Specific features of intracellular calcium signalling, distinctive for Wiskott-Aldrich syndrome patients

Abstract

Wiskott–Aldrich syndrome (WAS) is a rare X-linked immunodeficient disease accompanied by microthrombocytopenia, which leads to spontaneous/post-traumatic haemorrhages. It has been demonstrated that WAS is caused by gene mutation of WASP protein, which is participating in the processes of actin polarization and actin cytoskeleton re-organisation. It is yet unknown how this mutation affects intracellular signalling and functional responses of platelets of patients with WAS. Assessment of the intracellular calcium signalling, shape change and fibrinogen binging by the platelets of WAS patients. The study was approved by the Independent Ethics Committee of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology, and Immunology. Three patients with WAS and three healthy volunteers were included in the study. Intracellular signaling and platelet functional responses were observed on a BD Facs Canto II flow cytometer. To measure the calcium concentration in the platelet cytosol, the Fura-Red fluorophore was used, platelet shape change upon activation was evaluated by side scattering of cells at a wavelength of 488 nm, platelet integrin activation was evaluated by binding of fluorescently-labeled fibrinogen. During activation, the platelet concentration was 1000 cells per ul to avoid the effects of secondary activation. In quescent state of platelets, an increased concentration of calcium in the cytosol of platelets of patients was observed compared with platelets of healthy donors. In response to stimulation, the highest achievable calcium concentrations were comparable in both cases. The binding of fibrinogen to platelets in patients was not significantly changed compared to healthy donors. On the other hand, the change in the shape of the cells in response to activation, expressed as a percentage, was more significant in patients than the change in the shape of the platelets of healthy donors. With similar maximum responses to stimulation by all agonists, the concentration of calcium in resting platelets, as well as the change in the platelet shape of patients with WAS is significantly higher than that of healthy platelet donors. These results can be explained by the increased ratio of the platelet membrane area to their volume.