Wojciech P. Mielicki

Activation of blood coagulation and the activity of cancer procoagulant (EC 3.4.22.26) in breast cancer patients

The activity of cancer procoagulant (CP), prothrombin time (PT), activated partial thromboplastin time (APTT), the concentration of thrombin-antithrombin complexes (TAT) and the concentration of fibrinogen were analysed in blood of breast cancer patients scheduled for surgery. The serum level of CP activity was dependent on the stage of the disease. The CP activity was increased in 72% of patients with an early stage of cancer and in only 20% of patients with an advanced stage of the disease when compared to the baseline level for non-cancer controls. In all patients PT remained at normal levels (80-120%). There was no significant change in APTT (27-39 s) in early stage cancer patients. Only one patient with advanced cancer had APTT shortened to 23 s. Also one advanced stage patient had significantly elevated level of TAT (14.96 microg/l); in all other patients the concentration of TAT remained at normal levels (1-4.1 microg/l). Forty-four percent of early stage cancer patients and 22% of advanced cancer patients had an elevated level of fibrinogen (Fg) ( > 350 mg%). However, there was no correlation between the level of Fg and the CP activity (P > 0.05). The data suggest that: (1) serum CP activity increases at the early stage of breast cancer and decreases down to the normal level in the advanced stage of the disease; (2) there is no evidence of blood clotting activation in the early stage breast cancer patients; and (3) CP does not facilitate the activation of coagulation in the breast cancer patients or the level of such activation is below the sensitivity of assays used in the experiment.

Cancer procoagulant and blood platelet activation.

The effects of cancer procoagulant (CP), cysteine protease (EC 3.4.22.26), on the pig blood platelet secretory process and platelet aggregation have been studied. The response of platelets to CP was compared with the response of these cells to thrombin. The obtained results show that blood platelets treated with CP (0.5, 1, 2.5, and 5 microg/ml, 2-30 min, 37 degrees C) released adenine nucleotides (P < 0.05) and proteins (P < 0.05). The secretion of compounds from blood platelets after incubation with CP does not correlate with the release of platelet lactic dehydrogenase activity (marker of cell lysis) into the extracellular medium. In comparison with thrombin action, CP stimulates secretory process to a smaller extent than thrombin alone. In the presence of CP, the thrombin action is suppressed (P < 0.05). We noticed that CP does not induce platelet aggregation.

Impact of lithium alone or in combination with haloperidol on oxidative stress parameters and cell viability in SH-SY5Y cell culture.

Background It has been reported that lithium may inhibit lipid peroxidation and protein oxidation. Lithium salts also appear to stimulate cell proliferation, increase neurogenesis, and delay cell death. Oxidative stress and neurodegeneration may play an important role in the pathophysiology of bipolar disorder and the disease course thereof. The aim of this research is to estimate the influence of lithium (alone and in combination with haloperidol) on the parameters of oxidative stress and viability of SH-SY5Y cell lines in neutral and pro-oxidative conditions. Methods The evaluated oxidative stress parameter was lipid peroxidation. The viability of the cell lines was measured utilising the MTT test. Results In neutral conditions, higher levels of thiobarbituric acid reactive substances were observed in those samples which contained both haloperidol and lithium than in other samples. However, these differences were not statistically significant. Cell viability was significantly higher in therapeutic lithium samples than in the controls; samples of haloperidol alone as well as those of haloperidol with lithium did not differ from controls. Conclusions The results of our study may indicate that lithium possess neuroprotective properties that may be partly due to antioxidative effects. The combination of lithium and haloperidol may generate increased oxidative stress.

