafficking defects. In platelets, the basal level of PI3P was only slightly affected by Vps34 loss, whereas the stimulation-dependent pool of PI3P was significantly decreased. Accordingly, a significant increase in the specific activity of Vps34 lipid kinase was observed after acute platelet stimulation. Similar to Vps34-deficient platelets, ex vivo treatment of wild-type mouse or human platelets with the Vps34-specific inhibitors, SAR405 and VPS34-IN1, induced abnormal secretion and affected thrombus growth at arterial shear rate, indicating a role for Vps34 kinase activity in platelet activation, independent from its role in MKs. In vivo, Vps34 deficiency had no impact on tail bleeding time, but significantly reduced platelet prothrombotic capacity after carotid injury. This study uncovers a dual role for Vps34 as a regulator of platelet production by MKs and as an unexpected regulator of platelet activation and arterial thrombus formation dynamics.