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Background Platelet glycoprotein VI (GPVI) receptor is essential for platelet adhesion and aggregation. Eltrombopag is as an effective treatment for chronic immune thrombocytopenia (ITP); yet, its effect on platelet function is not fully characterized. Aim This prospective study investigated the effect of eltrombopag therapy on platelet function through assessment of GPVI receptor expression and soluble GPVI levels among pediatric patients with persistent or chronic ITP. Methods Thirty-six children and adolescents with persistent or chronic ITP were divided equally into two groups either to receive eltrombopag therapy or the standard of care. All patients were followed-up for 12 months with assessment of bleeding score and complete blood count (CBC). Evaluation of GPVI expression using flow cytometry and measurement of its soluble form by ELISA was done at baseline and at 6 months. Results ITP patients on eltrombopag had significantly lower bleeding score after 6 months of therapy while the quality of life has significantly improved. Platelet count was significantly increased throughout the study. GPVI expression by flow cytometry and soluble GPVI levels were significantly increased after eltrombopag therapy. After 12 months, ITP patients on eltrombopag were able to maintain a good quality of life and low bleeding score. Conclusion Our data suggest that eltrombopag, through its effect on the GPVI receptor expression and its soluble form, might reduce bleeding manifestations and improve the quality of life of chronic and persistent ITP children independent of its effect on the platelet count.

Since iron overload is the commonest cause of morbidity and mortality in β thalassemia major (β-TM), it represents one major target in therapeutic management of the disease. The recently discovered erythroid regulator, erythroferrone (ERFE), governed by high levels of erythropoietin, was found to suppress hepcidin expression, thus increasing iron availability for developing erythroid progenitors. We aimed to investigate ERFE levels in Egyptian β-TM patients as an attempt to understand its role in the prediction of iron overload states. Our study included 70 β-TM patients, divided into two subgroups according to the degree of iron overload, and 30 sex and age-matched healthy subjects. ERFE gene expression was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR), and serum hepcidin was measured using enzyme-linked immunosorbent assay (ELISA) technique. Both ERFE gene expression levels and transferrin saturation (TS%) values were able to discriminate among cases with different degrees of iron overload, in contrast to hepcidin. TS% was acknowledged as the best predictor of iron overload (AUC 0.893) in comparison with serum hepcidin and ERFE gene levels (AUC 0.807 and 0.677, respectively), and ERFE gene expression was an independent predictor for the estimated TS%. In conclusion, we suggest that using the ERFE gene expression, combined with serum hepcidin estimation, can substantiate the role of estimated TS% as a promising tool in screening for iron overload in β-TM patients.

Rationale: T-type (Ca V 3.1/Ca V 3.2) Ca 2+ channels are expressed in rat cerebral arterial smooth muscle. Although present, their functional significance remains uncertain with findings pointing to a variety of roles. Objective: This study tested whether Ca V 3.2 channels mediate a negative feedback response by triggering Ca 2+ sparks, discrete events that initiate arterial hyperpolarization by activating large-conductance Ca 2+ -activated K + channels. Methods and Results: Micromolar Ni 2+ , an agent that selectively blocks Ca V 3.2 but not Ca V 1.2/Ca V 3.1, was first shown to depolarize/constrict pressurized rat cerebral arteries; no effect was observed in Ca V 3.2 −/− arteries. Structural analysis using 3-dimensional tomography, immunolabeling, and a proximity ligation assay next revealed the existence of microdomains in cerebral arterial smooth muscle which comprised sarcoplasmic reticulum and caveolae. Within these discrete structures, Ca V 3.2 and ryanodine receptor resided in close apposition to one another. Computational modeling revealed that Ca 2+ influx through Ca V 3.2 could repetitively activate ryanodine receptor, inducing discrete Ca 2+ -induced Ca 2+ release events in a voltage-dependent manner. In keeping with theoretical observations, rapid Ca 2+ imaging and perforated patch clamp electrophysiology demonstrated that Ni 2+ suppressed Ca 2+ sparks and consequently spontaneous transient outward K + currents, large-conductance Ca 2+ -activated K + channel mediated events. Additional functional work on pressurized arteries noted that paxilline, a large-conductance Ca 2+ -activated K + channel inhibitor, elicited arterial constriction equivalent, and not additive, to Ni 2+ . Key experiments on human cerebral arteries indicate that Ca V 3.2 is present and drives a comparable response to moderate constriction. Conclusions: These findings indicate for the first time that Ca V 3.2 channels localize to discrete microdomains and drive ryanodine receptor–mediated Ca 2+ sparks, enabling large-conductance Ca 2+ -activated K + channel activation, hyperpolarization, and attenuation of cerebral arterial constriction.

This study examined whether elevated intravascular pressure stimulates asynchronous Ca2+ waves in cerebral arterial smooth muscle cells and if their generation contributes to myogenic tone development. The endothelium was removed from rat cerebral arteries, which were then mounted in an arteriograph, pressurized (20–100 mmHg) and examined under a variety of experimental conditions. Diameter and membrane potential (VM) were monitored using conventional techniques; Ca2+ wave generation and myosin light chain (MLC20)/MYPT1 (myosin phosphatase targeting subunit) phosphorylation were assessed by confocal microscopy and Western blot analysis, respectively. Elevating intravascular pressure increased the proportion of smooth muscle cells firing asynchronous Ca2+ waves as well as event frequency. Ca2+ wave augmentation occurred primarily at lower intravascular pressures (

