Chemotherapy-induced thrombin generation via procoagulant endothelial microparticles is independent of tissue factor activity

Background: Cisplatin-based chemotherapy predisposes cancer patients to thromboembolic events.Objectives: To investigate whether endothelial damage, via formation of procoagulant endothelial microparticles (EMPs), contributes to cisplatin-related hypercoagulability.Methods: Cell viability and caspase-3/7 activities were assessed in two endothelial cell (EC) lines [human umbilical vein ECs (HUVECs) and human pulmonary microvascular ECs (HMVEC-Ls)] after exposure to cisplatin (1, 2.5, 5, 10 and 20 mu M) for up to 120 h. Counts and procoagulant activity of EMPs were measured by flow cytometry and a thrombin generation assay, respectively. Tissue factor (TF) antigen and TF-dependent procoagulant activity of EMP were determined by enzyme-linked immunosorbent assay and a novel functional assay.Results: By inducing apoptosis, cisplatin dose- and time-dependently decreased the viability of confluent HUVECs and HMVEC-Ls. Progression of EC death was accompanied by an increased release of EMPs (relative increase a mu M cisplatin for 48 h vs. control: HUVECs 6.5-fold, P < 0.001; HMVEC-Ls 18.4-fold, P < 0.001). EMPs were highly procoagulant (relative increase at 20 mu M cisplatin for 48 h vs. control: HUVECs 2.5-fold, P < 0.001; HMVEC-Ls 5.9-fold, P < 0.001). EMP-driven thrombin generation, however, was not dependent on TF: TF expression and TF procoagulant activity levels on microparticles were only marginal and EMP-associated thrombin generation remained unchanged when the extrinsic pathway was blocked by omission of factor VIIa and/or incubation with an anti-human TF antibody. In contrast, blocking of phospholipids by annexin V markedly diminished EMP-associated procoagulant activity.Conclusions: In vitro, cisplatin induced the release of EMPs that showed TF-independent procoagulant activity.