Platelet Production Research Articles

Rare cell-based liquid biopsy for improved measurement of low-grade chronic inflammation

Objectives and designLow-grade inflammation is a hallmark of chronic diseases. More sensitive tools for chronic low-grade inflammation detection are needed and herein presented as a proof of concept. Heightened sensitivity to inflammation may be achieved by analyzing the compensation mechanisms of hematopoiesis in response to stress. The production of red blood cells and platelets, which are particularly vulnerable to physiological imbalances, are especially important in this context due to their high turnover rates. The compensatory mechanisms involve the production or release of rare immature blood cell types that herein serve as important biomarker targets.MethodsA cell-based liquid biopsy platform, using negative selection, was used to detect circulating rare cells in comprehension, allowing simultaneous analysis of an immature cell panel from one sample. The concentration ranges under physiological conditions for each cell marker were evaluated on a self-reported healthy control cohort and prospectively tested on three individuals undergoing various interventions: one afflicted with early-stage breast cancer, another with atherosclerosis in follow-up, and a third healthy individual with cardiovascular disease risk.ResultsThe approach effectively identified rare cellular abnormalities in asymptomatic individuals who exhibited no abnormalities in their complete blood counts. This condition was designated as silent inflammation (SI). SI was effective in monitoring response to intervention and predicting inflammation state.ConclusionsThe detection of SI proved valuable in aiding inflammation differential diagnosis and for monitoring the response to interventions in all three subjects.

Trogocytosis: A potential pathogenic mechanism in immune thrombocytopenia leading to impaired megakaryocyte maturation and reduced platelet production

Immune thrombocytopenia (ITP) is an autoimmune disease due to increased peripheral platelets destruction and inappropriate bone marrow production of platelets by megakaryocytes (MKs). The pathophysiology of ITP is complex and multifactorial, which remains not fully understood. However, the production of antiplatelet autoantibodies and the destruction of antibody-coated platelets through antibody-dependent cellular phagocytosis by macrophages (Mθ) expressing Fc gamma receptor (FcγR) are regarded as the key mechanisms. Glycoprotein (GP) Ⅱb/Ⅲa is the main target of antiplatelet antibodies leading to platelet phagocytosis by splenic Mθ, through interactions with FcγR. As GPⅡb/Ⅲa is normally expressed both on platelets and the MKs from which they are derived, we inferred that partial phagocytosis (definitely trogocytosis) of MKs by bone marrow Mθ might exist, leading to the increased amount of granular MKs (or non-platelets-producing MKs) in ITP. And we further hypothesized that the “bitten” MKs cannot sustain their normal function of maturation. Such trogocytosis mechanism may shed new light on the understanding of morphology alterations, maturation defects and decreased platelets production of MKs in ITP, and provide appealing implications in therapeutic modifications targeting the interplay of MKs and Mθ in bone marrow.

Regulatory Effect of PDGF/PDGFR on Hematopoiesis.

Platelet-derived growth factor (PDGF) is a critical cytokine with substantial regulatory effects on hematopoiesis. Recent research highlights the essential role of PDGF in the modulation of hematopoietic stem/progenitor cells (HSPCs), megakaryocytes/platelets, and thrombopoietin (TPO) synthesis within the bone marrow microenvironment. PDGF directly stimulates the proliferation and differentiation of HSPCs while also inhibiting apoptosis. In addition, PDGF indirectly enhances the production of other growth factors, including granulocyte-macrophage colony-stimulating factors. Further, PDGF regulates TPO production and influences the bone marrow milieu, thus impacting hematopoiesis and platelet formation. Mechanistically, PDGF binds to its receptor, PDGF receptor (PDGFR), thus activating the PDGF/PDGFR signaling pathway. This pathway subsequently activates phosphoinositide 3-kinase/protein kinase B, leading to the activation of downstream cytokines, including c-Fos and NF-E2, while inhibiting caspase-3 activation. Collectively, these actions have prodifferentiation and antiapoptotic effects on megakaryocytes, thereby regulating platelet production. This review provides a comprehensive analysis of the regulatory role of the PDGF/PDGFR axis in hematopoiesis, with a particular focus on platelet production, by summarizing all studies on PDGF/PDGFR from our group and globally.

Myeloproliferative Neoplasms: Challenging Dogma.

