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

BACKGROUND: It 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. METHODS: We 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. RESULTS: There 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. CONCLUSIONS: In 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. Disclosures Tomassetti: Parexel: Research Funding; Novartis: Research Funding; Natera: Research Funding; Beigene: Research Funding; Rigel: Research Funding; Seagene: Research Funding.

Evaluation of Platelet Function Defects in Patients with Immune Thrombocytopenia

Background: Primary immune thrombocytopenia (ITP) is a megakaryocytic (MK)/platelet-specific autoimmune disorder characterized by platelet count <100×10 9/L with or without bleeding manifestations, and diagnosed by exclusion of other causes of thrombocytopenia. It is widely accepted the involvement of platelet autoantibodies on deterioration of platelets from patients with ITP. Moreover, an enhanced activity of neuraminidase may also reduce sialic acid from glycoside residues on platelet surface, especially from the highly glycosylated von Willebrand factor (vWF) receptor. Because controversial results regarding the functionality of platelets from ITP patients can be found in literature, we aimed to determine platelet ability to be stimulated by agonists. Moreover, we aimed to determine the way anti-platelet auto- antibodies (abs) and neuraminidase activity may affect the function of platelets derived from MKs of healthy controls. Methods: This observational, prospective and transversal study included 42 patients with chronic primary ITP and 55 healthy controls. Platelet fibrinogen and vWF receptors and activation markers (PAC1 binding to activated fibrinogen receptor and exposure of P-selectin after agonists treatment), were evaluated by flow cytometry. Presence of Antibodies (abs) against platelet's glycoproteins in ITP serum was analysed with a Luminex based assay (LifecodesPak Lx). Neuraminidase (NEU) activity in serum was determined with the substrate 20-(4-methylumbelliferyl)-a-D-N-(MUNANA). Human CD34 + cell-enriched population was obtained with CliniMACS (MiltenyiBiotec) from G-CSF mobilized peripheral blood of a healthy donor. For MK differentiation, CD34 + cells were cultured 12 days in StemSpan™ Serum-Free Expansion Medium II (SFEM II) with 50ng/ml of recombinant human thrompoietin. Then, 10% of serum from healthy controls (4) or ITP patients (4) were added to the culture of mature MKs and incubated for 3 days. Phenotypic analysis of MKs and culture derived-platelets was carried out using abs against CD34, CD41, CD42a and CD42b.Platelet-like particles were considered as CD41-positive events with a size (FSC) and granularity (SSC) scatter properties similar to blood platelets. Culture-derived platelets were stimulated with 100 µM TRAP and 10 µM ADP and activation markers were analyzed by flow cytometry. Results: Expression of fibrinogen receptor on platelets from ITP patients were similar to those from healthy controls but showed a reduced capacity to be activated. Impairment in platelet degranulation measured as exposition of P-selectin after agonist's stimulation was also observed in platelets from these patients (Figure 1). Of