This study was designed to investigate the dependency of the red blood cell deformability upon activation of extra- and intracellular signaling pathways. Exposures of red blood cells (RBCs) to catecholamines and to insulin led to positive change in the RBC deformability. When forskolin, a stimulator of adenylyl cyclase (AC), was added to RBC suspension, the RBC deformability was increased. Somewhat more significant deformability rise appeared after RBC incubation with dB-AMP. The inhibitors of phosphodiesterase (PDE) activity increased red cell deformability. These results revealed a considerable role of the AC-cAMP signaling system in the regulation of red blood cell deformability. The rise of the red blood cell Ca ²⁺ influx, stimulated by mechanical loading or A23187 was accompanied by a marked lowering of RBC deformability. At the same time blocking of Ca ²⁺ entry into RBC by verapamil or Ca ²⁺ chelating by EGTA led to significant deformability rise. The comparison of the effect of the different protein kinases on the red blood cell deformability showed that it was altered more considerable under PKA activation by forskolin or dB-cAMP than by other protein kinases. There was a lesser but quite statistically significant effect of tyrosine protein kinase (TPK) on RBC microrheology. Whereas the microrheological effect of PKC was not so considerable. The problem of the short-term regulation of red blood cell microrheology is examined. The latter includes: the modes of activation of extra- and intracellular molecular signaling pathways, ligand - receptor interaction, second messengers, membrane protein phosphorylation. On the whole the total data clearly show that the red cell deformability changes are connected with activation of different extra - and intracellular signaling pathways. It seems reasonable to suppose that red blood cell deformability changes were mainly associated with activation of the AC-cAMP-PKA pathway, and with decrease of Ca ²⁺ entry into cells.

An elevated concentration of fibrinogen in blood is a significant risk factor during many pathological diseases, as it leads to an increase in red blood cells (RBC) aggregation, resulting in hemorheological disorders. Despite the biomedical importance, the mechanisms of fibrinogen-induced RBC aggregation are still debatable. One of the discussed models is the non-specific adsorption of fibrinogen macromolecules onto the RBC membrane, leading to the cells bridging in aggregates. However, recent works point to the specific character of the interaction between fibrinogen and the RBC membrane. Fibrinogen is the major physiological ligand of glycoproteins receptors IIbIIIa (GPIIbIIIa or αIIββ3 or CD41/CD61). Inhibitors of GPIIbIIIa are widely used in clinics for the treatment of various cardiovascular diseases as antiplatelets agents preventing the platelets’ aggregation. However, the effects of GPIIbIIIa inhibition on RBC aggregation are not sufficiently well studied. The objective of the present work was the complex multimodal in vitro study of the interaction between fibrinogen and the RBC membrane, revealing the role of GPIIbIIIa in the specificity of binding of fibrinogen by the RBC membrane and its involvement in the cells’ aggregation process. We demonstrate that GPIIbIIIa inhibition leads to a significant decrease in the adsorption of fibrinogen macromolecules onto the membrane, resulting in the reduction of RBC aggregation. We show that the mechanisms underlying these effects are governed by a decrease in the bridging components of RBC aggregation forces.

Exposure of red blood cells (RBCs) to catecholamines (epinephrine, phenylephrine, an agonist of α1 -adrenergic receptors, clonidine, an agonist of α2 -adrenergic receptors and isoproterenol, an agonist of β-adrenergic receptors) led to change in the

There are evidences that red blood cell (RBC) deformation and aggregation change under their incubation with catecholamines and it is connected with activation of intracellular signaling pathways. The present study was designed to explore the adenylyl cyclase signaling pathway and Ca ²⁺ regulatory mechanism of RBCs together with their microrheological changes. The washed RBCs were resuspended in PBS. In each of the three research sessions RBC suspensions were divided into two aliquots: 1) control (without drug) and 2) with an appropriate drug. After cell incubation RBC deformability (RBCD) and aggregation (RBCA) were estimated. RBC incubation with catecholamines resulted in RBCD changes by 18–30%. RBCs incubation with forskolin facilitated an increase of RBCD by 17% (p < 0.05). A significant deformability rise under dB-AMP incubation was found by 27% (p < 0.01). Ca ²⁺ cell influx, stimulated by A23187, was accompanied by an increase of RBCA; whereas red cell deformability was changed only slightly. On the other hand, Ca ²⁺ entry blocking into the cells by verapamil has led to significant RBCA decrease and RBCD rise. The obtained results make us believe that RBCD change was closely associated with Ca ²⁺ control mechanisms. An effect of Ca ²⁺ concentration increase on RBC microrheology was removed, if it was preliminary added to incubation medium EGTA as Ca ²⁺ chelator. It was found that all four PDE inhibitors: IBMX, vinpocetine, rolipram, pentoxifylline decreased RBCA significantly and, quite the contrary, they increased red cell deformability. Our data have shown that Ca ²⁺ entry increase was accompanied by red cell aggregation rise, while adenylyl cyclase-cAMP system stimulation led to red cell deformability increase and its aggregation lowered. The crosstalk between two intracellular signaling systems is probably connected with phosphodiesterase activity.

This study was designed to evaluate hemorheological changes in patients with cerebrovascular disease (CVD) and peripheral arterial disease (PAD) after 4 weeks of pentoxifylline therapy as well as to study red blood cell microrheological variables after the cell incubation with pentoxifylline and some phosphodiesterase (PDE) activity inhibitors. The patients with CVD (n = 50) and PAD (n = 33) were treated with pentoxifylline (400 mg, thrice a day) for 4 weeks. Before and after drug therapy the hemorheological measurements including plasma and whole blood viscosity, red blood cell aggregation (RBCA) and deformability (RBCD) were completed. In vitro study RBCs were incubated with: 1) Vinpocetine – inhibitor PDE-1, 10 μM; 2) Rolipram – PDE-4, 10 μM; 3) Isobutyl-methylxanthine (IBMX) – nonselective PDE inhibitor, 100 μM and with pentoxifylline, 10 μM The cell incubation was performed at 37°C for 15 min. There were the positive changes of hemorheological profile after 4 weeks of the pentoxifylline therapy both in CVD and PAD patients. The marked RBCD changes were observed after the in vitro cell pentoxifylline treatment as well. Perhaps it is connected with the inhibition of the phosphodiesterase activity in RBCs. An application of drugs and chemicals that can inhibit the PDE activity resulted in RBCD rise and RBCA decrease. The experiments with the use of selective PDE inhibitors have revealed the similar red cell deformability changes. Vinpocetine increased RBCD significantly (p < 0.05). PDE-4 inhibitor – Rolipram stimulated RBCD by 15% (p < 0.05). Some more effective was IBMX. After cell incubation with it a significant rise of the deformability (by 27%; p < 0.05) was found. All drugs, having PDE activity decreased RBCA, but the most pronounced effect had Vinpocetine (50%; p < 0.05). Thus, administered pentoxifylline, daily (1200 mg), during four weeks improves hemorheological profile and especially its microrheological part as well as the blood transport capacity in subjects with cerebral and peripheral vascular disorders. It is most probably red cell microrheological control mechanisms may be associated with the phosphodiesterase activity alterations.