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.