Circulating Nucleosomes Reflect Disease Activity in Patients with Thrombotic Microangiopathies.

Thrombotic microangiopathies (TMAs) including thrombotic thrombocytopenic purpura (TTP) associated with acquired or congenital severe ADAMTS13 deficiency, typical diarrhea-associated (D+) and atypical (D-) hemolytic uremic syndrome (HUS), and other TMAs associated with disseminated neoplasia, some drugs, pregnancy and several infectious or autoimmune conditions are life-threatening diseases characterized by microvascular thrombosis with organ dysfunction, consumptive thrombocytopenia and hemolytic anemia with red blood cell fragmentation. Severe autoantibody-induced ADAMTS13 deficiency is found in about 60% of patients diagnosed with acquired TTP and severe constitutional ADAMTS13 deficiency due to ADAMTS13 mutations is found in almost all patients with hereditary TTP. However, some patients with severe acquired or hereditary ADAMTS13 deficiency may be asymptomatic for prolonged time intervals and some triggering factor(s) may be needed to bring about acute disease. Extracellular exposure of DNA and histones is a recently discovered mechanism linking inflammation with thrombosis. Chromatin fibers released from activated neutrophils (Neutrophil Extracellular Traps, NETS) bind platelets, erythrocytes and DNA/histone complexes stimulate platelet aggregation and coagulation. We investigated stored plasma samples from patients with various forms of TMA, both during acute disease and during remission, for circulating nucleosomes, i.e. subunits of chromatin composed of DNA wrapped around a histone core.

First, 29 patients with acute TMA (6 acquired TTP with severe ADAMTS13 deficiency, 4 D+ HUS, 8 neoplasia-associated TMAs and 11 TMAs of undetermined etiology) were investigated and compared to 10 healthy controls. Second, serial samples from 8 patients with TTP and acquired ADAMTS13 deficiency obtained during acute disease, during plasma exchange (PE) treatment and during remission were analyzed as were samples from 3 hereditary TTP patients during acute disease and during remission.

Plasma DNA was quantified, nucleosomes were measured using an antibody against DNA complexed with histones H2A/H2B. Plasma lactate dehydrogenase and myeloperoxidase were assayed using commercial kits.

Significantly elevated levels of DNA and histones organized in nucleosomes were found in patients with acute TMAs. Increase in nucleosome levels was paralleled by elevation of plasma myeloperoxidase and calgranulin pointing to neutrophils as the origin of nucleosomes. All patients achieving clinical remission from acute acquired TTP showed normalized levels of nucleosomes indistinguishable from those in healthy controls. Transient disease flare-ups with worsening platelet counts in acute acquired TTP patients subjected to daily PE therapy were often associated with a transient increase of the nucleosome levels. One patient who died after 10 days of PE had extremely high nucleosome levels prior to death. Three patients with hereditary severe ADAMTS13 deficiency had variably elevated nucleosome levels during active disease and normal levels in remission maintained by regular plasma infusion (n=2) or mildly increased levels in remission without maintenance plasma therapy (n=1).

Circulating nucleosomes may reflect the degree of systemic inflammation and seem – at least in some cases - to reflect the second hit precipitating acute disease in patients at risk for acute TMA, such as those with acquired or hereditary severe ADAMTS13 deficiency.

Kremer Hovinga:Baxter Bioscience: Consultancy. Lämmle:Baxter Bioscience: Consultancy.