note, surface content of CD42b subunit of vWF receptor was reduced (Figure 1). To determine whether diminished platelet function might be due to a plasma component, we induced platelet production from MK of healthy controls as referred in Methods. Abs against platelets and neuraminidase activity were determined in serum samples. Serum from 4 healthy controls or from 4 ITP patients (1 with anti-CD42b, 1 with anti-GPIa-IIa and 2 with undetectable abs) were added to MKs culture. No differences existed in MK differentiation and platelet production between MKs incubated with serum from healthy controls or from ITP patients, but similarly as observed in platelets from ITP patients, MK-derived platelets had an impaired ability to be activated (Table 1). Platelets derived from MKs incubated with ITP serum with anti-platelet abs had also a diminished exposure of CD42b (73±8% of controls). Moreover, neuraminidase content of these samples was slightly higher than that from ITP samples without abs (130 vs 100 % of controls). Conclusion: Platelets from ITP patients had a diminished ability to be stimulated. In vitro study showed that megakaryopoiesis was normal in presence of ITP serum, but released platelets had a lower ability to be activated. Involvement of abs in this effect cannot be ruled out despite we detected abs only in 2 of the tested sera because efficiency of method to detect these abs is ~ 50%. On the other hand, reduced levels of CD42b might be due to the increased activity of neuraminidase. Reduction of sialic acid from CD42b might initiate its metalloproteinase-mediated cleavage or change affinity of the ab used for its detection. Research funded by ISCIII-Fondos FEDER PI19/00772 and Platelet Disorder Support Association Figure 1 Figure 1. Disclosures Alvarez Román: Pfizer: Consultancy, Honoraria, Research Funding; Octapharma: Consultancy, Honoraria, Research Funding; Sobi: Consultancy, Honoraria, Research Funding; Grifols: Consultancy, Honoraria, Research Funding; Biomarin: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; CSL-Behring: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Novo-Nordisk: Consultancy, Honoraria, Research Funding. García Barcenilla: Roche: Speakers Bureau; Takeda: Speakers Bureau; Bayer: Speakers Bureau; SOBI: Speakers Bureau. Canales: Janssen: Consultancy, Honoraria, Speakers Bureau; Celgene/Bristol-Myers Squibb: Consultancy, Honoraria; Gilead/Kite: Consultancy, Honoraria; Eusa Pharma: Consultancy, Honoraria; Incyte: Consultancy; Karyopharm: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Sanofi: Consultancy; iQone: Honoraria; Sandoz: Honoraria, Speakers Bureau; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Speakers Bureau; Takeda: Consultancy, Honoraria, Speakers Bureau. Jiménez-Yuste: Grifols: Consultancy, Honoraria, Research Funding; NovoNordisk: Consultancy, Honoraria, Research Funding; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; CSL Behring: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; BioMarin: Consultancy; Sobi: Consultancy, Honoraria, Research Funding; Octapharma: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Research Funding. Butta: Novo-Nordisk: Speakers Bureau; Takeda: Research Funding, Speakers Bureau; Roche: Speakers Bureau; CSL-Behring: Research Funding.