Myeloproliferative neoplasms, polycythemia vera, essential thrombocytosis, and primary myelofibrosis are a unique group of clonal hematopoietic stem cell neoplasms that share somatic, gain-in-function driver mutations in JAK2, CALR, and MPL. As a consequence, these disorders exhibit similar phenotypic features, the most common of which are the ceaseless production of normal erythrocytes, myeloid cells, platelets alone or in combination, extramedullary hematopoiesis, myelofibrosis, and a potential for leukemic transformation. In the case of polycythemia vera and essential thrombocytosis, however, prolonged survival is possible. With an incidence value in the range of 0.5-2.0/100,000, myeloproliferative neoplasms are rare disorders, but they are not new disorders, and after a century of scrutiny, their clinical features and natural histories are well-defined, though their individual management continues to be controversial. With respect to polycythemia vera, there has been a long-standing dispute between those who believe that the suppression of red blood cell production by chemotherapy is superior to phlebotomy to prevent thrombosis, and those who do not. With respect to essential thrombocytosis, there is a similar dispute about the role of platelets in veinous thrombosis, and the role of chemotherapy in preventing thrombosis by suppressing platelet production. Linked to these disputes is another: whether therapy with hydroxyurea promotes acute leukemia in disorders with a substantial possibility of longevity. The 21st century revealed new insights into myeloproliferative neoplasms with the discovery of their three somatic, gain-of-function driver mutations. Almost immediately, this triggered changes in the diagnostic criteria for myeloproliferative neoplasms and their therapy. Most of these changes, however, conflicted with prior well-validated, phenotypically driven diagnostic criteria and the management of these disorders. The aim of this review is to examine these conflicts and demonstrate how genomic discoveries in myeloproliferative neoplasms can be used to effectively complement the known phenotypic features of these disorders for their diagnosis and management.

Inhibition of FcRn with rozanolixizumab in adults with immune thrombocytopenia: Two randomised, double‐blind, placebo‐controlled phase 3 studies and their open‐label extension

SummaryPrimary immune thrombocytopenia (ITP) is an antiplatelet‐antibody‐mediated disorder with accelerated platelet clearance and decreased platelet production. Rozanolixizumab, a monoclonal IgG4 anti‐FcRn antibody, blocks IgG recycling and decreases IgG levels. We report efficacy and safety of rozanolixizumab in adults with persistent/chronic ITP in 24‐week phase 3 studies (TP0003; TP0006), and their 52‐week open‐label extension (OLE). Primary end‐point was durable clinically meaningful platelet response (DCMPR) of ≥50 × 109/L for 8/12 weeks during Weeks 13–25 in the double‐blind studies. Operational delays and evolving ITP treatment landscape led the sponsor to terminate these studies early; thus, only 21 and 12 (TP0003) and 20 and 10 (TP0006) patients were randomised to rozanolixizumab or placebo. Forty‐three patients enrolled in the OLE: 42 started on every 2‐week dosing; 21 later switched to weekly dosing. More rozanolixizumab‐treated than placebo‐treated patients achieved DCMPR: 4/21 versus 0 (TP0003) and 1/20 versus 0 (TP0006). Platelet increases to ≥50 × 109/L were observed on Day 8 in 52.4% (TP0003; 2/12 placebo) and 45.0% (TP0006; 1/10 placebo) of rozanolixizumab‐treated patients. OLE platelet increases were maintained while on weekly dosing. The most frequent treatment‐emergent adverse events overall were headache, pyrexia and nausea, as seen previously. Weekly dosing appears more efficacious than every 2‐week dosing.

Immunological face of megakaryocytes.

Megakaryocytes (MKs), which are traditionally known for their role in platelet production, are now emerging as unique immune cells with diverse capabilities. They express immune receptors, participate in pathogen recognition and response, phagocytose pathogens, contribute to antigen presentation, and interact with various immune cell types. When encountering inflammatory challenges, MKs exhibit intricate immune functions that can either promote or inhibit inflammation. These responses are mediated through mechanisms, such as the secretion of either anti-inflammatory or pro-inflammatory cytokines and release of immunomodulatory platelets according to specific conditions. This intricate array of responses necessitates a detailed exploration to determine whether the immune functions of MKs are carried out by the entire MK population or by a specific subpopulation. Breakthroughs in single-cell RNA sequencing have uncovered a unique "immune MK" subpopulation, revealing its distinct characteristics and immunoregulatory functions. This review provides latest insights into MKs' immune attributes and their roles in physiological and pathological contexts and emphasizes the discovery and functions of "immune MKs".

