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Regulation of platelet activation in vitro by the c-Mpl ligand,
thrombopoietin
J Chen, L Herceg-Harjacek, JE Groopman and J Grabarek
Department of Medicine, Deaconess Hospital, Boston, MA 02215, USA.
Thrombopoietin (TPO) is a recently identified growth factor that regulates
megakaryocytopoiesis. Its receptor, c-Mpl, is expressed in megakaryocyte
progenitors, mature megakaryocytes, and human blood platelets. We have
observed that TPO treatment of human platelets resulted in tyrosine
phosphorylation of several cellular proteins, including the c-Mpl receptor
and the 85-kD subunit of phosphatidylinositol 3-kinase (PI3-K). TPO
stimulated this tyrosine phosphorylation in a time-dependent manner,
reaching a maximum in 5 minutes. The tyrosine phosphorylation of PI 3-K was
dependent on the concentration of TPO and reached a maximum at
concentrations between 50 and 100 ng/mL. This phosphorylation was
independent of extracellular fibrinogen and ligation of the alpha IIb beta
3 integrin. In contrast, TPO, in the presence of exogenous fibrinogen,
induced concentration- dependent platelet aggregation, which was blocked by
the soluble c-Mpl receptor. Increasing TPO concentrations modulated the
degree of the primary wave of aggregation and the lag phase, but not the
slope or maximum of the secondary wave of aggregation. This secondary
aggregation was controlled by the addition of apyrase, suggesting an
adenosine diphosphate (ADP)-dependent mechanism. Treatment of platelets
with TPO resulted in augmented binding of 125I-fibrinogen to intact
platelets, with a 50% effect (EC50) occurring between 5 and 10 ng/mL.
TPO-induced binding of fibrinogen to platelets was comparable in degree
with that observed by stimulation with 10 mumol/L ADP. In an immobilized
collagen-platelet adhesion assay, a significant increase in the attachment
of TPO-stimulated platelets was observed. This effect was dependent on the
concentration of TPO. At 50 ng/mL of TPO, platelet attachment to collagen
increased threefold compared with the buffer control. Furthermore, the
presence of fibrinogen did not significantly alter TPO augmentation of the
platelet-collagen interaction. This interaction was mediated by the
Arg-Gly-Asp (RGD) adhesion recognition sequence, as it was completely
abolished by 100 mumol/L of the RGDS peptide. A fraction of the
TPO-dependent platelet attachment to a collagen-coated surface was
insensitive to treatment with prostaglandin E1. Furthermore, antibody to
alpha IIb integrin partially inhibited platelet attachment to collagen,
suggesting that the integrin alpha IIb beta 3 participates in this
association. These data indicate that TPO might function not only as a
cytokine in megakaryocyte growth and differentiation, but may also
participate in direct platelet activation and modulate
platelet-extracellular matrix interactions.
Volume 86,
Issue 11,
pp. 4054-4062,
12/01/1995
Copyright © 1995 by The American Society of Hematology
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