Chemotherapy-induced thrombin generation via procoagulant endothelial microparticles is independent of tissue factor activity.
BACKGROUND
Cisplatin-based chemotherapy predisposes cancer patients to thromboembolic events.


OBJECTIVE
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 microm) 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 at 20 microm 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 microm 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.
Lechner
D
D
Department of Medicine I, Medical University of Vienna, Waehringer Guertel 18-20, Vienna, Austria.
Kollars
M
M
Gleiss
A
A
Kyrle
P A
PA
Weltermann
A
A
D000970:Antineoplastic Agents / Q000633:toxicity*; D017209:Apoptosis / Q000187:drug effects; D001777:Blood Coagulation / Q000187:drug effects*; D053148:Caspase 3 / Q000378:metabolism; D053179:Caspase 7 / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002945:Cisplatin / Q000633:toxicity*; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D015942:Factor VIIa / Q000378:metabolism; D006801:Humans; D010743:Phospholipids / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism*; D013925:Thromboplastin / Q000378:metabolism*; D013997:Time Factors; D022161:Transport Vesicles / Q000187:drug effects* / Q000378:metabolism
D000970:Antineoplastic Agents; D010743:Phospholipids; D013925:Thromboplastin; D015942:Factor VIIa; D013917:Thrombin; C505520:CASP7 protein, human; D053148:Caspase 3; D053179:Caspase 7; D002945:Cisplatin