Inhibition of PAR4 Signaling in Ethanol-Attenuation of Platelet Function.

Background: Moderate consumption of alcohol beverages reduces the morbidity from coronary heart disease. Previous studies describing of inhibitory activity of ethanol (EtOH) on platelet function have substantiated this observation. However, the effects of EtOH on thrombin-related platelet activation remains to be fully elucidated, though platelet activation by thrombin is essential for normal hemostasis as well as relevant to pathophysiological conditions of thrombosis.

Objectives: The aim of this study is to elucidate the effect of EtOH on α-thrombin-related platelet function by measuring platelet aggregation and intracellular calcium ([Ca²⁺]_i).

Materials and Methods: A dual-wavelength spectrofluorometer was used for measurement. α-thrombin, PAR1-activating peptide (AP) (10 μM) or PAR4-AP (25 μM) was added to fura2-AM loaded washed platelet preincubated with or without EtOH (40, 80, 160 and 320 mM).

Results and Interpretations: First, the effects of EtOH on 0.5 nM of thrombin-induced platelet activation was assessed. The concentration 0.5 nM used is conceived to activate platelets only via PAR-1. EtOH did not affect platelet aggregation. EtOH inhibited rise of [Ca²⁺]_i dose-dependently. [Ca²⁺]_i peak time at which maximal rise of [Ca²⁺]_i delayed in a dose-dependent manner.

Secondly, 10 nM of thrombin was used as an agonist. Stimulation by high concentrations of thrombin (〉 5nM) results in cleavage of both PAR1 and PAR4. The changes in [Ca²⁺]_i showed double-phase curve composed of transient spike and prolonged peak in the absence of EtOH. Although EtOH inhibited neither platelet aggregation nor the first phase of [Ca²⁺]_i increasing, it reduced the second prolonged elevation of [Ca²⁺]_i dose-dependently.

To elucidate the inhibiting mechanism of EtOH more precisely, the effects of EtOH on PAR1-AP-induced platelet function were examined. Rise of [Ca²⁺]_i gave a spike form and was almost unchanged even in the presence of high concentrations of EtOH, whereas platelet aggregation was reduced and dissociated in the presence of EtOH. Lastly, the effects of EtOH on PAR4-AP-induced platelet function was examined. Aggregation of PRP was quenched by high concentrations of EtOH but dissociation was not observed contrary to that observed in PAR1-AP-induced aggregation. Further, EtOH inhibited [Ca²⁺]_i rise and delayed [Ca²⁺]_i peak time dose-dependently.

Our results provided a possible mechanism by which EtOH inhibits platelet activation. Reduction of the prolonged elevation of [Ca²⁺]_i by high concentrations of thrombin suggested that EtOH inhibits PAR4 signaling not PAR1 since the second prolonged phase of [Ca²⁺]_i is mediated by PAR4. Inhibition of PAR4-induced aggregation and [Ca²⁺]_i elevation by EtOH supported the findings and EtOH might reduce Ca²⁺ influx through inhibition of PAR4.

Furethermore, the difference between the platelet activation mechanisms of low concentrations of thrombin and PAR1-AP was suggested. PAR1-AP can aggregate platelets at least but might fail to activate phospholipase A₂ required for sustaining stable aggregation since EtOH abolishes phospholipase A₂ and thereby reduces thromboxane A₂ generation. On the other, thrombin at low concentrations might have another pathway for activating platelet differently than PAR1-AP.

Further characterization of the mechanisms involved in inhibition of platelet activation by EtOH may help develop new strategies to control thrombin-mediated platelet activation.