The Effects of Glycoprotein IIb/IIIa Antagonists and Chloride Channel Blockers on Platelet Cytoplasmic Free Calcium.

Objective To explore the effects and interactions of GPIIb/IIIa antagonists and chloride channel blockers on the platelet cytoplasmic free calcium ([Ca2+]i ). Methods We washed and suspended fresh platelets with Hepes buffer containing 0.1% bovine serum albumin (BSA), then loaded platelets with 5μmol/L Fura-3/AM. Then RGDS, the GPIIb/IIIa antagonists, and the chloride channel blockers 4,4′-diisothiocyano-2,2′-disulfonic acid stilbene(DIDS) or niflumic acid(NFA) were added to the platelet suspension. After 2 minutes incubating, we observed the effects and interactions of GPIIb/IIIa antagonists and chloride channel blockers on platelet [Ca2+]i by measuring the Fura-3 fluorescence intensity. Results 1. Effects of GPIIb/IIIa antagonists and chloride channel blockers on platelet [Ca2+]i. The fluorescence intensity of resting platelet [Ca2+]i were 369.6±62.2, 381.9±72.4, 392.8 ±69.9 after adding RGDS(250 μmol/L), DIDS(100 μmol/L) or NFA( 100 μmol/L) respectively. every agent had no significant effect on resting [Ca2+]i (p>0.05). After thrombin(0.03 U/ml) stimulating and adding RGDS(250 μmol/L), DIDS(100 μmol/L) or NFA( 100 μmol/L), the platelet [Ca2+]i were 883.9±107.0, 789.8±99.8, 564.1±79.4. Compare with the control(977.9±108.8), the three agents could inhibit the elevation of [Ca2+]i stimulated by thrombin (p<0.05). The inhibiting rates were (9.37±7.5)%, (18.7±10.4)% and (41.8±10.1)% respectively. 2. Combined effects of GPIIb/IIIa antagonists and chloride channel blockers The fluorescence intensity of resting platelet [Ca2+]i was 383.9±67.9 after incubated with RGDS and DIDS. That had no significant effects. When platelets were stimulated by thrombin (0.03 U/ml), the combined inhibition rate was (24.4±10.8)%, RGDS and DIDS couldn’t weaken or enhance each other on thrombin-induced elevation of [Ca2+]i (p>0.05). Neither RGDS nor NFA had significant combined effects on resting [Ca2+]i(p>0.05). The combined inhibition rate was (46.0±7.3)%, they had no interactions too(p>0.05). Conclusion The GPIIb/IIIa antagonists RGDS and the chloride channel blockers DIDS or NFA have no effect on resting platelet [Ca2+]i. All of them can inhibit the elevation of platelet [Ca2+]i induced by thrombin. There are no interactions between GPIIb/IIIa antagonists RGDS and chloride channel blockers (DIDS or NFA) in resting platelet [Ca2+]i and elevation of platelet [Ca2+]i induced by thrombin, and their effects were independent.

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.