Dual effects of DLG5 (disks large homolog 5 gene) modulation on chemotherapy-induced thrombocytopenia and nausea/vomiting via the hippo signalling pathway.

The CAPEOX (combination of oxaliplatin and capecitabine) chemotherapy protocol is widely used for colorectal cancer treatment, but it can lead to chemotherapy-induced adverse effects (CRAEs). To uncover the mechanisms and potential biomarkers for CRAE susceptibility, we performed whole-genome sequencing on normal colorectal tissue (CRT) before adjuvant chemotherapy. This is followed by in vivo and in vitro verifications for selected gene and CRAE pair. Our analysis revealed specific germline mutations linked to Grade 2 (or higher) chemotherapy-induced thrombocytopenia (CIT) and nausea/vomiting (CINV). Notably, both CRAEs were associated with mutations in the DLG5 gene. We found that DLG5 mutations related to CIT were associated with increased gene expression, while those associated with CINV were linked to suppressed gene expression, as indicated by the Genotype-Tissue Expression (GTEX) database. In megakaryocytes, overexpression of human DLG5 suppressed the hippo signalling pathway and induced YAP expression. In zebrafish, overexpression of human DLG5 not only reduced platelet production but also inhibited thrombus formation. Subsequent qPCR analysis revealed that DLG5 overexpression affected genes involved in cytoskeleton formation and alpha-granule formation, which could impact the normal generation of proplatelets. We identified a series of germline mutations associated with susceptibility to CIT and CINV. Of particular interest, we demonstrated that induced and suppressed DLG5 expression is respectively related to CIT and CINV. These findings shed light on the involvement of the hippo signalling pathway and DLG5 in the development of CRAEs, providing valuable insights into potential targets for therapeutic interventions.

Microfluidics in Assessing Platelet Function.

Microfluidics incorporate physiologically relevant substrates and flows that mimic the vasculature and are, therefore, a valuable tool for studying aspects of thrombosis and hemostasis. At high-shear environments simulating arterial flow, a microfluidic assay facilitates the study of platelet function, as platelet-rich thrombi form in a localized stenotic region of a flow channel. Utilizing devices that allow for small sample volume can additionally aid in evaluating platelet function under flow from volume-limited patient samples or animal models. Studying trauma patient samples or samples following platelet product transfusion may aid in directing therapeutic strategies for patient populations in which platelet function is critical. Effects of platelet inhibition via pharmacological agents can also be studied in this model. The objective of this protocol is to establish a microfluidic platform that incorporates physiologic flow, biological surfaces, and relevant hemostatic mechanisms to assess platelet function with implications for the study of trauma induced coagulopathy and transfusion medicine.

Agarose Micro/Nanostructured Surfaces: A Step Toward an Innovative Solution for Platelet Storage Bags

AbstractIn addressing the critical limitations associated with platelet storage, the study investigates an innovative solution aimed at preventing the unwanted activation of platelets within storage bags. This activation is a key factor that currently restricts the shelf life of platelet products to only 72 h, leading to extreme waste production and high costs. Here, highly effective surfaces are identified for minimizing surface‐induced platelet activation. Using thermal nanoimprint lithography (T‐NIL), a new method is demonstrated for patterning reproducible agarose micro/nanostructures (a natural hydrogel with anti‐platelet adhesive properties) including dots, chains, pills, and squares. The agarose (3%) structured surfaces displayed outstanding flexibility and hydrophilic behavior that prevented platelet adhesion as confirmed by confocal microscopy. Importantly, pill‐shaped structures effectively maintained their ability to prevent platelet adhesion, even after a long cell‐contacting duration. Atomic force microscopy indicated that the effectiveness of dampening platelet adherence is determined by the shape, size, height, and aspect ratio of the structures. A model is provided to explain how the different shapes affect wettability and thereby hinder platelet adherence. The developed anti‐adhesive agarose structured surfaces show promise to revolutionize platelet storage, provide vital insights into biomaterials research, and demonstrate the potential of tailored agarose surfaces in biomedical applications.

