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Purinergic signaling, the oldest evolutionary transmitter system, has been increasingly studied as a pivotal target for novel anti‐cancer therapies. In the present work, the developed nanobiocatalytic system consisting of adenylate kinase immobilized on graphene oxide (AK‐GO) was characterized in terms of its physicochemical and biochemical properties. We put special emphasis on the AK‐GO influence on purinergic signaling components, that is, ecto‐nucleotides concentration and ecto‐enzymes expression and activity in human lung carcinoma epithelial (A549) cells. The immobilization‐dependent modification of AK kinetic parameters allowed for the removal of ATP excess while maintaining low ATP concentrations, efficient decrease in adenosine concentration, and control of the nucleotide balance in carcinoma cells. The cyto‐ and hemocompatibility of developed AK‐GO nanobiocatalytic system indicates that it can be successfully harnessed for biomedical applications. In A549 cells treated with AK‐GO nanobiocatalytic system, the significantly decreased adenosinergic signaling results in reduction of the proliferation and migration capability of cancer cells. This finding is particularly relevant in regard to AK‐GO prospective anti‐cancer applications. [ABSTRACT FROM AUTHOR]

Background : Recent studies indicate that aquaporin (AQP) water channels have a regulatory function in human platelet secretion and in procoagulant response of murine platelets. However, the engagement of AQPs in morphological changes, procoagulant response, and thrombus formation in human blood has never been investigated. Methods : Confocal microscopy was used to study platelet spreading, filopodia formation, ballooning, and thrombus formation under flow. Flow cytometry was utilized to assess platelet phosphatidylserine (PS) exposure and microparticles shedding. Kinetics of clot formation in vitro was evaluated by thromboelastometry. Mouse model of ferric chloride (III) (FeCl3)-induced thrombosis was used to investigate thrombus formation in vivo. Results : We found that chloroauric(III) acid (HAuCl4), a classical AQP inhibitor (10–100 μM), reduced spreading of human platelets on collagen-coated surfaces and inhibited filopodia formation in a fluid phase. Under flow conditions, HAuCl4 (100 μM) attenuated thrombi growth on collagen, platelet secretion, and PS exposure. Thrombus formation was restored by the addition of exogenous adenosine diphosphate (ADP). Collagen-evoked platelet procoagulant response (evaluated as PS exposure, shedding of microparticles, platelet-dependent thrombin generation, and membrane ballooning) was distinctly reduced by HAuCl4 (25–200 μM), as well as the dynamics of clot formation. In mouse model of thrombosis, reduction of surface of PS-positive cells within thrombus was observed in the presence of HAuCl4 (1–10 mg/kg). Conclusion : These results suggest that in human platelets AQPs are crucial for agonist-evoked morphological changes, thrombus formation under flow, and in development of procoagulant response. Antithrombotic effect in vivo suggests that nontoxic inhibitors of AQPs may be considered as potential candidates for a novel class of antiplatelet drugs. [ABSTRACT FROM AUTHOR]

Polycythemia vera (PV) is associated with an increased frequency of thrombotic complications. This study was undertaken to evaluate the hemostatic balance in the blood of PV patients by means of thromboelastography (TEG). The effect of isovolemic erythrocytapheresis (ECP) on the hemostasis of PV patients was also studied. We assessed the coagulation status of 76 PV patients undergoing ECP and 50 of healthy controls. TEG measurements were performed immediately before and after the ECP procedure. Coagulation was triggered by recalcification in freshly collected citrated blood. We recorded clotting time ( R), alpha angle, and maximum amplitude (MA) of the clot. The results presented here show that, compared with healthy controls, PV patients demonstrated an increase in alpha angle ( p < 0.005) and in MA ( p = 0.14). In the subgroup of PV patients with high (>440 × 109 l−1) platelet (PLT) count, differences in MA ( p < 0.01) and alpha angle ( p < 0.001) were more significant. Following ECP procedure, a significant ( p ≤ 0.01) reduction of R time, a rise of alpha angle, and MA were observed, indicating augmentation of a hypercoagulable state. In PV patients, the rise in alpha angle positively correlated ( r = 0.549) with platelet count but not with the number of erythrocytes and leukocytes. Following ECP, this correlation was reduced ( r = 0.382). Dilution (with saline) of blood from PV patients and of healthy controls, to a degree similar to that used during the ECP procedure, resulted in reduction of R and rise of the alpha angle. In conclusion, TEG measurements show that the majority of PV patients demonstrate abnormal hemostasis in which a major role is played by platelets rather than plasma factors. The hypercoagulable state in PV patients is significantly augmented following the ECP and may be related to the hemodilution intrinsically included in this procedure. TEG may help to assess the thrombotic risk in individual PV patients. [ABSTRACT FROM AUTHOR]

