REGULATION OF PLATELET ACTIVATION IN-VITRO BY THE C-MPL LIGAND, THROMBOPOIETIN

Thrombopoietin (TPO) is a recently identified growth factor that regul ates megakaryocytopoiesis. Its receptor, c-Mpl, is expressed in megaka ryocyte progenitors, mature megakaryoytes, and human blood platelets. We have observed that TPO treatment of human platelets resulted in tyr osine phosphorylation of several cellular proteins, including the c-Mp l receptor and the 85-kD subunit of phosphatidylinositol 3-kinase (PI 3-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 max imum 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 f ibrinogen, induced concentration-dependent platelet aggregation, which was blocked by the soluble c-Mpl receptor. Increasing TPO concentrati ons modulated the degree of the primary wave of aggregation and the la g phase, but not the slope or maximum of the secondary wave of aggrega tion. This secondary aggregation was controlled by the addition of apy rase, suggesting an adenosine diphosphate (ADP)-dependent mechanism. T reatment of platelets with TPO resulted in augmented binding of I-125- fibrinogen to intact platelets, with a 50% effect (EC(50)) occurring b etween 5 and 10 ng/mL. TPO-induced binding of fibrinogen to platelets was comparable in degree with that observed by stimulation with 10 mu mol/L ADP. In an immobilized collagen-platelet adhesion assay, a signi ficant increase in the attachment of TPO-stimulated platelets was obse rved. This effect was dependent on the concentration of TPO. At 50 ng/ mL of TPO, platelet attachment to collagen increased threefold compare d with the buffer control. Furthermore, the presence of fibrinogen did not significantly alter TPO augmentation of the platelet-collagen int eraction. This interaction was mediated by the Arg-Gly-Asp (RGD) adhes ion recognition sequence, as it was completely abolished by 100 mu mol /L of the RGDS peptide. A fraction of the TPO-dependent platelet attac hment to a collagen-coated surface was insensitive to treatment with p rostaglandin E(1). Furthermore, antibody to alpha(IIb) integrin partia lly inhibited platelet attachment to collagen, suggesting that the int egrin alpha(IIb)beta(3) participates in this association. These data i ndicate that TPO might function not only as a cytokine in megakaryocyt e growth and differentiation, but may also participate in direct plate let activation and modulate platelet-extracellular matrix interactions . (C) 1995 by The American Society of Hematology.