Feasibility of plasma proteomics in patients with immune thrombocytopenia.

ITP is an acquired autoimmune bleeding disorder characterized by an isolated thrombocytopenia. The pathophysiology is highly multifactorial and involves antibody- and/or cytotoxic T cell-mediated killing of platelets and disruption of megakaryocyte function hampering platelet production. ITP remains a diagnosis of exclusion, and due to the high degree of variability between patients, it remains challenging to predict disease courses and responses to therapeutic agents. Hence, diagnostic and therapeutic laboratory biomarkers are highly warranted. To address this issue, in their paper, Jiang and colleagues have performed plasma proteomics in ITP patients (n = 40), in comparison to patients with thrombocytopenia due to other causes than ITP (non-ITP thrombocytopenia, n = 19) and healthy controls (n = 18). The data underscore that patients with ITP have a distinct plasma proteomic signature compared to non-ITP thrombocytopenia patients and healthy individuals. The findings should be further validated and investigated but suggest that the application of plasma proteomics is feasible and promising with respect to the search for potential biomarkers in patients with ITP. Commentary on: Jiang etal. Targeted proteomics profiling reveals valuable biomarkers in the diagnosis of primary immune thrombocytopenia. Br J Haematol 2024 (Online ahead of print). doi: 10.1111/bjh.19760.

LMAN1 serves as a cargo receptor for thrombopoietin.

Thrombopoietin (TPO) is a plasma glycoprotein that binds its receptor on megakaryocytes (MK) and MK progenitors, resulting in enhanced platelet production. The mechanism by which TPO is secreted from hepatocytes remains poorly understood. LMAN1 and MCFD2 form a complex at the endoplasmic reticulum membrane, recruiting cargo proteins into COPII vesicles for secretion. In this study, we showed that LMAN1 deficient mice (with complete germline LMAN1 deficiency) exhibited mild thrombocytopenia, whereas the platelet count was entirely normal in mice with approximately 7% Lman1 expression. Surprisingly, mice deleted for Mcfd2 did not exhibit thrombocytopenia. Analysis of peripheral blood from LMAN1 deficient mice demonstrated normal platelet size and normal morphology of dense and alpha granules. LMAN1 deficient mice exhibited a trend toward reduced MK and MK progenitors in the bone marrow. We next showed that hepatocyte-specific but not hematopoietic Lman1 deletion results in thrombocytopenia, with plasma TPO level reduced in LMAN1 deficient mice, despite normal Tpo mRNA levels in LMAN1 deficient livers. TPO and LMAN1 interacted by co-immunoprecipitation in a heterologous cell line and TPO accumulated intracellularly in LMAN1 deleted cells. Altogether, these studies confirmed the hepatocyte as the cell of origin for TPO production in vivo and were consistent with LMAN1 as the endoplasmic reticulum cargo receptor that mediates the efficient secretion of TPO. To our knowledge, TPO is the first example of an LMAN1-dependent cargo that is independent of MCFD2.

Comparative Evaluation of Hematological Parameters and Instrument Performance in Single and Double Plateletpheresis Procedures Using Haemonetics MCS+ and Trima Accel Systems

ObjectivesPlateletpheresis (PP) has become increasingly prevalent due to its cost-effectiveness and fewer immunological and infectious complications for recipients. This study compares hematological indices of platelet donors and instrument-related parameters in high-yield PP donors using Haemonetics MCS+ and Trima Accel. MethodsEligible and healthy PP donors meeting the platelet donation criteria were randomly selected.19 single-dose platelet (SDP), and 26 double-dose platelet (DDP) donors underwent PP using the Haemonetics MCS+, while 21 SDP and 21 DDP donors were processed using the Trima Accel system. Complete Blood Count (CBC) and hematological indices of donors between groups with both devices were measured with the cell counter. Platelet yield, collection efficiency (CE), and collection rate (CR) were evaluated for both devices. Results were reported using R-4.3.2 software and a p-value<0.05 was considered statistically significant ResultsThe Trima Accel processed significantly more blood volume and had shorter procedure times than MCS+. Platelet yield in the SDP group with Trima Accel was significantly higher than the Haemonetics MCS+. The Trima Accel demonstrated a significantly higher CR and CE than the MCS+ in both SDP and DDP groups. Post-PP lymphocyte counts significantly decreased with the Trima compared to the MCS+ in the SDP group. However, post-PP hematocrit (HCT), mean corpuscular volume (MCV), and mean platelet volume (MPV) in the DDP group with the MCS+ were significantly lower than Trima. ConclusionDouble-dose plateletpheresis (DDP) offers advantages in cost-effectiveness and platelet production, and although it reduces some hematological indices, these remain within normal limits. The Trima Accel may offer superior efficiency and processing times compared to the MCS+. However, careful monitoring of DDP donors following AABB standards remains essential.

