Increased activity of procoagulant factors in patients with small cell lung cancer

Abstract

Small cell lung cancer (SCLC) patients have augmented risk of developing venous thromboembolism, but the mechanisms triggering this burden on the coagulation system remain to be understood. Recently, cell-derived microparticles carrying procoagulant phospholipids (PPL) and tissue factor (TF) in their membrane have attracted attention as possible contributors to the thrombogenic processes in cancers. The aims of this study were to assess the coagulation activity of platelet-poor plasma from 38 SCLC patients and to provide a detailed procoagulant profiling of small and large extracellular vesicles (EVs) isolated from these patients at the time of diagnosis, during and after treatment compared to 20 healthy controls. Hypercoagulability testing was performed by thrombin generation (TG), procoagulant phospholipid (PPL), TF activity, Protein C, FVIII activity and cell-free deoxyribonucleic acid (cfDNA), a surrogate measure for neutrophil extracellular traps (NETs). Our results revealed a coagulation activity that is significantly increased in the plasma of SCLC patients when compared to age-related healthy controls, but no substantial changes in coagulation activity during treatment and at follow-up. Although EVs in the patients revealed an increased PPL and TF activity compared with the controls, the TG profiles of EVs added to a standard plasma were similar for patients and controls. Finally, we found no differences in the coagulation profile of patients who developed VTE to those who did not, i.e. the tests could not predict VTE. In conclusion, we found that SCLC patients display an overall increased coagulation activity at time of diagnosis and during the disease, which may contribute to their higher risk of VTE.

Fig 1. EV confirmation and validation.

Immunoelectron microscopy (IEM) and Western blot analysis of extracellular vesicle marker CD9 performed on a pool of isolated vesicles from all donors. A) 20K pel CD9 positive vesicles. B) Both CD9 positive and negative vesicles isolated for the 100K pel. C) 100K pel CD9 positive vesicle. D) Western blot analysis against CD9 for the 20K and 100K EV pellets from healthy controls (HC) and small cell lung cancer patients (SCLC).

Fig 2. Procoagulant profiling of EVs isolated from healthy age-related controls and SCLC patients at baseline, during (prior to cycle 3) and after treatment (follow-up).

A) Thrombin generation curves from standard plasma (SP) and SP-containing vesicles isolated from patients and controls. B) EV-associated activity of procoagulant phospholipids (PPL) depicted as a clotting time. In both A) and B) SP represents the coagulation activity of the standard pooled plasma into which the isolated vesicles have been added. C) difference in tissue factor (TF) activity associated with EVs D) concentration and size distribution of isolated vesicles in both pellet types i.e. 20K pel and 100K pel.

Fig 3. Coagulation profile of SCLC patients who developed a VTE during the study period compared to all other patients.

Data is presented as mean ± SD where applicable. Patients 3 and 4 did not donate all blood samples and for P1, thrombin generation during treatment could not be assessed due to treatment with anticoagulants.