Stefanich E, Senn T, Widmer R, Fratino C, Keller G A, Fielder P J
Department of Pharmacokinetics and Metabolism, Genentech Inc, S San Francisco, CA 94080, USA.
Blood. 1997 Jun 1;89(11):4063-70.
Previous in vivo studies have established that plasma thrombopoietin (TPO) levels are regulated by binding to c-Mpl on platelets and that, in vitro, platelets bind and degrade TPO. To determine if the in vivo metabolism of TPO was specific and saturable, we injected normal CD-1 mice IV with trace amounts of 125I-rmTPO with or without a saturating concentration of rmTPO. The amount of radioactivity present in the spleen, blood cell fraction, platelet fraction, tibia/fibula, and femur was significantly greater in the mice receiving 125I-rmTPO alone. Conversely, the amount of radioactivity present in the plasma was significantly greater in the mice receiving both 125I-rmTPO and rmTPO, thus suggesting the uptake of rmTPO by the spleen, platelets, and bone marrow in vivo was saturable. Platelet and spleen homogenates from animals receiving 125I-rmTPO alone showed a degradation pattern of 125I-rmTPO similar to that observed in vitro using mouse platelet rich plasma. To determine the in vivo binding dynamics for rmTPO, mice were injected with 125I-rmTPO alone or with increasing concentrations of rmTPO; spleen and blood cell-associated radioactivity was determined at 2 hours postinjection. A 4-parameter curve fit of the data indicated that the "in vivo binding affinity" for rmTPO was approximately 6.4 microg/kg. These data indicate that after a dose of approximately 6.4 microg/kg, 50% of all c-Mpl receptors will be saturated with rmTPO. Electron microscopy indicated that radioactivity was present bound to and within megakaryocytes and platelets in both sternum and spleen and platelets in circulation. Together these data demonstrate that in vivo, 125I-rmTPO is mainly metabolized by platelets and to a small extent by cells of the megakaryocyte lineage, via a specific and saturable mechanism.
以往的体内研究已经证实，血浆血小板生成素（TPO）水平通过与血小板上的c-Mpl结合来调节，并且在体外，血小板会结合并降解TPO。为了确定TPO在体内的代谢是否具有特异性和饱和性，我们给正常的CD-1小鼠静脉注射微量的125I-重组人血小板生成素（rmTPO），同时注射或不注射饱和浓度的rmTPO。单独接受125I-rmTPO的小鼠脾脏、血细胞部分、血小板部分、胫腓骨和股骨中的放射性量显著更高。相反，同时接受125I-rmTPO和rmTPO的小鼠血浆中的放射性量显著更高，这表明体内脾脏、血小板和骨髓对rmTPO的摄取是可饱和的。单独接受125I-rmTPO的动物的血小板和脾脏匀浆显示出125I-rmTPO的降解模式，类似于在体外使用富含小鼠血小板的血浆所观察到的模式。为了确定rmTPO在体内的结合动力学，给小鼠单独注射125I-rmTPO或注射浓度不断增加的rmTPO；在注射后2小时测定脾脏和与血细胞相关的放射性。对数据进行的四参数曲线拟合表明，rmTPO的“体内结合亲和力”约为6.4微克/千克。这些数据表明，在剂量约为6.4微克/千克后，所有c-Mpl受体的50%将被rmTPO饱和。电子显微镜显示，放射性存在于胸骨和脾脏中的巨核细胞和血小板以及循环中的血小板上和内部。这些数据共同表明，在体内，125I-rmTPO主要通过血小板代谢，在较小程度上通过巨核细胞系细胞代谢，通过一种特异性和可饱和的机制。