L-type Ca2+channels are broadly expressed in arterial smooth muscle cells, and their voltage-dependent properties are important in tone development. Recent studies have noted that these Ca2+channels are not singularly expressed in vascular tissue and that other subtypes are likely present. In this study, we ascertained which voltage-gated Ca2+channels are expressed in rat cerebral arterial smooth muscle and determined their contribution to the myogenic response. mRNA analysis revealed that the α1-subunit of L-type (Cav1.2) and T-type (Cav3.1 and Cav3.2) Ca2+channels are present in isolated smooth muscle cells. Western blot analysis subsequently confirmed protein expression in whole arteries. With the use of patch clamp electrophysiology, nifedipine-sensitive and -insensitive Ba2+currents were isolated and each were shown to retain electrical characteristics consistent with L- and T-type Ca2+channels. The nifedipine-insensitive Ba2+current was blocked by mibefradil, kurtoxin, and efonidpine, T-type Ca2+channel inhibitors. Pressure myography revealed that L-type Ca2+channel inhibition reduced tone at 20 and 80 mmHg, with the greatest effect at high pressure when the vessel is depolarized. In comparison, the effect of T-type Ca2+channel blockade on myogenic tone was more limited, with their greatest effect at low pressure where vessels are hyperpolarized. Blood flow modeling revealed that the vasomotor responses induced by T-type Ca2+blockade could alter arterial flow by ∼20–50%. Overall, our findings indicate that L- and T-type Ca2+channels are expressed in cerebral arterial smooth muscle and can be electrically isolated from one another. Both conductances contribute to myogenic tone, although their overall contribution is unequal.

This study examined whether elevated intravascular pressure stimulates asynchronous Ca2+ waves in cerebral arterial smooth muscle cells and if their generation contributes to myogenic tone development. The endothelium was removed from rat cerebral arteries, which were then mounted in an arteriograph, pressurized (20–100 mmHg) and examined under a variety of experimental conditions. Diameter and membrane potential (VM) were monitored using conventional techniques; Ca2+ wave generation and myosin light chain (MLC20)/MYPT1 (myosin phosphatase targeting subunit) phosphorylation were assessed by confocal microscopy and Western blot analysis, respectively. Elevating intravascular pressure increased the proportion of smooth muscle cells firing asynchronous Ca2+ waves as well as event frequency. Ca2+ wave augmentation occurred primarily at lower intravascular pressures (

This study examined whether elevated intravascular pressure stimulates asynchronous Ca(2+) waves in cerebral arterial smooth muscle cells and if their generation contributes to myogenic tone development. The endothelium was removed from rat cerebral arteries, which were then mounted in an arteriograph, pressurized (20-100 mmHg) and examined under a variety of experimental conditions. Diameter and membrane potential (V(M)) were monitored using conventional techniques; Ca(2+) wave generation and myosin light chain (MLC(20))/MYPT1 (myosin phosphatase targeting subunit) phosphorylation were assessed by confocal microscopy and Western blot analysis, respectively. Elevating intravascular pressure increased the proportion of smooth muscle cells firing asynchronous Ca(2+) waves as well as event frequency. Ca(2+) wave augmentation occurred primarily at lower intravascular pressures (60 mmHg) and ryanodine, a plant alkaloid that depletes the sarcoplasmic reticulum (SR) of Ca(2+), eliminated these events. Ca(2+) wave generation was voltage insensitive as Ca(2+) channel blockade and perturbations in extracellular [K(+)] had little effect on measured parameters. Ryanodine-induced inhibition of Ca(2+) waves attenuated myogenic tone and MLC(20) phosphorylation without altering arterial V(M). Thapsigargin, an SR Ca(2+)-ATPase inhibitor also attenuated Ca(2+) waves, pressure-induced constriction and MLC(20) phosphorylation. The SR-driven component of the myogenic response was proportionally greater at lower intravascular pressures and subsequent MYPT1 phosphorylation measures revealed that SR Ca(2+) waves facilitated pressure-induced MLC(20) phosphorylation through mechanisms that include myosin light chain phosphatase inhibition. Cumulatively, our findings show that mechanical stimuli augment Ca(2+) wave generation in arterial smooth muscle and that these transient events facilitate tone development particularly at lower intravascular pressures by providing a proportion of the Ca(2+) required to directly control MLC(20) phosphorylation.

This study examined the role of the actin cytoskeleton in Rho-kinase-mediated suppression of the delayed-rectifier K+ (KDR) current in cerebral arteries. Myocytes from rat cerebral arteries were enzymatically isolated, and whole cell KDR currents were monitored using conventional patch-clamp electrophysiology. At +40 mV, the KDR current averaged 19.8 ± 1.6 pA/pF (mean ± SE) and was potently inhibited by UTP (3 × 10−5 M). This suppression was observed to depend on Rho signaling and was abolished by the Rho-kinase inhibitors H-1152 (3 × 10−7 M) and Y-27632 (3 × 10−5 M). Rho-kinase was also found to concomitantly facilitate actin polymerization in response to UTP. We therefore examined whether actin dynamics played a role in the ability of Rho-kinase to suppress KDR current and found that actin disruption using either cytochalasin D (1 × 10−5 M) or latrunculin A (1 × 10−8 M) prevented current modulation. Consistent with our electrophysiological observations, both Rho-kinase inhibition and actin disruption significantly attenuated UTP-induced depolarization and constriction of cerebral arteries. We propose that UTP initiates Rho-kinase-mediated remodeling of the actin cytoskeleton and consequently suppresses the KDR current, thereby facilitating the depolarization and constriction of cerebral arteries.