Characterizing the Metabolic Determinants of Thromboinflammation in Myeloproliferative Neoplasms

BACKGROUND: Myeloproliferative neoplasms (MPNs) are a group of clonal hematopoietic disorders characterized by an overproduction of terminally differentiated myeloid elements. Venous and arterial thrombotic events represent the leading causes of morbidity and mortality in patients with JAK2 positive MPNs (essential thrombocythemia [ET] and polycythemia vera [PV]) in chronic phase. Platelet hyperreactivity is a hallmark finding of MPNs but the biological mechanisms remain to be elucidated. We have previously identified that platelets from patients with PV and ET exhibit dysmorphic mitochondria, however, the detailed bioenergetic and metabolomic implications of such alterations in mitochondrial mass and morphology remain to be investigated. Here in, we aim to identify the metabolic determinants that underpin thromboinflammation in patients with JAK2 positive MPNs in chronic phase. METHODS: Platelets from sex and age-matched healthy control subjects and individuals with either JAK2 positive PV or ET were isolated and washed following standard protocols. Platelet activation by flow cytometry was determined at baseline conditions. Platelet activated fibrinogen binding site (αIIbβIII), P-selectin, and phosphatidylserine (PS) surface marker expression (as measured by mean fluorescence intensity [MFI]) was determined with PAC-1 and P-selectin antibodies and lactadherin, respectively. The bioenergetic profile of washed platelets was determined by the 24-well format Seahorse extracellular flux analyzer. Metabolomic profile was developed with the Vanquish UHPLC system coupled online to a Q Exactive mass spectrometer. Statistical analyses were performed using the unpaired 2-tailed Student t test (GraphPad Software v9.1.2). Data expressed as mean plus or minus standard error of the mean (SEM). Significance was determined at p < 0.05. RESULTS: Platelets from patients with MPNs had higher surface marker expression of PS under resting conditions. A trend of higher surface marker expression of αIIbβIII and P-selectin was seen (Figure 1). The platelet bioenergetic profile in individuals with MPN demonstrated decreased basal respiration (p = 0.0008), ATP-linked respiration (p = 0.001), and basal extracellular acidification rate (ECAR) (p = 0.09) (Figure 2). Liquid chromatography-mass spectrometry-based metabolomics revealed elevated pentose phosphate pathway metabolites (pentose phosphates and 6-Phospho-D-gluconate), and diminished adenosine diphosphate (ADP) and adenosine triphosphate (ATP) pools (Figure 3). CONCLUSION: The findings from this study suggest that platelets from patients with JAK2 positive PV and ET have a procoagulant phenotype given the increased expression of PS, an anionic phospholipid known to facilitate the generation of thrombin. The decreased basal respiration, ATP-linked respiration, basal ECAR (a surrogate marker of glycolysis), and energy pools (ADP and ATP) in platelets from MPN patients suggest a hypometabolic phenotype. Additionally, it appears there is preferential shunting of glucose into the pentose phosphate pathway. Such metabolic switch in conjunction with the hypometabolic state of platelets in MPN, could potentially represent a compensatory mechanism of platelets against oxidative stress. The bioenergetic dysregulation of platelets in MPNs may precipitate a cascade of events that leads to the loss of plasma membrane integrity (increased PS exposure) therefore increasing its procoagulant potential. Higher amounts of platelet PS could underly the elevated incidence of thrombosis observed in patients with JAK2 positive ET and PV. Further studies are underway to identify the inciting factor(s) that lead to the platelet bioenergetic failure and elevated PS exposure seen in patients with MPNs. These investigations will provide significant mechanistic insight for identifying therapies aimed at preventing thrombotic events in patients with MPNs. Figure 1 Figure 1. Disclosures Nemkov: Omix Thecnologies: Other: Co-founder. D'Alessandro: Omix Thecnologies: Other: Co-founder; Rubius Therapeutics: Consultancy; Forma Therapeutics: Membership on an entity's Board of Directors or advisory committees.