An Investigation of Size Distribution and Calcium Signaling in Human Platelets.

Abstract

Background Platelets are thin disc-shaped blood cells that play a major role in hemostasis, maintenance of vascular integrity, and blood coagulation. Large platelets are more reactive and seen in patients with cardiovascular disease. This study aims to analyze the changes in platelet size of ex vivo activated platelets which phenotypically simulates that of a patient at risk of cardiovascular disease and elucidate the calcium signaling pathway responsible for this change. Methodology Platelets were isolated from adult human blood by differential centrifugation. Calcium was mobilized into platelets by treatment with calcium ionophore A23187 in the presence of Ca2+. Platelet size distribution was analyzed using Coulter Counter Multisizer 4. The following signaling parameters were studied: intracellular Ca2+ measurement (using Fura-2/AM by fluorescence spectrophotometry), Ca2+-dependent thiol protease calpain assay (using fluorogenic substrate t-butoxycarbonyl-Leu-metchloromethylcoumarin in fluorescence microplate reader), platelet-derived microparticles (using FACS Calibur flow cytometry), and cytoskeletal protein talin expression (by western immunoblotting). Results When adult platelets were treated with A23187 and Ca2+, two subcellular populations (<2 µm and between 2-4 µm) were noted. The mean size of the second cell population was significantly higher than that of resting platelets (2.94 ± 0.13 µm vs. 2.82 ± 0.15 µm, t = 4.605, p = 0.00). A23187 treatment led to elevated intracellular Ca2+, release of platelet-derived microparticles, increase in calpain activity, and cytoskeletal talin degradation. These events were inhibited by calpeptin (a specific calpain inhibitor). Conclusions Elevated calcium caused talin degradation by calpain activity. Breakdown of this cytoskeletal protein leads to relative swelling of cells reflected by the increase in platelet size.