Thrombopoietin enhances the alpha(IIb)beta(3)-dependent adhesion of megakaryocytic cells to fibrinogen or fibronectin through PI 3 kinase

The effect of thrombopoietin (TPO) on the functional activity of surface alpha(IIb)beta(3) (GPIIbIIIa) was investigated in both primary human megakaryocytic cells, derived from peripheral blood CD34(+) cells, and HEL hematopoietic cell line. TPO (100 ng/mL) induced a sixfold to ninefold enhancement of adhesion of both primary megakaryocytic and HEL cells to plates coated with either fibrinogen or fibronectin and a parallel increase of immunoreactivity to the PAC1 monoclonal antibody (MoAb) and fluorescein isothiocyanate-fibrinogen, both of which recognize an activated state of alpha(IIb)beta(3). The enhanced adhesion to fibrinogen or fibronectin was mediated by the Arg-Gly-Asp (RGD) recognition sequence of alpha(IIb)beta(3) as if was abolished by pretreatment of cells with saturating concentrations of RGDS peptide. A MoAb specific for the alpha(IIb) subunit of alpha(IIb)beta(3) also inhibited cell attachment to fibrinogen or fibronectin, while MoAb to anti-alpha(v) beta(3) or anti alpha 5 integrins were completely ineffective, clearly indicating that alpha(IIb)beta(3) participates in this association. A role for PI 3 kinase (PI 3-K) in the TPO-mediated increase in alpha(IIb)beta(3) function in megakaryocytic cells was suggested by the ability of the PI 3-K inhibitor wortmannin (100 nmol/L) and antisense oligonucleotides directed against the p85 regulatory subunit of PI 3-K to completely block the TPO-induced increase in alpha(IIb)beta(3) integrin activity upon TPO stimulation. The modulation of adhesiveness to extracellular matrix proteins containing the RGD motif mediated by TPO likely plays a physiologic role in megakaryocytopoiesis, as pretreatment of CD34(+) cells with RGDS or anti-alpha(IIb) MoAb significantly reduced the number of megakaryocytic colonies obtained in a fibrinclot semisolid assay. (C) 1997 by The American Society of Hematology.