Dysregulated Autophagy in Platelets from JAK2^V617F-Positive Polycythemia Vera and Essential Thrombocythemia Underlies Bioenergetic Failure As a Possible Mechanism for Thrombohemorrhagic Complications

Background: JAK2^V617F-positive polycythemia vera (PV) and essential thrombocythemia (ET) are the most common Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs). ET/PV patients have high levels of inflammation and are at increased risk of thrombosis. Platelets from patients with ET/PV are characterized by hyperreactivity (low activation threshold), contributing to the increased incidence of thrombosis in the MPNs. We previously described that platelets from PV patients accumulate dysmorphic mitochondria.

Autophagy is a housekeeping and an inducible cellular process that eliminates damaged proteins and organelles (such as mitochondria and endoplasmic reticulum), providing biosynthetic and bioenergetic substrates for the cell. Dysregulated autophagy has been described in cancer, aging, and neurodegenerative, cardiac, and muscular diseases. We hypothesized that impairment of the platelet autophagic flux in ET/PV Jak^2V617F alters platelet metabolism and function at the early and late stages of platelet activation.

Methods: Platelets from sex and age-matched healthy subjects and patients with PV were analyzed. Platelets were incubated for two hours with chloroquine (CQ) [50 and 100 µM] to inhibit autophagy. CQ blocks the late stage of the autophagy process; therefore, functional autophagy was assessed by quantifying the accumulation of autophagic-related proteins by western blot. Oxidative stress (4-hydroxynonenal protein adducts) was measured by western blot. Platelet bioenergetics was studied by the Seahorse extracellular flux analyzer. Semi-quantitative metabolomics was performed using the Vanquish UHPLC system. Clot retraction was assessed by measuring the weight of serum extruded from the clot.

Results: Platelets from healthy patients showed basal and inducible autophagy characterized by the accumulation of autophagic markers such as LC3II (p<0.0024), GABARAP, the autophagic cargo receptor p62 and the mitochondrial marker TOM20 (p<0.021). However, ET/PV platelets showed dysfunctional basal and inducible autophagy. Moreover, PV platelets and platelets treated with CQ had higher levels of ROS-damaged proteins (p<0.0001) compared to healthy platelets. Platelets from PV patients exhibited hyperreactivity by flow cytometry (PAC-1 binding, p<0.05) and under flow conditions (p<0.03). Moreover, platelets from ET/PV patients showed marked bioenergetic failure characterized by increased mitochondrial depolarization, higher intracellular calcium, depressed mitochondrial respiration, and lower levels of ADP and ATP.

Pharmacological inhibition of autophagy of platelets from healthy individuals with CQ recapitulated the bioenergetic and functional profiles of platelets from patients with ET/PV. Bioenergetically, autophagy inhibition resulted in depressed basal (p<0.03) and ATP-linked respiration (p<0.01). Functionally, pharmacological inhibition of the autophagic flux led to significantly decreased clot retraction, as observed with the platelets from patients with ET/PV.

Conclusion: Our results suggest that impairment of autophagy in platelets from ET/PV patients might exert a negative bioenergetic effect on platelet function, promoting platelet hyperreactivity and dysfunctional clot retraction, which would likely favor the formation of unstable clots. Our findings may explain why patients with Jak2^V617F ET and PV are at increased risk for both thrombosis and hemorrhage and suggest that targeting autophagy might lower this risk.

Nemkov:Omix Technologies: Other: Co-Founder of Omix Technologies Inc.; Altis Biosciences LLC: Other: Co-Founder of Altis Biosciences LLC.