Donatella Balducci

Thrombin generation and activated protein C resistance in patients with essential thrombocythemia and polycythemia vera

We used the thrombin generation assay to evaluate the hypercoagulable state according to JAK2(V617F) mutational status in essential thrombocythemia (ET) and polycythemia vera (PV) patients. Thrombin generation was determined in the presence and absence of activated protein C (APC), and APC resistance was expressed as normalized APC sensitivity ratio (nAPCsr). Tissue factor pathway inhibitor (TFPI), total and free protein S (PS), prothrombin (FII), factor V (FV), and neutrophil elastase were measured in plasma; CD11b was measured on neutrophils. Compared with normal controls, patients had a lower endogenous thrombin potential in the absence of APC but had a higher endogenous thrombin potential in the presence of APC, showing the occurrence of APC resistance. The nAPCsr increased in JAK2(V617F) carriers compared with noncarriers and was highest in JAK2(V617F) homozygous patients. FII, FV, free PS, and TFPI levels were reduced in patients, mainly in JAK2(V617F) carriers. Multiple regression analysis indicated the low free PS level as major determinant of the increased nAPCsr. Elastase was increased in patients and inversely correlated with free PS. In conclusion, these data indicate the occurrence of acquired APC resistance in ET and PV patients, probably because of a reduction in free PS levels. The APC-resistant phenotype is influenced by the JAK2(V617F) mutational load.

All trans-retinoic acid modulates the procoagulant activity of human breast cancer cells

All trans-retinoic acid (ATRA) induces apoptosis and/or differentiation in solid tumors, including breast cancer, and has become a therapeutic tool in this disease. In human promyelocytic leukemia ATRA reduces the expression of cellular procoagulant activities (PCA), i.e. tissue factor (TF) and cancer procoagulant (CP). There are no studies on the effects of ATRA on the PCA of solid tumors, i.e. breast cancer cells. We analyzed different human breast cancer cell lines in order to: 1. characterize the expression of TF and CP; 2. evaluate whether these activities are affected by ATRA; and 3. verify whether a reduction in tumor cell procoagulants may occur in association to apoptosis and growth inhibition induced by ATRA. Two estrogen receptor positive (ER-positive; i.e. MCF7 and ZR75.1) and one estrogen receptor negative (ER-negative; i.e. MDA.MB.231) cell lines were included into the study. The results show that ATRA affected TF in a dose-dependent fashion only in ER-positive cell lines. In particular, at 1 uM ATRA, TF significantly (p < 0.05) decreased by 57%, 44% in MCF7, ZR75.1 cells, respectively. Differently the results show that ATRA dose-dependently affected CP expression in all three cell lines. Specifically, at 1 uM ATRA, CP significantly decreased by 44%, 50% and 25% in MCF7, ZR75.1, and MDA.MB.231. Only in ER-positive cell lines, there was a dose-dependent inhibition of cell growth that became statistically significant at 1 uM ATRA, which was associated to a slight but significant increase in the percentage of apoptotic cells. In conclusion, this study demonstrates for the first time that ATRA downregulates the expression of TF and CP in breast cancer cells. Due to the pivotal role of coagulation activation in tumor progression, the capacity of ATRA to affect also tumor procoagulants, in parallel to cell apoptosis, open new perspectives in tumor therapy.