[Role of protein kinases of human red cell membrane in deformability and aggregation changes].

The proteomic analysis has showed that red cell membrane contains several kinases and phosphatases. Therefore the aim of this study was to investigate the role of protein kinases of human red cell membrane in deformability and aggregation changes. Exposure of red blood cells (RBCs) to some chemical compounds led to change in the RBC microrheological properties. When forskolin (10 microM), an adenylyl cyclase (AC) and a protein kinase A (PKA) stimulator was added to RBC suspension, the RBC deformability (RBCD) was increased by 20% (p < 0.05). Somewhat more significant deformability rise appeared after RBC incubation with dB-AMP (by 26%; p < 0.01). Red cell aggregation (RBCA) was significantly decreased under these conditions (p < 0.01). Markedly less changes of deformability was found after RBC incubation with protein kinase stimulator C (PKC)--phorbol 12-myristate 13-acetate (PMA). This drug reduced red cell aggregation only slightly. It was inhibited red cell tyrosine phosphotase activity by N-vanadat and was obtained a significant RBCD rise and RBCA lowering. The similar effect was found when cells were incubated with cisplatin as a tyrosine protein kinase (TPK) activator. It is important to note that a selective TPK inhibitor--lavendustin eliminated the above mention effects. On the whole the total data clearly show that the red cell aggregation and deformation changes were connected with an activation of the different intracellular signaling pathways.

Red blood cell aggregation changes are depended on its initial value: Effect of long-term drug treatment and short-term cell incubation with drug.

This study was designed to investigate whether the red cell aggregation depends on its initial level under drug therapy or cell incubation with bioactive chemical compounds. Sixty six subjects were enrolled onto this study, and sub-divided into two groups: the first group of patients (n = 36) with cerebral atherosclerosis received pentoxifylline therapy (400 mg, thrice daily) for 4 weeks. The patients of the second group were initially treated with Epoetin beta 10,000 units subcutaneously thrice a week, for 4 weeks. The second group - adult anemic patients (n = 30) with the confirmed diagnosis of solid cancer (Hb < 100 g/L). After 4 weeks of pentoxifylline treatment the red cell aggregation increased (p < 0.05) in the patients with initially low RBCA. On the other hand in the patients with initially high RBCA treatment with pentoxifylline reduced it markedly (p < 0.01). In vitro experiments with pentoxifylline RBC incubation resulted in a decrease of the initially high RBCA by 47% (p < 0.01), whereas in the sub-group with initially low RBCA it increased. It was observed that after 4 weeks of epoetin-beta treatment 75% the anemic patients with initially high RBCA had an aggregation lowering. The drop of aggregation was about 34% (p < 0.01). At the same time 25% of the study patients had a significant RBCA increase (p < 0.05) after treatment. The initially low red cell aggregation after incubation with epoetin-beta was markedly increased by 122% (p < 0.05). On the contrary initially high RBCA was reduced by 47% (p < 0.05). When forskolin (10 µM) was added to the RBC suspensions the RBCA was increased in sub-group of subjects with initially low aggregation and it was decreased in sub-group with initially high one. The similar RBCA changes were observed when RBC suspensions were incubated with vinpocetine, calcium ionophore (A23187), Phorbol 12-myristate 13-acetate (PMA) as a protein kinase C (PKC) stimulator. A major finding of this study is that the red cell aggregation effects of some drugs depend markedly on the initial, pre-treatment aggregation status of the patients. These results demonstrate that the different red blood cell aggregation responses to the biological stimuli depend strongly on the initial, pre-treatment status of the subject and the most probably it is connected with the crosstalk between the adenylyl cyclase signaling pathway and Ca2+ regulatory mechanism.

[Techniques for experimental and clinical studies of erythrocyte deformability].

Deformability is an important property of red blood cells and ensures the efficiency of their transport in the microcirculatory vascular system, as well as tissue oxygenation. A study of erythrocyte deformability uses different methods: filtration through about 5.0-microm pores, laser difractometry techniques and a focus of attention has recently been on a procedure for measuring the deformability of erythrocytes in a flow microchamber. The authors have developed a procedure for the recording of erythrocyte deformability and for the computer-aided treatment of an obtained image to have an accurate quantitative assessment of this important microrheological property of the cells. Comparison of the suspension viscosity of erythrocytes at high shift rates and the erythrocyte lengthening index (ELI) in the flow microchamber yielded a high correlation (p = -0.917; p < 0.01). That of ELI in the fractions of young and old cells also supported the high sensitivity of the procedure. Analysis of changes in the degree of erythrocyte deformability (in accordance with ELI), by applying three values of shift strain, revealed an almost linear relationship of the lengthening of fixed cells to the applied shift pressure. The known rheomodulator pentoxiphylline caused a significant erythrocyte deformability increase that was recorded by both methods (in the flow microchamber and at viscosimetry of erythrocyte suspensions with the hematocrit being equal to 40%). Thus, findings suggest that the method for the recording of erythrocyte deformability in the flow microchamber with the computer-aided analysis of images allows one to adequately measure this property of red blood cells.

Comparative efficiency and hemorheological consequences of hemotransfusion and epoetin therapy in anemic cancer patients.

The aim of our study was to compare hemorheological consequences of hemotransfusion and recombinant human erythropoetin treatment in anemic cancer patients. Forty anemic patients with solid nonmyeloid malignancies were enrolled in this prospective, open-label study. Both prior to and following treatment (epoetin beta, 10,000 units subcutaneously thrice weekly, for four weeks and transfusion of 400 ml of erythrocyte mass) hemorheological measurements including blood and plasma viscosity, hematocrit (Hct), hemoglobin, red blood cell aggregation (RBCA) and deformability were completed. It was found an increase of Hb from 76.07+/-3.68 g/l to 87.86+/-4.26 g/l after the transfusion. It was accompanied by Hct rise by 25% (from 23.67+/-1.85 to 29.50+/-1.96%, p<0.05). Under these conditions the whole blood viscosity (BV) was increased by 19% (p<0.05). Plasma viscosity did not change markedly. Therefore the main cause of the whole blood viscosity rise was an increase of Hct. After erythrocyte mass transfusion there were some increases of red cell deformability and aggregation (by 7%, p>0.05). Under these conditions the Hct/BV ratio as an index of oxygen transport efficiency was changed after transfusion only slightly. While after four weeks of epoetin treatment the hematocrit/viscosity ratio was raised by 14% (p<0.05), in spite of the high blood viscosity. In addition RBCA decreased (p<0.01) and their deformability was increased by 14% (p<0.05). In vitro microrheological data permit to suggest that epoetin has a direct effect on the microrheological properties of red cells due to activation of the cellular signal transduction system including the tyrosine kinases and phosphatases. Thus, Epoetin beta administered s.c. thrice weekly, during four weeks, increased hemoglobin levels, improved hemorheological profile and especially its microrheological part as well as the blood transport capacity in anemic cancer patients who were receiving chemotherapy and its hemorheological effect was more positive than under hemotransfusion.