The role of PALLD-STAT3 interaction in megakaryocyte differentiation and thrombocytopenia treatment.

Impaired differentiation of megakaryocytes constitutes the principal etiology of thrombocytopenia. The signal transducer and activator of transcription 3 (STAT3) is a crucial transcription factor in regulating megakaryocyte differentiation, however the precise mechanism of its activation remains unclear. PALLD, an actin-associated protein, has been increasingly recognized for its essential functions in multiple biological processes. This study revealed that megakaryocyte/platelet-specific knockout of Palld in mice exhibited thrombocytopenia due to diminished platelet biogenesis. In megakaryocytes, PALLD deficiency led to impaired proplatelet formation and polyploidization, ultimately weakening their differentiation for platelet production. Mechanistic studies demonstrated that PALLD bound to STAT3 and interacted with its DNA-binding domain and Src homology 2 domain via immunoglobulin domain 3. Moreover, the absence of PALLD attenuated STAT3 Y705 phosphorylation and impeded STAT3 nuclear translocation. Based on the PALLD-STAT3 binding sequence, we designed a peptide C-P3, which can facilitate megakaryocyte differentiation and accelerate platelet production in vivo. In conclusion, this study highlights the pivotal role of PALLD in megakaryocyte differentiation and proposes a novel approach for treating thrombocytopenia by targeting the PALLD-STAT3 interaction.

Analysis of platelet transfusion efficacy during extracorporeal membrane oxygenation (ECMO) treatment in pediatric patients post-cardiac surgery-a retrospective cohort study.

Postoperative extracorporeal membrane oxygenation (ECMO) may be necessary for pediatric patients following cardiac surgery, with associated risks of thrombocytopenia and bleeding. Prophylactic platelet transfusions are utilized to mitigate these risks, but the effectiveness of platelet transfusion cannot be reliably predicted. The aim of this study was to investigate the effect of platelet transfusion during postoperative treatment with ECMO in children undergoing cardiac surgery and to explore the optimal transfusion thresholds to reduce the number of platelet transfusions in patients and reduce the risk of death. We included in our study patients from the Pediatric Cardiac Surgery Department at the First Affiliated Hospital of Tsinghua University who underwent cardiac surgery and received ECMO treatment from January 1, 2019, to December 31, 2023, and received platelets transfusion at least once during the ECMO therapy. The platelet counts were determined both before and 24 hours posttransfusion of the platelet product. The corrected count increment (CCI) was calculated for the effectiveness estimation of platelet transfusion. The research subjects were divided into 3 groups based on the platelet count before transfusion (pretransfusion platelet count ≤30×109/L was the low-threshold group, pretransfusion count 31-50×109/L was the medium-threshold group, and ≥51×109/L was the high-threshold group) and the effective rates of each group were calculated. A total of 11 patients received 47 platelet transfusions, an average of 4.27 ± 1.67 per patient. According to the 24-hour postinfusion platelet (Plt) corrected critical control increase index (24-hour CCI) ≥4500, the infusion was considered to be effective, and ineffective when the CCI was <4500. Out of these, 22 transfusions (46.8%) proved effective, whereas 25 (53.2%) were deemed ineffective. The effective transfusion rates across the 3 groups were 69.2%, 50%, and 27.7%, respectively. The efficacy of platelet transfusion may be higher if a low threshold of platelet transfusion is chosen during ECMO treatment, on the premise of ensuring life safety.

Desialylation and Apoptosis in Immune Thrombocytopenia: Implications for Pathogenesis and Treatment.

