Human platelets as a model for the binding and degradation of thrombopoietin.
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.
Fielder
P J
PJ
Department of Pharmacokinetics and Metabolism, Genentech, Inc, South San Francisco, CA 94080, USA.
Hass
P
P
Nagel
M
M
Stefanich
E
E
Widmer
R
R
Bennett
G L
GL
Keller
G A
GA
de Sauvage
F J
FJ
Eaton
D
D
D000328:Adult; D001792:Blood Platelets / Q000378:metabolism*; D003720:Densitometry; D010450:Endopeptidases / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D009363:Neoplasm Proteins; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D018121:Receptors, Cytokine; D053628:Receptors, Thrombopoietin; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013926:Thrombopoietin / Q000378:metabolism*
D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins; D018121:Receptors, Cytokine; D053628:Receptors, Thrombopoietin; D011993:Recombinant Fusion Proteins; C082218:MPL protein, human; D013926:Thrombopoietin; D010450:Endopeptidases