The Effects of Chloride Channel Blockers on Platelet Cytoplasmic Free Calcium Concentration and Platelet Aggregation.

Objective To explore the effects of chloride channels on the regulations of platelet cytoplasmic free calcium concentration ([Ca²⁺]i) and platelet aggregation (PAG).

Methods Platelet were separated freshly and then activated by thrombin; The chloride channel blockers 4,4′-diisothiocyano-2, 2′-disulfonic acid stilbene (DIDS) or niflumic acid (NFA), and calcium channel blockers 1-{β-[3-(4-methoxyphenyl)propoxy]- 4-methoxyphenethyl}- 1H - imidazole hydrochloride (SK&F96365) or Nifedipine were added to react with the activated platelets. The effects of each agent on platelet [Ca²⁺]i and PAG were detected. The combine effects and the interactions among chloride channel blockers (DIDS, NFA) and calcium channel blockers (SK&F96365, Nifedipine) were also investigated.

Results Both DIDS and NFA [the concentration were12.5, 25, 50, 100 and 200μmol•L⁻¹ respectively] could inhibit the PAG induced by thrombin (1U/ml) and the effect was dose-dependent. Compared with the control, they had no significant effects on resting [Ca²⁺]i. Compare with the control group, DIDS (100μmol•L⁻¹⁾, SK&F96365 (100μmol•L⁻¹) and Nifedipine (100μmol•L⁻¹) could significantly reduce the PAG, Ca²⁺ release and Ca²⁺ influx activated by thrombin in platelet (P<0.05). DIDS (100μmol•L⁻¹) and SK&F96365 (100μmol•L⁻¹) could enhance each other’s effect on reducing the PAG, Ca²⁺ release and Ca²⁺ influx (P<0.05). DIDS (100μmol•L⁻¹) and Nifedipine (100μmol•L⁻¹) could enhance each other’s effect on reducing Ca²⁺ release (P<0.05). NFA (100μmol•L⁻¹) and SK&F96365 (100μmol•L⁻¹) could weaken each other’s effect on Ca²⁺ release (P<0.05). NFA (100μmol•L⁻¹) and Nifedipine (100μmol•L⁻¹) could weaken each other’s effect on PAG, Ca²⁺ release and Ca²⁺ influx activated by thrombin in platelet (P<0.05).

Conclusion The chloride channel blockers DIDS and NFA have no effect on the resting [Ca²⁺]i and the leak calcium influx of platelet. DIDS can inhibit the Ca²⁺ release, Ca²⁺ influx and PAG of platelet induced by thrombin, while NFA can only inhibit the Ca²⁺ release of platelet induced by thrombin. There are interactions between chloride channel blockers and calcium channel blockers in resting [Ca²⁺]i and PAG of platelet. The opening of chloride channel can influence the cellular calcium movement of platelet.