A Novel Allogeneic Cell Therapy That Targets Pathogenic Autoantibodies for the Treatment of Immune Thrombocytopenia

Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by low platelet counts (<100x10 9/L) and increased risk of bleeding. ITP is predominantly driven by immunoglobulin G (IgG)-autoantibodies directed against platelet surface antigens resulting in platelet destruction by the spleen and/or impaired platelet production by bone marrow megakaryocytes. Approximately 60% of ITP patients have measurable levels of anti-platelet autoantibodies in plasma and about 70% of them have autoantibodies directed against the fibrinogen receptor glycoprotein (GP) IIbIIIa. Current treatment strategies for ITP include non-specific immunosuppression (steroids, rituximab), inhibition of platelet clearance (immunoglobulins, splenectomy, anti-D immunoglobulin, and the Syk inhibitor fostamatinib) and stimulation of platelet production (thrombopoietin receptor agonists). In addition, therapeutics targeting the neonatal Fc receptor, responsible for IgG recycling, are under clinical investigation for ITP. Despite these treatment options, some patients with ITP are refractory or have inadequate responses to existing therapy. Here we describe a novel allogeneic cellular therapy, entitled platelet-like cells (PLC), that specifically targets pathogenic IgG for the treatment of thrombocytopenia in ITP. PLC are produced by differentiating a human induced pluripotent stem cell (hiPSC) line into megakaryocyte-like cells (MLC) which are further processed in a proprietary bioreactor that mimics the bone marrow environment and induces the release of anucleate PLC which are then cryopreserved. Analyses of PLC demonstrate sizes ranging between 65 nm and 10 μm. Expression of GPIIbIIIa protein as measured by enzyme-linked immunosorbent assay (ELISA) is evident in all particle sizes. PLC were administered intravenously (IV) into NOD-scid IL2Rgamma null (NSG) mice to evaluate their pharmacokinetics and biodistribution. PLC were rapidly cleared from the mouse circulation and predominantly distributed to the mouse liver where they colocalized primarily with Kupffer cells. PLC clearance through the liver was significantly inhibited by pre-treating mice with liposomes expressing phosphatidylserine (PS), indicating that one mechanism of PLC clearance is dependent on PS exposure on the PLC outer plasma membrane. The ability of PLC to bind anti-GPIIbIIIa autoantibodies was evaluated in an in vitro assay where PLC were incubated with ITP patient plasmas and subsequently removed by centrifugation and filtration. The concentration of anti-GPIIbIIIa autoantibodies remaining in the plasmas decreased following PLC treatment (96.6% ± 2.4%). The ability of PLC to clear anti-human GPIIbIIIa antibodies in circulation was evaluated by using a passive mouse model of ITP. Here, a fluorescently labeled mouse anti-human GPIIbIIIa monoclonal antibody (PAB-1) was dosed intravenously (IV) into NSG mice to achieve a circulating level of antibody comparable to those observed in ITP patients. The GPIIbIIIa antibody persisted in circulation for over 24 hours following a single dose in untreated mice and did not induce clearance of mouse platelets. Following administration of PLC in the passive ITP model, a decrease in antibody fluorescence in blood was observed within 2 minutes and full antibody clearance was observed at 3 hours post dosing. Furthermore, clearance of the antibody was observed to be both dose responsive and specific since PLC treatment did not affect the antibody concentration of an isotype matched control antibody. PLC activity in clearing the anti-GPIIbIIIa antibody was observed in all particle sizes. Fluorescence quantification of liver and spleen tissue demonstrated that PLC driven anti-GPIIbIIIa antibody clearance occurred preferentially in the liver. Taken together, these data indicate that by specifically binding and rapidly removing anti-platelet antibodies from circulation, PLC may disrupt the platelet degradation mechanisms underlying ITP pathogenesis and restore platelet counts. Disclosures Patel-Hett: PlateletBio: Current Employment. Giannini: Platelet Biogenesis: Current Employment. Peters: Platelet Biogenesis: Current Employment. Dykstra: Platelet Biogenesis: Current Employment. Lehmann: Platelet Biogenesis: Current Employment. Russo: Platelet Biogenesis: Current Employment. Kohnke: Platelet Biogenesis: Current Employment. Carpenter: Platelet Biogenesis: Current Employment. Tomczak: Platelet Biogenesis: Current Employment. Lazarus: Platelet Biogenesis: Consultancy. Masuko: Platelet Biogenesis: Current Employment. Leung: Platelet Biogenesis: Current Employment. Meredith: Platelet Biogenesis: Current Employment. Pete: Platelet Biogenesis: Current Employment. Lee: Platelet Biogenesis: Current Employment. Falb: Platelet Biogenesis: Current Employment.

Manipulation of Fatty Acid Metabolism Impairs Megakaryocyte Differentiation and Platelet Production

