Alterations in platelet Ca2+ signalling in diabetic patients is due to increased formation of superoxide anions and reduced nitric oxide production

Increased aggregation of platelets might contribute to the development of v ascular complication in diabetes mellitus. In this study release of superox ide anions, intracellular Ca2+ signalling and nitric oxide formation stimul ated by the receptor-dependent agonist adenosine 5'-diphosphate (ADP) and t he receptor-independent stimulus thapsigargin, were compared ill platelets isolated from patients with Type II (non-insulin-dependent) diabetes mellit us and healthy control subjects. Diabetes augmented intracellular Ca2+ rele ase and Ca2+ entry to ADP by 40 and 44% (control subjects: n = 11; diabetic : n = 6), while the median effective concentration (EC50) of ADP to initiat e Ca2+ signalling was similar in both groups. The effect of thapsigargin on Ca2+ concentration was increased by 69% in diabetic patients (control subj ects: n = 22; diabetic patients: n = 9). In addition, release of superoxide anions was 70% greater in diabetic patients (control subjects: n = 9; diab etic patients: n = 6). Treatment of platelets from control subjects with th e superoxide anion-generating mixture xanthine oxidase and hypoxanthine or buthioninesulphoximine (BSO) mimicked the effect of diabetes on platelet Ca 2+ signalling. The antioxidant glutathione normalized enhanced Ca2+ respons e in the diabetic group (control subjects: n = 5; diabetic patients: n = 6) . Basal and thapsigargin evoked nitric oxide synthase activity was reduced in the diabetic group by 85 and 64%, respectively (control subjects: n = 13 ; diabetic subjects: n = 13). The nitric oxide-donor 2-(N,N-diethylamino)-d iazenolate-2-oxide sodium (DEA/NO) normalized enhanced Ca2+ signalling in p latelets preincubated with xanthine oxidase and hypoxanthine (n = 12) and i n those from diabetics (control subjects: n = 6; diabetic patients: n = 6). Inhibition of nitric oxide synthase by N-nitro-L-arginine (L-NA) augmented thapsigargin induced Ca2+ signalling by 51% (n = 8). These data indicate t hat in diabetes platelet Ca2+ signalling might be enhanced by excessive sup eroxide production and an attenuated negative direct or indirect feedback c ontrol by nitric oxide, due to its reduced production.