thromboplastin and Carcinoma--Small-Cell

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.

Topics: Adult; Aged; Aged, 80 and over; Blood Coagulation Tests; Carcinoma, Small Cell; Centrifugation; Cysteine Endopeptidases; DNA; Extracellular Vesicles; Female; Humans; Lung Neoplasms; Male; Microscopy, Immunoelectron; Middle Aged; Nanoparticles; Neoplasm Proteins; Pulmonary Embolism; Risk Factors; Thrombin; Thrombophilia; Thromboplastin; Venous Thromboembolism

Small cell lung cancer (SCLC) is a very malignant tumor known to grow aggressively and to metastasize early. It is well established that metastasis generally involves both tumor cell adhesion and proteolytic degradation of the extracellular matrix. However, SCLC cells cultured in vitro, such as the classic SCLC cell line NCI-H69, grow in floating aggregates and express only negligible proteolytic activity. In this report, we show that NCI-H69 cells can be selected for adherent growth. In contrast to parental suspension cells, the adherent cells were found to express tissue factor as well as gelatinolytic activity, attributable to matrix metalloproteinases 2 and 9. Such a switch of tumor cell characteristics, if it could occur in SCLC patients, might add to the understanding of the steps involved in the spreading of this highly metastatic type of lung cancer.

Topics: Carcinoma, Small Cell; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Matrix Metalloproteinases; Thromboplastin; Tumor Cells, Cultured

Several tumour-derived factors have recently been identified which induce tissue factor (TF) expression in endothelial cells in vitro. However, there is only limited evidence that endothelial cells lining tumour blood vessels express elevated procoagulant activity (PCA) in vivo. We have investigated the effects of human breast and small cell lung cancer cell lines on the PCA of human micro- and macrovessel endothelial cell monolayers using a one-stage clotting assay, as well as detection of TF mRNA by RT-PCR. Only conditioned medium from the MDA-MB-231 breast adenocarcinoma cell line produced a consistent although transient increase in endothelial cell surface PCA, which was maximal by 6-9 hr. TF mRNA was detectable in the endothelial cells after 1 hr incubation with MDA-MB-231-conditioned medium and subsequently fell below detectable levels. Following 24 hr stimulation, nearly half the endothelial cell PCA was due to the presence of TF-containing membrane vesicles shed by the MDA-MB-231 cells. Consistent with these findings, the MDA-MB-231 cell line expressed high levels of cell surface-associated TF activity. Co-culture of MDA-MB-231 and endothelial cells for up to 5 days increased (approx. 118-fold) PCA associated with endothelial cell monolayers, due mainly to sequestration of shed tumour cell vesicles. Our results suggest that induction of TF de novo is not a common feature in the supporting endothelium of these tumour types.

Topics: Adenocarcinoma; Biological Factors; Blood Coagulation Tests; Breast; Breast Neoplasms; Capillaries; Carcinoma, Small Cell; Cells, Cultured; Coculture Techniques; Culture Media, Conditioned; Endothelium, Vascular; Female; Humans; Lung Neoplasms; Polymerase Chain Reaction; Thromboplastin; Tumor Cells, Cultured; Umbilical Veins

We have studied the effects of two human pancreatic cancer and two human small cell lung cancer cell lines on clotting and platelet aggregation. Both pancreatic lines markedly shortened recalcification times and induced platelet aggregation. The lung cancer lines produced little shortening of recalcification times and no platelet aggregation. The clotting and aggregation activities of the pancreatic lines were further characterized. Recalcification times following the addition of cancer cell line material to plasmas deficient in factors VII and X were markedly prolonged, suggesting that the activity is due to tissue factor. Hirudin, an inhibitor of thrombin from the saliva of leeches, and rabbit polyclonal immunoglobulin G anti-bovine brain tissue factor inhibited both procoagulant and aggregation activities. Apyrase (an enzyme degrading ADP), diisopropylfluorophosphate (a serine protease inhibitor) and L-trans-epoxysuccinylleucylamido(4-guanidino)butane (a cysteine protease inhibitor) failed to inhibit these activities. Increasing concentrations of heparin inhibited platelet aggregation. Subcellular fractionation studies showed these activities to be localized to the plasma membrane. The association between mucin and the acceleration of clotting has been well described. The absence of mucin in electron micrographs of these pancreatic whole cells, membrane fractions, and shed microvesicles, as well as the failure of chaotropic agents (i.e., agents stripping material extrinsic to the cell membrane such as mucin) to abrogate this activity support these activities being intrinsic to the plasma membrane. These data strongly suggest that these activities are due to tissue factor which appears to be released as microvesicles in vitro. The release of tissue factor via microvesicles in vivo is one possible mechanism for the coagulopathy sometimes seen in patients with pancreatic carcinoma.

Topics: Adenocarcinoma; Blood Coagulation; Calcium; Carcinoma, Small Cell; Cell Membrane; Humans; Lung Neoplasms; Microscopy, Electron; Pancreatic Ducts; Pancreatic Neoplasms; Platelet Aggregation; Thromboplastin; Tumor Cells, Cultured

Fibrin was detected by specific immunofluorescence in tissue obtained from five of six cases of small cell carcinoma of the lung. Dense specific fluorescence was observed in the connective tissue stroma surrounding metastatic tumor nodules and frequently in the scant extracellular stroma surrounding individual viable tumor cells and small tumor cell clusters. When observed by electron microscopy, the fibrin hugged tumor cell plasma membranes and, in some areas, seemed to envelop the cells. Fluorescent staining of tumor cells, but not the stroma, was observed with an antibody to tissue factor. These findings suggest that local activation of coagulation occurs in small cell carcinoma of the lung. Deposited fibrin may contribute to the growth and spread of this particular type of cancer.

Topics: Antigens; Carcinoma, Small Cell; Fibrin; Fluorescent Antibody Technique; Humans; Lung Neoplasms; Microscopy, Electron; Thromboplastin