ROS-Driven Structural and Functional Fibrinogen Modifications Are Reverted By JAK Inhibition in Myeloproliferative Neoplasms

Abstract

Myeloproliferative neoplasms (MPNs) are bone marrow diseases characterized by excess clonal hematopoiesis resulting in elevated peripheral blood counts. Subtypes include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). They share clinical features (e.g., splenomegaly, thrombotic complications, risk of leukemic transformation), and a common molecular basis: the dysregulation of the JAK-STAT pathway, which confers a hypersensitivity of hematopoietic stem cells (HSC) to the action of growth factors and cytokines, and, as a result, an overproduction of blood cells. Genetic analysis of patients with MPNs revealed that >95% of patients with PV and about 50% of patients with ET or PMF carry an activating point mutation in the JAK2 gene (JAK2-V617F). Despite clinical evidence indicating a higher susceptibility of MPN patients to develop thrombotic disease, the underlying pathogenetic mechanisms are not fully understood. It appears that thrombosis is the result of a severe inflammatory response associated to remarkable oxidative stress in the presence of endothelial dysfunction and a procoagulant environment. Hypercoagulability is a consequence of disrupted hemostasis and fibrinolysis due to damage of the endothelium and defective fibrinolysis. Evidence suggests that overproduction of ROS and a deprived antioxidant system play a major role in the pathogenesis of MPN, as well as in the resistance to tyrosine-kinase inhibitor therapy. Moreover, systemic inflammation is thought to be the main trigger of thrombosis and seems to be mainly mediated by pro-inflammatory cytokines along with endothelial cell dysfunction. Recent studies have provided evidence that fibrinogen plays a multifaceted role in inflammatory responses and autoimmunity. The ability of fibrinogen to participate in the inflammatory response depends on its specific interaction with integrins, the leukocyte cell surface adhesion receptors expressed on neutrophils, monocytes, macrophages, and several subsets of lymphocytes. In MPN patients, endothelial cell dysfunction, increased reactive oxygen species (ROS) production, and proinflammatory cytokines have been reported, together with impaired fibrinolysis. We also investigated the potential role of oxidative stress and inflammation in the promotion of thrombosis. In order to study the mechanisms of inflammation-induced thrombosis in MPN patients, the aim of the present study was to investigate the role of oxidation-induced structural and functional fibrinogen modifications in MPN patients. The eventual counteracting effects of ruxolitinib, a first-in-class JAK inhibitor, were also assessed. 40 MPN patients and 40 age- and sex-matched healthy control subjects were included in the study. We measured specific oxidative stress markers andtheir modifications in plasma, and functional and structural parameters in purified fibrinogen fractions. Marked signs of oxidative stress in plasma (P<0.01 versus controls) and, correspondingly, an increased extent of fibrinogen oxidation (P<0.01 versus controls) were observed in patients. Furthermore, fibrinogen fractions purified from MPN patients exhibited significantly reduced clotting ability and decreased susceptibility to plasmin-induced lysis (P<0.01 versus controls). Alterations in fibrinogen secondary structure, as suggested by circular dichroism spectroscopy, and in fibrin clot architecture were also identified. Importantly, clot structure obtained by stimulated emission depletion (STED) super-resolution microscopy indicated modifications in fiber diameter and clot porosity in MPN patients (P<0.01 versus controls). In patient samples, fibrinogen clot analysis and oxidative-dependent changes reveal novel structural and functional fibrinogen modifications which may favor thrombotic complications in MPNs. In patients receiving ruxolitinib, a significant improvement in redox status was observed, accompanied by a significant improvement in fibrinogen structural and functional features, suggesting a potential impactof ruxolitinib on cardiovascular prevention in MPNs.