Fielder P J, Hass P, Nagel M, Stefanich E, Widmer R, Bennett G L, Keller G A, de Sauvage F J, Eaton D
Department of Pharmacokinetics and Metabolism, Genentech, Inc, South San Francisco, CA 94080, USA.
Blood. 1997 Apr 15;89(8):2782-8.
Recent studies have shown that plasma thrombopoietin (TPO) levels appear to be directly regulated by platelet mass and that removal of plasma TPO by platelets via binding to the c-Mpl receptor is involved in the clearance of TPO in rodents. To help elucidate the role of platelets in the clearance of TPO in humans, we studied the in vitro specific binding of recombinant human TPO (rhTPO) to human platelet-rich plasma (PRP), washed platelets (WP), and cloned c-Mpl. Using a four-parameter fit and/or Scatchard analysis, the approximate affinity of rhTPO for its receptor, which was calculated from multiple experiments using different PRP preparations, was between 128 and 846 pmol/L, with approximately 25 to 224 receptors per platelet. WP preparations gave an affinity of 260 to 540 pmol/L, with approximately 25 to 35 receptors per platelet, and erythropoietin failed to compete with 125I-rhTPO for binding to WP. Binding and dissociation studies conducted with a BiaCore apparatus yielded an affinity of 350 pmol/L for rhTPO binding to cloned c-Mpl receptors. The ability of PRP to bind and degrade 125I-rhTPO was both time- and temperature-dependent and was blocked by the addition of excess cold rhTPO. Analysis of platelet pellets by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that 125I-rhTPO was degraded into a major fragment of approximately 45 to 50 kD. When 125I-rhTPO was incubated with a platelet homogenate at pH = 7.4, a degradation pattern similar to intact platelets was observed. Together, these data show that human platelets specifically bind rhTPO with high affinity, internalize, and then degrade the rhTPO.
最近的研究表明，血浆血小板生成素（TPO）水平似乎直接受血小板数量的调节，并且在啮齿动物中，血小板通过与c-Mpl受体结合来清除血浆TPO。为了阐明血小板在人类TPO清除中的作用，我们研究了重组人TPO（rhTPO）与人富血小板血浆（PRP）、洗涤血小板（WP）以及克隆的c-Mpl的体外特异性结合。使用四参数拟合和/或Scatchard分析，通过使用不同PRP制剂的多次实验计算得出，rhTPO与其受体的近似亲和力在128至846 pmol/L之间，每个血小板约有25至224个受体。WP制剂的亲和力为260至540 pmol/L，每个血小板约有25至35个受体，促红细胞生成素不能与125I-rhTPO竞争与WP的结合。使用BiaCore仪器进行的结合和解离研究得出rhTPO与克隆的c-Mpl受体结合的亲和力为350 pmol/L。PRP结合和降解125I-rhTPO的能力既与时间有关也与温度有关，并被添加过量的冷rhTPO所阻断。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳对血小板沉淀进行分析表明，125I-rhTPO被降解为约45至50 kD的主要片段。当125I-rhTPO在pH = 7.4的条件下与血小板匀浆孵育时，观察到了与完整血小板相似的降解模式。这些数据共同表明，人血小板以高亲和力特异性结合rhTPO，将其内化，然后降解rhTPO。
