Ariyoshi H, Shiba E, Kambayashi J, Sakon M, Kawasaki T, Yoshida K, Mori T
Department of Surgery II, Osaka University Medical School, Fukushima, Japan.
Cell Calcium. 1993 Jun;14(6):455-63. doi: 10.1016/0143-4160(93)90004-p.
To clarify the possible role of calpain (calcium activated neutral protease; EC 3.4.22.17) in Ca2+ homeostasis of human platelets, we investigated the effects of cell permeable calpain inhibitors, calpeptin and E-64d (EST), on the restoration of cytoplasmic Ca2+ ([Ca2+]i) in both Fura-2 and aspirin (ASA) loaded platelets. Although neither calpeptin (30 microM) nor EST (250 microM) altered the increase of [Ca2+]i in thrombin (1 U/ml) stimulated platelets, both calpain inhibitors delayed the decrease of [Ca2+]i back towards the basal level. These observations suggested that calpain might be involved in Ca2+ restoration. Then, the activity of Ca(2+)-ATPase was examined in thrombin (2 U/ml) stimulated platelets. Thrombin produced a rapid rise in Ca(2+)-ATPase activity by 2-fold at 8 s of incubation, which then returned to below the basal activity within 2 min. Calpeptin inhibited transient Ca(2+)-ATPase activation induced by thrombin in a dose related manner. Ca(2+)-ATPase of isolated platelet membranes was digested by purified human platelet calpain-I and Ca(2+)-ATPase activity was investigated. With a short incubation (8-15 s), Ca(2+)-ATPase activity was increased about 2-fold and then it decreased below the basal level at longer incubations or at a higher calpain/membrane ratio. The initial rate of Ca2+ uptake was also increased by about 2-fold with a short incubation (8-15 s). For molecular characterization of the Ca(2+)-ATPase, the formation of the enzyme-phosphate complex (EP) was investigated. The membrane bound intact 105 kD Ca(2+)-ATPase was converted by calpain to a fragment of approximately 50 kD.(ABSTRACT TRUNCATED AT 250 WORDS)
为阐明钙蛋白酶（钙激活中性蛋白酶；EC 3.4.22.17）在人血小板钙稳态中的可能作用，我们研究了细胞可渗透的钙蛋白酶抑制剂钙肽素和E-64d（EST）对Fura-2和阿司匹林（ASA）负载血小板中细胞质钙（[Ca2+]i）恢复的影响。尽管钙肽素（30 microM）和EST（250 microM）均未改变凝血酶（1 U/ml）刺激的血小板中[Ca2+]i的增加，但两种钙蛋白酶抑制剂均延迟了[Ca2+]i向基础水平的下降。这些观察结果表明钙蛋白酶可能参与钙的恢复。然后，在凝血酶（2 U/ml）刺激的血小板中检测了Ca(2+)-ATP酶的活性。凝血酶在孵育8秒时使Ca(2+)-ATP酶活性迅速升高2倍，然后在2分钟内恢复到基础活性以下。钙肽素以剂量相关的方式抑制凝血酶诱导的瞬时Ca(2+)-ATP酶激活。用纯化的人血小板钙蛋白酶-I消化分离的血小板膜的Ca(2+)-ATP酶，并研究Ca(2+)-ATP酶活性。短时间孵育（8 - 15秒）时，Ca(2+)-ATP酶活性增加约2倍，然后在较长孵育时间或较高钙蛋白酶/膜比例下降至基础水平以下。短时间孵育（8 - 15秒）时钙摄取的初始速率也增加约2倍。为对Ca(2+)-ATP酶进行分子表征，研究了酶 - 磷酸复合物（EP）的形成。膜结合的完整105 kD Ca(2+)-ATP酶被钙蛋白酶转化为约50 kD的片段。（摘要截于250字）
