Gende Oscar A
Centro de Investigaciones Cardiovasculares, Facultad de Ciencias Médicas, Universidad Nacional de La Plata, 60 Y 120, 1900 La Plata, Argentina.
Thromb Res. 2003 Apr 15;110(1):33-7. doi: 10.1016/s0049-3848(03)00289-5.
Hypertonic solutions are useful for the management of hypovolemic shock but cause impairment in platelet function. This effect would reduce the ischemia/reperfusion damage caused by activated platelets, but it could be the cause of aggravating blood loss in case of uncontrolled hemorrhage. In this paper, it was studied if osmotic shrinkage of platelets affects the changes in intracellular calcium concentration (Ca(2+)) induced by thrombin or adenosine 5' diphosphate (ADP). Furthermore, it was investigated if hypertonic solutions change the mobilization from intracellular stores or the calcium entry. Previous reports from our laboratory described that the capacitative calcium entry is increased by alkalization and also that hyperosmolarity has an alkalinizing effect on human platelets so it was hypothesized that hyperosmolarity would be able to modify agonist-induced calcium entry.
To study the response to agonists, platelets loaded with 2'7'-bis(carboxyethyl)-5(6)-carboxy-fluorescein (FURA 2) were incubated at 37 degrees C for 500 s in a N-2-hidroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered solution with 1 mM CaCl(2). The osmolarity of the solution was elevated by the addition of 200 mM mannitol or sucrose and after the stimulation with 0.1 IU/ml of thrombin or 100 microM ADP, Ca(2+) increases were compared. Platelets incubated in zero calcium/EGTA were stimulated with these agonists or with 0.1 microM thapsigargin to separately study the effect of hyperosmolarity on both calcium mobilization from intracellular stores and extracellular calcium entry.
It was found that hypertonic solutions decrease the Ca(2+) peak induced by the agonist: The increase of Ca(2+) in the presence of 200 mM mannitol produced by 100 microM ADP was 62+/-6% and response to 0.1 IU/ml thrombin was 74+/-7% of the increase in isotonic control solution. In the case of ADP, both mobilization and calcium entry were reduced to 66+/-3% and 65+/-6% of isotonic control, respectively. In the case of thrombin, only the mobilization showed a significant change (79+/-2% of parallel control). In platelets depleted by thapsigargin, the capacitative calcium entry was increased in hypertonic mannitol (174+/-25% of the isotonic control). Similar results were obtained with hypertonic sucrose solutions.
In spite of the stimulatory effect of hyperosmolarity on capacitative calcium entry observed in platelets in which the calcium stores were depleted with thapsigargin, the full response of intracellular calcium to the agonists tested (ADP and thrombin) was reduced by an increase in osmolarity. The decreased Ca(2+) mobilization observed may play a role in the reduction in hypertonic media of accompanying platelets responses such as aggregation or shape change.
高渗溶液可用于治疗低血容量性休克，但会损害血小板功能。这种作用会减少活化血小板引起的缺血/再灌注损伤，但在出血未得到控制的情况下，可能会加重失血。本文研究了血小板的渗透性收缩是否会影响凝血酶或腺苷5'-二磷酸（ADP）诱导的细胞内钙浓度（[Ca(2+)]i）变化。此外，还研究了高渗溶液是否会改变细胞内钙库的动员或钙的内流。我们实验室之前的报告描述了碱化会增加容量性钙内流，并且高渗对人血小板有碱化作用，因此推测高渗能够改变激动剂诱导的钙内流。
为了研究对激动剂的反应，将负载有2'7'-双（羧乙基）-5（6）-羧基荧光素（FURA 2）的血小板在37℃下于含有1 mM氯化钙的N-2-羟乙基哌嗪-N'-2-乙磺酸（HEPES）缓冲溶液中孵育500秒。通过添加200 mM甘露醇或蔗糖提高溶液的渗透压，在用0.1 IU/ml凝血酶或100 microM ADP刺激后，比较[Ca(2+)]i的增加情况。将血小板在零钙/乙二醇双四乙酸（EGTA）中孵育，并用这些激动剂或0.1 microM毒胡萝卜素刺激，以分别研究高渗对细胞内钙库动员和细胞外钙内流的影响。
发现高渗溶液可降低激动剂诱导的[Ca(2+)]i峰值：100 microM ADP在200 mM甘露醇存在下引起的[Ca(2+)]i增加为等渗对照溶液增加量的62±6%，对0.1 IU/ml凝血酶的反应为74±7%。对于ADP，动员和钙内流分别降至等渗对照的66±3%和65±6%。对于凝血酶，只有动员显示出显著变化（平行对照的79±2%）。在用毒胡萝卜素耗尽的血小板中，高渗甘露醇中容量性钙内流增加（等渗对照的174±25%）。高渗蔗糖溶液也得到了类似的结果。
尽管在毒胡萝卜素耗尽钙库的血小板中观察到高渗对容量性钙内流有刺激作用，但渗透压的增加会降低细胞内钙对所测试激动剂（ADP和凝血酶）的完全反应。观察到的Ca(2+)动员减少可能在高渗介质中伴随的血小板反应如聚集或形状改变的减少中起作用。
