Characterization of the thrombin generation potential of leukemic and solid tumor cells by calibrated automated thrombography

Abstract

Background: Thrombin, the final enzyme of blood coagulation, is a multifunctional serine protease also involved in the progression of cancer. Tumor cells may activate blood coagulation proteases through the expression of procoagulant activities. However, specific information about the thrombin generation potential of malignant tissues is lacking. In this study we applied a single global coagulation test, the calibrated automated thrombogram assay, to characterize the specific procoagulant phenotypes of different tumor cells.

Design and methods: Malignant hematologic cells (i.e. NB4, HEL, and K562) or solid tumor cells (i.e. MCF-7 breast cancer and H69 small cell lung cells) were selected for the study. The calibrated automated thrombo-gram assay was performed in normal plasma and in plasma samples selectively deficient in factor VII, XII, IX or X, in the absence or presence of a specific anti-tissue factor antibody. Furthermore, cell tissue factor levels were characterized by measuring antigen, activity and mRNA expression.

Results: In normal plasma, NB4 induced the highest thrombin generation, followed by MCF-7, H69, HEL, and K562 cells. The anti-tissue factor antibody, as well as deficiencies of factors VII, IX and XII affected the thrombin generation potential of malignant cells to different degrees, allowing differentiation of the two different pathways of blood clotting activation - by tissue factor or contact activation. The thrombin generation capacity of NB4 and MCF-7 cells was tissue factor-dependent, as it was highly sensitive to inhibition by anti-tissue factor antibody and factor VII deficiency, while the thrombin generation capacity of H69, HEL and K562 was contact activation-dependent, as no thrombin was generated by these cells in factor XII-deficient plasma.

Conclusions: This study demonstrates that the calibrated automated thrombogram assay is capable of quantifying, characterizing, and comparing the thrombin generation capacity of different tumor cells. This provides a useful tool for understanding the key factors determining the global pro-coagulant profile of tumors, which is important for addressing specific targeted therapy for the prevention of thrombosis and for cancer.

Figure 1.

Expression of TF by the different tumor cell lines. Cells were collected after 24 h of seeding, washed and lysed in the appropriate buffer for the different TF assays, as described in the Design and Methods section. (A) TF activity measured by a chromogenic assay. (B) TF antigen measured by an ELISA, (C) TF mRNA levels expressed as a percentage of the ratio between GAPDH and TF cDNA measured in PCR experiments.

Figure 2.

Influence of increasing cell numbers on thrombin generation parameters. Thrombin generation in platelet-free plasma was induced by lysates from NB4 (panel A) and H69 (panel B) cell lines. Three different concentrations (i.e. 1, 2, 3 ×10⁶ cells/mL) of cells were tested and compared to 1 and 5 pM TF standard preparations. Results are the mean±SD of three experiments performed in triplicate.

Figure 3.

Thrombin generation curves. The graph shows the thrombin generation curves in platelet-free plasma induced by the different tumor cell lines or buffer (phosphate-buffered saline, dashed line).

Figure 4.

Effect of anti-TF antibody on thrombin generation in platelet-free plasma (PFP). Cell lysates were preincubated with 0.09 mg/mL anti-TF antibody or negative antibody and then tested for thrombin generation in PFP. Inhibition of TF determined a more pronounced reduction of thrombin generation in NB4 and MCF-7 cells than in HEL, K562 and H69 cells. Results represent the mean ± SD of three experiments performed in triplicate. *P<0.05 vs. cotrol antibody.

Figure 5.

Thrombin generation in platelet-free plasma and in FVII-deficient plasma. FVII depletion significantly reduced the thrombin generation capacity of MCF-7 and NB4 cell lines. H69, HEL and K562 cell lines were poorly affected by FVII depletion. Results represent the mean±SD of three experiments performed in triplicate. *P<0.05 vs. PFP.

Figure 6.

Thrombin generation in FXII- and FIX-deficient plasma. All the cell lines were tested in both FXII- and FIX-deficient plasma. Only MCF-7 and NB4 cells retained the capacity to induce thrombin generation in FXII-deficient plasma (A). In FIX-deficient plasma only the NB4 cell line could generate thrombin (B).