Magnier C, Corvazier E, Aumont M C, Le Jemtel T H, Enouf J
U 348 INSERM, Hôpital Lariboisière, Paris, France.
Biochem J. 1995 Sep 1;310 ( Pt 2)(Pt 2):469-75. doi: 10.1042/bj3100469.
Although the interrelationship between the two messengers Ca2+ and cyclic AMP in platelet function is well documented, its mechanism of action still remains to be established. We investigated here the question of the regulation of platelet Ca(2+)-ATPases by cyclic AMP through the phosphorylation of the Rap1 protein using a pathological model. We first found experimental conditions where Ca(2+)-transport by platelet membrane vesicles appeared to be dependent on the phosphorylation of the Rap1 protein. Then, we studied platelets of patients with congestive heart failure for their expression of the potential 97 kDa Ca(2+)-ATPase target of regulation through the Rap1 protein as well as the phosphorylation of the Rap1 protein using the catalytic subunit of the cyclic AMP-dependent protein kinase (C. Sub.). In the first patients studied, we found no significant modification in the expression of the 97 kDa Ca(2+)-ATPase by Western blotting using the PL/IM 430 monoclonal antibody which specifically recognized this isoform. In contrast, the Rap1 protein was differentially phosphorylated when using 15 micrograms/ml of the C. Sub. These results allowed us to use these pathological platelets to study the relationship between the expression of Rap1 protein and the regulation of Ca2+ transport by selecting a patient with severe heart failure. We could show a decrease in the expression as well as in the phosphorylation of Rap1 protein and demonstrate a lower effect of C. Sub. on Ca2+ transport. Finally, by studying a further series of patients, we could confirm that the decrease in Rap1 protein expression in heart failure, whatever its extent, was variable, and could strictly correlate the expression of Rap1 protein with the stimulatory effect of C. Sub. on Ca2+ transport. Besides the evidence for regulation of the expression of the Rap1 protein in platelets from patients with heart failure, these findings constitute a new approach in favour of the regulation of platelet Ca2+ transport through the phosphorylation of the Rap1 protein.
尽管钙离子（Ca2+）和环磷酸腺苷（cyclic AMP）这两种信使分子在血小板功能中的相互关系已有充分记录，但其作用机制仍有待确定。我们在此使用病理模型研究了环磷酸腺苷通过Rap1蛋白磷酸化对血小板Ca(2+)-ATP酶的调节问题。我们首先发现了血小板膜囊泡的Ca(2+)转运似乎依赖于Rap1蛋白磷酸化的实验条件。然后，我们研究了充血性心力衰竭患者的血小板，以了解通过Rap1蛋白调节的潜在97 kDa Ca(2+)-ATP酶靶点的表达以及使用环磷酸腺苷依赖性蛋白激酶催化亚基（C. Sub.）对Rap1蛋白的磷酸化情况。在首批研究的患者中，我们使用特异性识别该异构体的PL/IM 430单克隆抗体进行蛋白质印迹分析，发现97 kDa Ca(2+)-ATP酶的表达没有显著变化。相比之下，当使用15微克/毫升的C. Sub.时，Rap1蛋白发生了不同程度的磷酸化。这些结果使我们能够利用这些病理血小板，通过选择一名重度心力衰竭患者来研究Rap1蛋白表达与Ca2+转运调节之间的关系。我们可以证明Rap1蛋白的表达和磷酸化均有所下降，并表明C. Sub.对Ca2+转运的作用较低。最后，通过研究更多系列的患者，我们可以确认心力衰竭中Rap1蛋白表达的下降，无论其程度如何，都是可变的，并且可以将Rap1蛋白的表达与C. Sub.对Ca2+转运的刺激作用严格关联起来。除了有证据表明心力衰竭患者血小板中Rap1蛋白表达受到调节外，这些发现构成了一种支持通过Rap1蛋白磷酸化调节血小板Ca2+转运的新方法。
