Metabolism of thrombopoietin (TPO) in vivo: determination of the binding dynamics for TPO in mice.
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.
Stefanich
E
E
Department of Pharmacokinetics and Metabolism, Genentech Inc, S San Francisco, CA 94080, USA.
Senn
T
T
Widmer
R
R
Fratino
C
C
Keller
G A
GA
Fielder
P J
PJ
D000818:Animals; D001665:Binding Sites; D005260:Female; D051379:Mice; D008854:Microscopy, Electron; D011994:Recombinant Proteins / Q000378:metabolism / Q000493:pharmacokinetics; D013926:Thrombopoietin / Q000378:metabolism* / Q000493:pharmacokinetics; D014018:Tissue Distribution
D011994:Recombinant Proteins; D013926:Thrombopoietin