Activation of Inflammatory and Coagulation Pathways in Patients with Graft-Versus-Host Disease

Graft-versus-host disease (GvHD) remains a major complication in patients with hematological malignancies undergoing hematopoietic stem cell transplantation, driven by interconnected neutrophil activation, endothelial dysfunction, inflammation, and dysregulated coagulation. However, characterization of these pathways in GvHD patients remains limited. We used ELISA and flow cytometry to quantify biomarkers of neutrophil extracellular traps (NETs), cytokines, endothelial activation, fibrinolysis, coagulation, and immune cell subsets in peripheral blood of GvHD patients, and Western blot to assess NF-κB, STAT3, and p38 MAPK signaling. GvHD patients showed a thromboinflammatory profile, with elevated NET markers (MPO, cell-free DNA), increased cytokines (IL-1β, IL-8, MCP-1, TNF-α), and amplified platelet activation (P-selectin). Coagulation and fibrinolytic activity were enhanced, reflected by increased tissue factor (TF) and urokinase plasminogen activator (uPA), alongside higher frequencies of TF-expressing monocytes and platelet-monocyte aggregates. In vitro, TNF-α activated NF-κB, STAT3, and p38 MAPK signaling and increased ICAM-1- and VCAM-1-positive mononuclear cells, indicating a proinflammatory, adhesive phenotype, while transendothelial migration and nitric oxide levels were reduced. Leukemia patients with GvHD thus show a coordinated thromboinflammatory response involving NET activation, platelet-monocyte interactions, inflammatory signaling, endothelial dysfunction, and coagulation-fibrinolytic dysregulation, providing mechanistic insight into GvHD pathophysiology and highlighting circulating biomarkers as candidates for early detection, monitoring, and therapeutic intervention.