High glucose levels enhance platelet activation: involvement of multiple mechanisms.

Diabetes mellitus (DM) and hyperglycaemia are associated with platelet activation. The present study was designed to investigate how high glucose levels influence platelet function. Fasting human blood was incubated with different concentrations of D-glucose (5, 15 and 30 mmol/l) and other sugars without or with in vitro stimuli. Platelet activation was monitored by whole blood flow cytometry. High glucose levels enhanced adenosine diphosphate (ADP)- and thrombin receptor-activating peptide (TRAP)-induced platelet P-selectin expression, and TRAP-induced platelet fibrinogen binding. Similar effects were seen with 30 mmol/l L-glucose, sucrose and galactose. Hyperglycaemia also increased TRAP-induced platelet-leucocyte aggregation. Protein kinase C (PKC) blockade did not counteract the enhancement of platelet P-selectin expression, but abolished the enhancement of TRAP-induced platelet fibrinogen binding by hyperglycaemia. Superoxide anion scavenging by superoxide dismutase (SOD) attenuated the hyperglycaemic enhancement of platelet P-selectin expression, but did not counteract the enhancement of TRAP-induced platelet fibrinogen binding. Hyperglycaemia did not alter platelet intracellular calcium responses to agonist stimulation. Blockade of cyclo-oxygenase (COX), phosphotidylinositol-3 (PI3) kinase, or nitric oxide synthase, or the addition of insulin did not influence the effect of hyperglycaemia. In conclusion, high glucose levels enhanced platelet reactivity to agonist stimulation through elevated osmolality. This occurred via superoxide anion production, which enhanced platelet P-selectin expression (secretion), and PKC signalling, which enhanced TRAP-induced fibrinogen binding (aggregablity).

Influence of extracellular calcium on single platelet activation as measured by whole blood flow cytometry.

The influence of extracellular calcium concentrations ([Ca(2+)]) on single platelet activation and platelet aggregation was evaluated. Platelet fibrinogen binding and P-selectin expression were monitored by whole blood flow cytometry in the presence of EDTA or 0.125, 1.25, 2.5, 5, or 10 mM [Ca(2+)] and in the absence or presence of the thromboxane A(2) (TXA(2)) blockade. Platelet aggregation was measured in citrated and hirudinized platelet-rich plasma (PRP). Platelet fibrinogen binding was slightly increased at >/=2.5 mM [Ca(2+)] in unstimulated samples. ADP-induced platelet fibrinogen binding was, however, higher at 0.125 mM, but lower at 5 and 10 mM [Ca(2+)], as compared to 1.25 mM [Ca(2+)]. Platelet P-selectin expression was not affected by extracellular [Ca(2+)], except mild increases of ADP-induced platelet P-selectin expression in the presence of EDTA. TXA(2) blockade by ICI 192.605 influenced above flow cytometric analyses little. Using Born aggregometry, ADP induced more intense platelet aggregation in citrated PRP than in hirudinized PRP. TXA(2) blockade did not affect platelet aggregation in hirudinized PRP, but reduced aggregation in citrated PRP to approximately 85% of that in hirudinized samples. ADP also induced a more marked and sustained elevation of intracellar [Ca(2+)] in the presence of extracellular [Ca(2+)]. Thus, extracellular [Ca(2+)] has little influence on flow cytometric analysis of platelet P-selectin expression. High [Ca(2+)] enhances spontaneous platelet fibrinogen binding, but reduces ADP-induced platelet fibrinogen binding, while low [Ca(2+)] enhances ADP-induced platelet fibrinogen binding. Physiological [Ca(2+)] supports more intense platelet aggregation when effect of artificial TXA(2) synthesis is blocked.