Dalla Via L, Stimamiglio M, Scapin M, Cesaro L, Deana R
Department of Biological Chemistry, University of Padova, Italy.
Cell Calcium. 1996 Nov;20(5):431-40. doi: 10.1016/s0143-4160(96)90006-8.
Addition of the calcium-ionophore ionomycin to acetylsalicylate-treated platelets suspended in a low Ca2+ concentration-containing medium (about 0.1 microM), induced a dose-dependent (range 0.25-3 microM) and transient increase in the cytosolic Ca2+ concentration ([Ca2+]c). Less than 10% of the maximal releasable amount of serotonin was secreted at [Ca2+]c lower than 1 microM, whereas secretion was almost maximal at [Ca2+]c higher than 2 microM. In all cases the secretion stopped after about 1 min even if the [Ca2+]c was kept constant by repeated small additions of CaCl2 (25-40 microM). A rapid phosphorylation of pleckstrin (47 kDa) and myosin light chain (20 kDa) was found in all cases, whereas a weak phosphorylation of a 27 kDa protein occurred at [Ca2+]c lower than 1.5 microM. Addition of 0.2 mM CaCl2 to platelets pretreated for 4 min with 0.5-1 microM ionomycin brought about a serotonin secretion remarkably lower than obtained by the simultaneous addition of CaCl2 and ionophore. Platelets suspended in a low calcium-containing medium and exposed to ionomycin showed a major increase in tyrosine phosphorylation of 60 and 72 kDa proteins and a slight increment in tyrosine phosphorylation of 115 and 130 kDa proteins. Subsequent addition of 0.2 mM CaCl2 induced a widespread phosphotyrosine dephosphorylation, particularly evident in the 60 kDa protein identified as p60c-src kinase. The protein kinase inhibitor genistein caused, together with a marked prevention of the protein tyrosine phosphorylation, a remarkable increase in the ionomycin-elicited secretory activity of platelets All together these results indicate that protein kinase C-dependent pleckstrin phosphorylation is a prerequisite of platelet secretion, but that the latter process is apparently regulated by a network of phosphoproteins, in particular the serine/threonine phosphorylation of 27 and 68 kDa proteins and the tyrosine phosphorylation of the p60c-src were found to be associated with a decrease in the secretory activity.
在悬浮于低钙浓度（约0.1微摩尔）培养基中的乙酰水杨酸处理的血小板中加入钙离子载体离子霉素，可诱导胞质钙离子浓度（[Ca2+]c）呈剂量依赖性（范围为0.25 - 3微摩尔）且短暂升高。当[Ca2+]c低于1微摩尔时，分泌的5 - 羟色胺量不到最大可释放量的10%，而当[Ca2+]c高于2微摩尔时，分泌几乎达到最大。在所有情况下，即使通过反复少量添加氯化钙（25 - 40微摩尔）使[Ca2+]c保持恒定，分泌在约1分钟后也会停止。在所有情况下均发现血小板-2蛋白（47 kDa）和肌球蛋白轻链（20 kDa）迅速磷酸化，而当[Ca2+]c低于1.5微摩尔时，一种27 kDa蛋白发生微弱磷酸化。向用0.5 - 1微摩尔离子霉素预处理4分钟的血小板中加入0.2毫摩尔氯化钙，导致5 - 羟色胺分泌明显低于同时加入氯化钙和离子霉素时的分泌量。悬浮于低钙培养基中并暴露于离子霉素的血小板显示60和72 kDa蛋白的酪氨酸磷酸化大幅增加，115和130 kDa蛋白的酪氨酸磷酸化略有增加。随后加入0.2毫摩尔氯化钙会引起广泛的磷酸酪氨酸去磷酸化，在鉴定为p60c-src激酶的60 kDa蛋白中尤为明显。蛋白激酶抑制剂染料木黄酮与显著抑制蛋白酪氨酸磷酸化一起，使离子霉素诱导的血小板分泌活性显著增加。所有这些结果共同表明，蛋白激酶C依赖性的血小板-2磷酸化是血小板分泌的先决条件，但后者过程显然受一组磷蛋白网络调节，特别是发现27和68 kDa蛋白的丝氨酸/苏氨酸磷酸化以及p60c-src的酪氨酸磷酸化与分泌活性降低有关。