Background: Obesity is associated with an altered plasma lipid composition that impacts platelet activation, indicating that lipids directly affect platelet function and reactivity. However, very little is known about which essential lipids are important to enable platelet production from their precursors, megakaryocytes (MKs), and whether obesity influences lipid composition and consequently affects thrombopoiesis. Aim: We aimed to determine which lipids and associated pathways play a significant role in MK differentiation and platelet production in health and upon obesity. Our long-term goal is to determine if we can manipulate MK maturation and platelet production through lipid incorporation or inhibition. Methods: In order to determine the lipid profile of MKs through maturation, we performed an extensive lipidomics screen on primary MK progenitors, mature MKs, and platelets. Based on our findings, we then targeted de novo fatty acid (FA) synthesis in MKs to determine whether manipulation of this pathway attenuates MK differentiation and platelet formation. In parallel, we investigated megakaryo- and thrombopoiesis in an experimental setting of diet-induced obesity (DIO) in mice to examinate how altered lipid compositions within the plasma affect MK maturation. Results: Lipidomic data revealed increased polyunsaturated FA and plasmalogen content in the late stages of MKs maturation, suggesting that lipid composition is undergoing changes that may enable platelet production. To validate and extend these results, we inhibited multiple enzymes in de novo lipogenesis and FA synthesis. Using two different sources of primary murine hematopoietic stem cells (HSCs), we found that MK differentiation and maturation from HSCs was significantly decreased up to 60% after inhibition of acetyl-coA carboxylase (ACC) and 80% after fatty acid synthetase (FASN) inhibition, both enzymes from the de novo lipogenesis pathway. Moreover, we also found that MK differentiation and maturation were highly decreased (up to 70%) after the inhibition of acyl-coA synthetase (ACS). To further examine the role of fatty acids incorporation on platelet production, we next treated mature MKs with an ACS inhibitor to investigate the direct effect of FA incorporation on proplatelet formation; we observed a significant, multifoldreduction in proplatelet area. While dihydroceramides were altered in the lipidomic data, impairment of the de novo ceramide synthesis pathway did not affect either MK maturation or platelet production. Moreover, we observed an increased abundance of HSCs, myeloid cells, and MK progenitors in the bone marrow of DIO mice compared to control mice on a chow diet. This further supports an important role for lipids in megakaryopoiesis and suggests that differences in platelet reactivity during obesity may be caused by altered MK maturation and/or platelet production. Conclusions: Our results elucidate an important and previously unrecognized role for fatty acid synthesis in megakaryopoiesis and platelet production. The findings imply that an altered lipid content, as observed in patients with obesity, may not only impact platelet reactivity but also MK maturation and function. Consequently, platelets with an altered lipid content might originate from pathogenic MKs in obesity, which makes lipid-regulating proteins in MKs novel and viable therapeutic targets. Disclosures Machlus: KEROS Therapeutics: Consultancy, Honoraria; STRM.BIO: Consultancy, Honoraria.

Macrothrombocytopenia of Takenouchi-Kosaki syndrome is ameliorated by CDC42 specific- and lipidation inhibitors in MEG-01 cells

Macrothrombocytopenia is a common pathology of missense mutations in genes regulating actin dynamics. Takenouchi-Kosaki syndrome (TKS) harboring the c.191A > G, Tyr64Cys (Y64C) variant in Cdc42 exhibits a variety of clinical manifestations, including immunological and hematological anomalies. In the present study, we investigated the functional abnormalities of the Y64C mutant in HEK293 cells and elucidated the mechanism of macrothrombocytopenia, one of the symptoms of TKS patients, by monitoring the production of platelet-like particles (PLP) using MEG-01 cells. We found that the Y64C mutant was concentrated at the membrane compartment due to impaired binding to Rho-GDI and more active than the wild-type. The Y64C mutant also had lower association with its effectors Pak1/2 and N-WASP. Y64C mutant-expressing MEG-01 cells demonstrated short cytoplasmic protrusions with aberrant F-actin and microtubules, and reduced PLP production. This suggested that the Y64C mutant facilitates its activity and membrane localization, resulting in impaired F-actin dynamics for proplatelet extension, which is necessary for platelet production. Furthermore, such dysfunction was ameliorated by either suppression of Cdc42 activity or prenylation using chemical inhibitors. Our study may lead to pharmacological treatments for TKS patients.

Molecular mimicry between Spike and human thrombopoietin may induce thrombocytopenia in COVID-19

Thrombocytopenia, characterized by reduced platelet count, increases mortality in COVID-19 patients. We performed a computational investigation of antibody-induced cross-reactivity due to molecular mimicry between SARS-CoV-2 Spike protein and human thrombopoietin, the regulator of platelet production, as a mechanism for thrombocytopenia in COVID-19 infections. The presence of a common sequence motif with similar structure and antibody-binding properties for these proteins strongly indicate shared molecular mimicry. Recent reports of antibodies in COVID-19 patients and pre-pandemic samples against epitopes containing the motif offer additional support for the cross-reactivity. Altogether, this suggests cross-reactivity between an antibody with affinity for Spike protein and a human protein. Consideration of cross-reactivity for SARS-CoV-2 is important for therapeutic intervention and when designing the next generation of COVID-19 vaccines to avoid potential autoimmune interference.