Immune thrombocytopenia (ITP) is an autoimmune disease in which platelet autoantibodies play a significant role in its pathogenesis. Regulatory T cell dysfunction and T cell-mediated cytotoxicity also contribute to thrombocytopenia. Current therapies are directed towards immune suppression and modulation as well as stimulation of platelet production with thrombopoietin receptor agonists. Additional mechanisms of the pathogenesis of ITP have been suggested by recent experimental data. One of these processes, known as desialylation, involves antibody-induced removal of terminal sialic acid residues on platelet surface glycoproteins, leading to hepatic platelet uptake and thrombocytopenia. Apoptosis, or programmed platelet death, may also contribute to the pathogenesis of ITP. The extent of the impact of desialylation and apoptosis on ITP, the relative proportion of patients affected, and the role of antibody specificity are still the subject of investigation. This review will discuss both historical and new evidence of the influence of desialylation and apoptosis in the pathogenesis of ITP, with an emphasis on the clinical implications of these developments. Further understanding of both platelet desialylation and apoptosis might change current clinical practice and improve patient outcomes.

Newly diagnosed essential thrombocythemia leading to cardiogenic shock: a case report

BackgroundEssential thrombocythemia (ET) is a myeloproliferative neoplasm characterized by uninhibited platelet production. It can present with vasomotor symptoms, and less commonly, severe thrombotic events such as myocardial infarction. ST-segment elevation myocardial infarction (STEMI) secondary to the hypercoagulable state in ET is a diagnostic challenge as the complication is rare, especially outside the typical demographics affected by ET such as the female and elderly populations.Case presentationHere we report a case of a 32-year-old male found to have STEMI and a markedly elevated platelet count. Angiography revealed occlusion of the left anterior descending and left circumflex arteries, requiring percutaneous intervention and Impella support. The patient was later diagnosed with essential thrombocythemia, treated with hydroxyurea and antiplatelet therapy, and discharged with a wearable cardioverter defibrillator.ConclusionsThis case illustrates the potential for severe thrombotic complications such as STEMI due to ET. Severe thrombotic complications are less common manifestations of ET in general, particularly in young males. Recognition and diagnosis of ET are critical for the institution of appropriate therapy and prevention of STEMI and cardiogenic shock among other complications.

Clinical research progress of lusutrombopag for the treatment of thrombocytopenia in chronic liver disease

Thrombocytopenia is one of the common complications with the hematologic system in patients with chronic liver disease, which often causes poor quality of life and high risk of bleeding, thereby affecting their diagnosis, treatment, and prognosis. Platelet transfusion can improve the patient's declining platelet count to a certain extent, but it has problems such as high price and being easy to cause immune rejection. Thrombopoietin (TPO), as an important cytokine that affects platelet production, can bind to TPO receptors and activate megakaryocytes to produce platelets. Lusutrombopag is a newly developed small-molecule TPO receptor agonist that can induce bone marrow progenitor cells to differentiate into megakaryocytes and increase platelet production, posing characteristics of oral convenience, safety and effectiveness; thus, it has been used in many countries and regions for the treatment of patients with chronic liver diseases concurrent with thrombocytopenia. Herein, the pharmacological features and clinical research are reviewed.

