ACTIVATION OF CALPAIN-I IN THROMBIN-STIMULATED PLATELETS IS REGULATED BY THE INITIAL ELEVATION OF THE CYTOSOLIC CA2+ CONCENTRATION
ACTIVATION OF CALPAIN-I IN THROMBIN-STIMULATED PLATELETS IS REGULATED BY THE INITIAL ELEVATION OF THE CYTOSOLIC CA2+ CONCENTRATION
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DOI：
10.1042/bj2840755
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发表时间：
1992-06-15
影响因子：
4.1
通讯作者：
KAZAMA, M
中科院分区：
文献类型：
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作者：
ISHII, H;SUZUKI, Y;KAZAMA, M
在凝血酶刺激的血小板中，研究了激活钙蛋白酶I所需的Ca2+的来源和浓度。在含有fura-2-AM的血小板中测量胞质游离Ca2+ ([Ca2+]i)的浓度，并在0.05,0.1或0.5 NIH单位凝血酶/ml刺激后表现出双相反应。最初的短暂升高，主要依赖于Ca2+从内部储存释放到细胞质中，在刺激后15 s达到峰值，并且在初始升高后观察到二次持续升高，这是由于Ca2+内流。通过兔抗（人）钙蛋白酶I IgG免疫印迹法测定，未受刺激的血小板中钙蛋白酶I的含量约为540 ng/ 10(8)。在凝血酶刺激后10 s Calpain I被激活，这是通过免疫印迹上78kda和76kda形态的出现来确定的。calpain I的激活率计算为78 + 76 kDa形式的数量占总数（80 + 78 + 76 kDa）的百分比，并受到刺激后初始瞬时[Ca2+] I升高程度的影响。[Ca2+]i初始增加300 nm才能达到calpain 1的最大激活（60%），而半最大激活发生在160 nm-[Ca2+]i。这些结果表明血小板中钙蛋白酶I的激活是由凝血酶刺激后[Ca2+] I的初始升高调节的，并不一定需要Ca2+内流。
The source and concentration of Ca2+ required to activate calpain I were investigated in thrombin-stimulated platelets. The concentration of cytosolic free Ca2+ ([Ca2+]i) was measured in platelets containing fura-2-AM, and exhibited a biphasic response after stimulation with 0.05, 0.1 or 0.5 NIH units of thrombin/ml. An initial transient elevation, which was predominantly dependent upon Ca2+ released from the internal stores into the cytosol, peaked at 15 s after stimulation, and a secondary sustained elevation, which was due to Ca2+ influx, was observed following the initial elevation. Calpain I was present at about 540 ng/ 10(8) unstimulated platelets, as measured by immunoblotting using rabbit anti-(human calpain I) IgG. Calpain I was activated 10 s after thrombin stimulation, as determined by the appearance of the 78 kDa and 76 kDa forms on immunoblots. The activation ratio of calpain I was calculated as the amount of the 78 + 76 kDa forms as a percentage of the total (80 + 78 + 76 kDa), and was influenced by the extent of the initial transient [Ca2+]i elevation after stimulation. An initial increase in [Ca2+]i of 300 nm was required to achieve the maximal activation (60 %) of calpain 1, and half-maximal activation occurred at 160 nm-[Ca2+]i. These results suggest that the activation of calpain I in platelets is regulated by the initial elevation in [Ca2+]i after thrombin stimulation, and does not necessarily require a Ca2+ influx.