Regulation of Platelet Activation In Vitro by the c-Mpl Ligand, Thrombopoietin

• Published in: Blood Vol. 86; no. 11; pp. 4054 - 4062
• Main Authors: Chen, Jichun, Herceg-Harjacek, Lidija, Groopman, Jerome E., Grabarek, Jadwiga
• Format: Journal Article
• Language: English
• Published: Washington, DC Elsevier Inc 01-12-1995; The Americain Society of Hematology
• Subjects: Adenosine Diphosphate - metabolism; Amino Acid Sequence; Biological and medical sciences; Blood coagulation. Blood cells; Blood Platelets - drug effects; Blood Platelets - physiology; Fibrinogen - metabolism; Fundamental and applied biological sciences. Psychology; Humans; In Vitro Techniques; Megakaryocytes - drug effects; Megakaryocytes - physiology; Molecular and cellular biology; Molecular Sequence Data; Neoplasm Proteins; Oligopeptides - chemistry; Oligopeptides - pharmacology; Phosphatidylinositol 3-Kinases; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor) - metabolism; Platelet; Platelet Activation - drug effects; Platelet Activation - physiology; Platelet Aggregation - drug effects; Platelet Aggregation - physiology; Proto-Oncogene Proteins - drug effects; Proto-Oncogene Proteins - physiology; Receptors, Cytokine; Receptors, Immunologic - drug effects; Receptors, Immunologic - physiology; Receptors, Thrombopoietin; Thrombopoietin - pharmacology; Thrombopoietin - physiology; Tyrosine - metabolism; Human; Regulation(control); Platelet; Cytokine; Platelet function; Activation; In vitro; Thrombopoietin
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V86.11.4054.bloodjournal86114054

Thrombopoietin (TPO) is a recently identified growth factor that regulates megakaryocytopoiesis. Its receptor, c-MpI, is expressed in megakaryocyte progenitors, mature megakaryoytes, 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 (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 maximum at concentrations between 50 and 100 ng/mL. This phosphorylation was independent of extracellular fibrinogen and ligation of the αIIbβ3 integrin. In contrast, TPO, in the presence of exogenous fibrinogen, induced concentration-dependent platelet aggregation, which was blocked by the soluble c-MpI 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 125l-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 µmol/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 µmol/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 αIIb integrin partially inhibited platelet attachment to collagen, suggesting that the integrin αIIbβ3 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.