Cancer procoagulant (CP) analysis in human WM 115 malignant melanoma cells in vitro

Neoplastic cells produce procoagulants responsible for hypercoagulation states frequently observed in cancer patients. It is accepted that two major procoagulants from malignant tissue are tissue factor (TF) and a direct activator of coagulation factor X called cancer procoagulant (CP). Direct factor X-activating activity of cultured human malignant melanoma WM 115 cells has been analyzed in the cell extracts, whole cells and in the medium after the cell culture. The factor X-activating activity was detected in the malignant cell lysates but not in the cultured medium or intact malignant cells. The lysates contained no TF as determined by Western blotting and enzyme-linked immunosorbent assay (ELISA) using anti-TF monoclonal antibody. The enzymatic characteristics of the activity was typical for CP. The results suggest that cancer procoagulant is an intracellular protein.

Direct analysis reveals an absence of γ-carboxyglutamic acid in cancer procoagulant from human tissues

Additional carboxylation of glutamic acid by vitamin K-dependent γ-carboxylase is a common posttranslational modification of many proteins, including some of blood clotting factors. Vitamin K-antagonists, such as warfarin, are often included in the therapy of malignant disease, decreasing the blood coagulation potential. Cancer procoagulant, a direct blood coagulation factor X activator from malignant tissue, is considered as a vitamin K-dependent protein, so it could serve as one of possible targets for the therapy with warfarin. However, there is still no experimental data demonstrating directly the presence of γ-carboxyglutamic acid (Gla) in a cancer procoagulant molecule. The presence of Gla in cancer procoagulant isolated from human amnion–chorion membranes and from human malignant melanoma WM 115 cell line was analyzed directly, using specific anti-Gla monoclonal antibodies. There was no detectable amount of Gla in cancer procoagulant isolated from fetal or malignant tissue. Cancer procoagulant from human tissues does not contain Gla-rich domain. The finding indicates that cancer procoagulant is rather a poor target for warfarin therapy of malignant disease.

Impact of lithium alone or in combination with haloperidol on selected oxidative stress parameters in human plasma in vitro

Objectives: Lithium may inhibit lipid peroxidation (LP) and protein oxidation, stimulate cell proliferation, increase neurogenesis, and delay cell death. Oxidative stress (OxS) is a state of imbalance between oxidative processes and antioxidant defenses, which may play an important role in the pathophysiology and disease course of bipolar disorder (BD). The aim of this study was to estimate the influence of lithium, administered alone or in combination with haloperidol, on selected OxS parameters in human plasma in vitro. Methods: The OxS parameters evaluated were thiobarbituric acid reactive substances (TBARS) and total antioxidant capacity (TAC). Plasma samples from healthy volunteers were incubated with drug concentrations used in psychiatry. Results: Incubation of plasma with lithium or haloperidol alone did not produce statistically significant changes of TBARS levels in comparison with control samples. However, significantly higher TBARS levels were observed in samples incubated with haloperidol plus lithium compared to control, haloperidol, or lithium samples. The TAC value did not differ between samples. Conclusions: Lithium does not influence OxS parameters in human plasma in vitro during short-term observation when applied at concentrations used in psychiatry. However, lithium increased the TBARS level in the samples when given in combination with haloperidol, which may be one of the mechanisms behind the neurotoxicity associated with combined lithium and haloperidol administration.

Effect of immunisation with cancer procoagulant on the growth of Walker 256 carcinosarcoma cells in rats.

AbstractThe effect of preimmunisation with cancer procoagulant (CP) on the growth of Walker 256 carcinosarcoma cells in Wistar Lew/Han/IMP rats in vivo has been analysed. One group of rats was immunised with CP purified from human amnion-chorion membranes. The rest of the animals (control groups) were injected with 0.9% NaCl in Freund’s adjuvant or were not immunised at all. When the presence of polyclonal anti-CP antibody was detected in CP-immunised rats’ serum, all (CP-immunised and control) animals were injected i.p. with 4.8x105 Walker 256 cells per rat. After 5 days ascitic fluid was collected and viable cells were counted. The complete lack of viable Walker 256 carcinosarcoma cells in 24% of the CP-immunised rats was observed. However, the viable neoplastic cells were present in all control (NaCl-injected and nonimmunised) animals. It seems possible that CP plays an important role in tumour progression, so immunisation with CP can reduce the risk of development of malignant disease.