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2567 Inflammation Mediated By TNFα Disrupts the Autophagic Flux and Oxidative Phosphorylation of Megakaryocytes and Platelets through the Downregulation of STX17

Program: Oral and Poster Abstracts
Session: 301. Vasculature, Endothelium, Thrombosis and Platelets: Basic and Translational: Poster II
Hematology Disease Topics & Pathways:
Bleeding and Clotting, adult, bleeding disorders, elderly, platelet disorders, Diseases, thrombocytopenias, thrombotic disorders, young adult , Biological Processes, molecular biology, Technology and Procedures, multi-systemic interactions, Study Population, Human, Animal model, omics technologies
Sunday, December 10, 2023, 6:00 PM-8:00 PM

Guadalupe Rojas-Sanchez, PharmB, MSc, PhD1, Jorge Calzada-Martinez, MSc2*, Brandon McMahon, MD3, Orlando Esparza, MD4*, Giovanny Hernandez, BA4*, Dennis L Le, MBBS4*, Eric Wartchow, PhD5*, Tzu Phang, PhD6*, Travis Nemkov, PhD6*, Angelo D'Alessandro, PhD7, Paola Maycotte, PhD8*, Andrew Thorburn, PhD9*, Jose A. Lopez, M.D10 and Pavel Davizon-Castillo, MD11

1Bloodworks NW Research Institute, Seattle, WA
2Bloodworks Northwest, Seattle, WA
3University of Colorado School of Medicine, Aurora, CO
4Department of Pediatrics Hematology/Oncology and Bone Marrow Transplantation, University of Colorado Anschutz Medical Campus, Aurora, CO
5Electron Microscopy Department, Children’s Hospital Colorado, Aurora, CO
6University of Colorado Anschutz Medical Campus, Aurora, CO
7School of Medicine, University of Colorado Anschutz Campus, Aurora, CO
8Centro de Investigación Biomédica de Oriente, OOAD Puebla, IMSS, Puebla, PU, Mexico
9Department of Pharmacology, University of Colorado Anschutz Medical Campus, Aurora, CO
10University of Washington, Bloodworks Research Institute, Seattle, WA
111551 Eastlake Ave E #100, Bloodworks Northwest, Seattle, WA

Background. JAK2V617F-positive polycythemia vera (PV) is the most common Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs). Elevated levels of TNFα, and a significantly higher risk of thrombosis and bleeding (thrombohemorrhage) are hallmarks of JAK2V617F PV. Platelets from patients with PV are characterized by hyperreactivity (fast activation of the αIIbβ3 integrin), higher P-selectin exposure (thromboinflammation), and phosphatidylserine (procoagulant potential). We have also found that in addition to these phenotypes, platelets from PV patients have dysfunctional autophagy promoting mitochondrial dysfunction, accumulation of dysmorphic mitochondria, and decreased clot retraction. Therefore, we hypothesized that inflammation mediated by TNFα (like in some cancers) dysregulates autophagy in JAK2V617F PV by reprogramming (immunometabolism) metabolism of MKs and platelets.

Methods. Platelets from sex and age-matched healthy subjects and patients with PV were analyzed. Platelets were treated with chloroquine (CQ) to analyze the autophagic flux by western blot and electron microscopy. Platelet bioenergetics was analyzed using the Seahorse extracellular flux analyzer. Semi-quantitative metabolomics was performed using the Vanquish UHPLC system. Thrombus formation was measured by microfluidics using T-TAS and ATLAS equipment. Clot retraction was assessed by measuring the weight of serum extruded from the clot. Platelets for clot retraction were treated with CQ or EACC. The MEG-01 cell line was treated with TNFα to analyze its impact on the autophagic flux, mitochondrial metabolism, and transcription (RNAseq). Murine studies using platelets from wild type (WT), DARE (transgenic mice with constitutive expression of TNFα or a neutralizing antibody against TNFα were performed.

Results. Platelets from patients with PV have significantly impaired autophagic flux (p<0.01), mitochondrial dysfunction (p<0.002), and accumulation of dysmorphic mitochondria (p<0.007). Platelets from PV patients have significantly lower levels of STX17 (p<0.008), a key regulatory molecule of autophagosome and lysosome fusion (Figure 1). The inhibition of STX17 from healthy donor platelets decreased clot stability (p<0.004). Moreover, platelets from chronically (DARE) and acutely inflamed mice (WT mice treated with TNFα) exhibited the same autophagic defect (p<0.03 and 0.02, respectively) secondary to the downregulation of STX17 (p<0.01 and 0.02, respectively). In vitro experiments showed that megakaryocytes downregulate the transcription and translation STX17 (p<0.006 and p<0.03, respectively) in response to TNFα. Finally, in vivo treatment of inflamed mice with a neutralizing anti-TNFa antibody restored the expression of STX17 (p<0.0001) and the autophagic flux (p<0.002).

Conclusion. Using several in vitro and in vivo approaches, we demonstrated for the first time the immunometabolic effect of TNFα in platelets and megakaryocytes. TNFα is a key regulator of the autophagic flux in platelets inducing platelet hyperreactivity and decreasing clot stability. Our findings may explain why patients with JAK2V617F PV are at increased risk for both thrombo-hemorrhagic events and suggest that targeting autophagy might lower this risk.

Disclosures: McMahon: GSK, PharmaEssentia: Consultancy. Nemkov: Omix Technologies Inc: Current equity holder in private company. D'Alessandro: Macopharma: Consultancy; Omix Technologies Inc: Current equity holder in private company; Hemanext Inc: Consultancy.

*signifies non-member of ASH