钙指示剂Fura－2／AM对血小板功能及细胞内游离钙测定结果的影响

Effects of Fura-2/AM as a Calcium Indicator on Platelet Functions and Measurement of Cytosolic-Free Calcium Concentration

本实验结果表明，以Ｆｕｒａ－２／ＡＭ负载家兔洗脱血小板，当Ｆｕｒａ－２／ＡＭ的浓度低于２μｍｏｌ／Ｌ时，３μｍｏｌ／Ｌ和１０μｍｏｌ／Ｌ花生四稀酸（ＡＡ）诱导的血小板聚集（透光度增加：７２．５±７．８４％）及释放反应（ＡＴＰ释放：１．１２±０．２１μｍｏｌ／４×１０５血小板）不受影响，但随Ｆｕｒａ－２／ＡＭ浓度增加，ＡＡ的聚集和释放反应明显减弱（Ｐ＜０．０５或Ｐ＜０．０１）。Ｆｕｒａ－２／ＡＭ浓度对单一血小板内钙（［Ｃａ２＋］ｉ）的静息水平无影响，但Ｆｕｒａ－２／ＡＭ为２μｍｏｌ／Ｌ时，ＡＡ诱导的［Ｃａ２＋］ｉ动员最明显。另外，标本中血小板的数量也明显影响［Ｃａ２＋］ｉ的测定结果（Ｐ＜０．０５或Ｐ＜０．０１）。提示，Ｆｕｒａ－２／ＡＭ浓度过高，很可能导致过多的钙离子被螯合，降低了血小板的功能，同时，细胞内游离钙与Ｆｕｒａ－２／ＡＭ的结合过程存在着饱和性。

In the present experiments,the effects of different dose of Fura-2/AM on platelet functions, including aggregation and release reaction in the preparation of washed platelets, and cytosolic-free calcium concentration([Ca2+]i) measured in single washed platelets were evaluated. The data indicated that,after the incubation of washed platelet preparation with 2μmol·L-1 of Fura-2/AM, 10μmol·L-1 of arachidonic and(AA)-induced both aggregation (increase in light trasmission, 72.5±7.84%) and the amount of ATP released simutaeaously in the medium(1.12±0.21μmol·4×105platelets-1) were much higher, incubation of washed platelets with higher than of 2μmol·L-1 Fura-2/AM reduced the platelet functions, AA-induced aggregation and release reaction were decreased(P<0.05 and P<0.01 respectively).The results from[Ca2+]i measurement indicated that the concentration of Fura-2/AM did not affect the resting level of[ca2+]i, but 3 or 10 μmol·L-1 of AA-induced[Ca2+]i mobilization was the most significant when the preparations were incubated with 2 μmol·L-1 of Futa-2/AM before stimulation, this results were consistant with that of AA-induced aggregation and release reaction as mentioned above. In addition, when the preparations with different platelet count were incubated with 2 μmol·L-1 of Fura-2/AM, [Ca2+]i mobilizations by AA in each preparation(3×108·m1-1,6×108·ml-1 and 1.2 × 101· ml-1) were significantly diffefent(P<0.05 or P<0.01 ) although AA-induced aggregation and release reaction in three groups was no differences. In conclusions, the concentration of Fura-2/AM (higher 2 μmol·L-1) reduced the platelet functions because of chalating effect of Fura-2/AM, and Fura-2/AM as an indicator of cytosolic-free calcium existed the saturate effect and this was also associated with the platelet count in preparation.