Inflammation Mediated By TNFα Disrupts the Autophagic Flux and Oxidative Phosphorylation of Megakaryocytes and Platelets through the Downregulation of STX17
Authors: Rojas-Sanchez Guadalupe , Calzada-Martinez Jorge , McMahon Brandon , Esparza Orlando , Hernandez Giovanny , Le Dennis L , Wartchow Eric , Phang Tzu , Nemkov Travis , D'Alessandro Angelo , Maycotte Paola , Thorburn Andrew , Lopez Jose A. , Davizon-Castillo Pavel 
Published: December 2023

Background. JAK2 V617F-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 JAK2 V617F 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 JAK2 V617F 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&lt;0.01), mitochondrial dysfunction (p&lt;0.002), and accumulation of dysmorphic mitochondria (p&lt;0.007). Platelets from PV patients have significantly lower levels of STX17 (p&lt;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&lt;0.004). Moreover, platelets from chronically (DARE) and acutely inflamed mice (WT mice treated with TNFα) exhibited the same autophagic defect (p&lt;0.03 and 0.02, respectively) secondary to the downregulation of STX17 (p&lt;0.01 and 0.02, respectively). In vitro experiments showed that megakaryocytes downregulate the transcription and translation STX17 (p&lt;0.006 and p&lt;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&lt;0.0001) and the autophagic flux (p&lt;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 JAK2 V617F PV are at increased risk for both thrombo-hemorrhagic events and suggest that targeting autophagy might lower this risk.