Determination of Citrate Content of Commonly Transfused Blood Products

Abstract Background Citrate is a calcium-chelating anion used as an anticoagulant in the collection and manufacturing of blood products for transfusion. Overwhelming of normal hepatic catabolism of infused citrate, such as in liver failure or in massive transfusion, may cause citrate toxicity. Resultant hypocalcemia, impaired coagulation, and metabolic alkalosis results in increased patient mortality and morbidity. The citrate load per blood product unit is rarely known to the transfusionist, precluding precise calcium repletion. Manufacturing data of citrate content is available, but it is unreliable and often contradictory. The aim of this project is to directly measure the citrate concentrations of common blood products including packed red blood cells (pRBCs), plasma, and apheresis platelets. Methods Blood products (or samples thereof) were obtained, centrifuged, and assayed using a benchtop enzymatic citrate assay (Sigma-Aldrich). This technique generates a chromogenic product detectable via spectrophotometry at 570nm via conversion of citrate to a pyruvate intermediate. Blood products were serially diluted to the assay’s concentration measurement range based on estimations from the product manufacturing processes. Validation studies were performed to verify no significant interference from native pyruvate or from hemolysis at the dilutions used. All samples were run with a paired reference standard. Product type, storage duration, volume, and anticoagulant/preservative solution were recorded. Results Preliminary testing was performed on eleven pRBC units, twelve apheresis platelet units, and four whole blood(WB)-derived plasma. Measured citrate content of AS-1 and AS-5 units ranged from 43.56 - 114.5 mg while that of AS-3 units ranged from 327.23 – 816.82 mg, compared to manufacturing estimates of 188 mg and 883 mg, respectively. Measured citrate content of apheresis platelets ranged from 822-1533 mg compared to manufacturing estimates of 805 mg. Measured citrate content of WB-derived plasma also exceeded the manufacturing estimate of 1201 mg. Interference from pyruvate and hemolysis were insignificant at the dilutions used for testing. Validation of pRBC segment sampling is ongoing. Conclusion Varying guidelines exist for the correction of hypocalcemia during ongoing trauma resuscitation; however, guidance is limited regarding repletion by specific blood product. Our investigation affirms significant heterogeneity in pRBC citrate concentrations by collection method and identifies lower citrate concentrations than expected. This may be due to citrate metabolism by RBCs during storage. Platelet and FFP products contain significantly greater citrate masses by comparison, with a high degree of variation observed due to inherent product volume variability. Further sampling and testing are ongoing to establish reliable expected citrate loads for commonly transfused blood products.

The Establishment of a Novel Murine Model of Immune Thrombocytopenia in Pregnancy and the Impacts of Thrombopoietin Receptor Agonist on Platelet Production.

Objective Immune thrombocytopenia (ITP) is frequently associated with pregnancy. However, treatment options for ITP in pregnancy are limited, and there are few animal models available for the establishment of treatments. Here, we aimed to establish a novel murine pregnant model of ITP and to investigate the impacts of thrombopoietin receptor agonist (TPO-RA) on platelet production and reproductive outcomes. Methods Anti-glycoprotein Ib-alpha (GPIbα) antibody, which binds to megakaryocytes and platelets, was subcutaneously administered to pregnant mice in order to develop an ITP model (ITP group). TPO-RA was given in doses of 1 µg/kg, 10 µg/kg, and 100 µg/kg (low-dose group, mid-dose group, and high-dose group, respectively) for the treatment of ITP in pregnancy. Results The ITP group showed a significant reduction of platelet counts of less than 15% of healthy pregnant mice (control group) and also showed a significant increase in miscarriage rate (control group, 3.8%; ITP group, 44.4%; p < 0.05). Striking increases in platelet counts were observed in every TPO-RA group without any negative effects on fetal growth and placental pathology. No abnormality was noted in the external examination of fetal mice. Interestingly, a significant recovery of miscarriage rate was observed in the mid-dose group (23.5%) compared with the ITP group (p < 0.05). Conclusion A novel ITP model in pregnant mice was induced by injection of an anti-GPIbα antibody, and sufficient effects of TPO-RA on platelet production were observed in the present study. Furthermore, the positive impacts of TPO-RA on reproductive outcomes were revealed in the ITP model.

Identification of Nfel1a and Nfel3 as novel regulators for zebrafish thrombopoiesis

In mammalian hematopoiesis, megakaryocytes mature and become polyploid in the bone marrow before releasing platelets into circulation. In contrast, fish produce thrombocytes in kidney marrow, where young thrombocytes undergo maturation in circulation, akin to platelets. Despite morphological differences, our single-cell sequencing revealed significant gene expression similarities between fish thrombocytes and mammalian megakaryocytes, including Nfe2, which is crucial in platelet production. In addition to nfe2 expression, we found four nfe2-related genes, nfe2I1a, nfe2I1b, nfe2I2a, and nfe2I3, were expressed in mature thrombocytes. However, only nfe2, nfe2l2a, and nfe2l3 are expressed in young thrombocytes. Thus, we hypothesized that Nfe2-related factors may also be involved in thrombocyte production. To address this, we knocked down the four novel nfe2-related genes by the piggyback method and found both young and mature thrombocytes reduced when nfe2, nfe2l1a, and nfe2l3 were knocked down. They also exhibited greater gill bleeding and prolonged time to occlusion (TTO) compared to controls. In summary, our study shows Nfe2I1a and Nfe2l3 as novel transcription factors that are positive regulators influencing adult zebrafish thrombopoiesis.