Activated platelets release microvesicles, which express procoagulant activity. The mechanism by which vesicles are formed is not entirely clear. This study was undertaken to determine whether a link exists between the operation of the plasma membrane Na[sup +]/H[sup +] exchanger (NHE) and vesiculation. It was found, that platelets treated with NHE-simulating monensin and the sodium influx-inducing gramicidin (without concomitant H[sup +] efflux) produced vesicles demonstrating procoagulant activity. Alkalinization of platelet cytosol by NH[sub 4]Cl failed to evoke vesicle release. Collagen and phorbol ester (PMA)-evoked vesiculation was diminished in the presence of 5-(N-ethyl-N-isopropyl amiloride) (EIPA, inhibitor of NHE) or GF 109203X (inhibitor of protein kinase C). Vesicle formation induced by collagen, PMA, and the calcium ionophore A23187 was less pronounced in the absence of external Na[sup +]. In comparison with collagen, thrombin was a stronger inducer of vesiculation. Platelets stimulated by thrombin, collagen, and PMA accumulated Na[sup +], a phenomenon inhibited in the presence of EIPA. Collagen-evoked vesicle formation started with aggregation but culminated after its completion. The data indicate a significant contribution of the Na[sup +]/H[sup +] exchanger in the formation of microvesicles by porcine platelets. [ABSTRACT FROM AUTHOR]

Background : Recent studies indicate that aquaporin (AQP) water channels have a regulatory function in human platelet secretion and in procoagulant response of murine platelets. However, the engagement of AQPs in morphological changes, procoagulant response, and thrombus formation in human blood has never been investigated. Methods : Confocal microscopy was used to study platelet spreading, filopodia formation, ballooning, and thrombus formation under flow. Flow cytometry was utilized to assess platelet phosphatidylserine (PS) exposure and microparticles shedding. Kinetics of clot formation in vitro was evaluated by thromboelastometry. Mouse model of ferric chloride (III) (FeCl 3 )-induced thrombosis was used to investigate thrombus formation in vivo . Results : We found that chloroauric(III) acid (HAuCl 4 ), a classical AQP inhibitor (10-100 μM), reduced spreading of human platelets on collagen-coated surfaces and inhibited filopodia formation in a fluid phase. Under flow conditions, HAuCl 4 (100 μM) attenuated thrombi growth on collagen, platelet secretion, and PS exposure. Thrombus formation was restored by the addition of exogenous adenosine diphosphate (ADP). Collagen-evoked platelet procoagulant response (evaluated as PS exposure, shedding of microparticles, platelet-dependent thrombin generation, and membrane ballooning) was distinctly reduced by HAuCl 4 (25-200 μM), as well as the dynamics of clot formation. In mouse model of thrombosis, reduction of surface of PS-positive cells within thrombus was observed in the presence of HAuCl 4 (1-10 mg/kg). Conclusion : These results suggest that in human platelets AQPs are crucial for agonist-evoked morphological changes, thrombus formation under flow, and in development of procoagulant response. Antithrombotic effect in vivo suggests that nontoxic inhibitors of AQPs may be considered as potential candidates for a novel class of antiplatelet drugs., (Copyright © 2020 Misztal, Golaszewska, Branska-Januszewska, Marcinczyk, Chabielska, Tomasiak and Rusak.)