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 it was abolished by pretreatment of cells with saturating concentrations of RGDS peptide. A MoAb specific for the alpha IIb beta 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.
Zauli
G
G
Institute of Human Anatomy, University of Ferrara, Italy.
Bassini
A
A
Vitale
M
M
Gibellini
D
D
Celeghini
C
C
Caramelli
E
E
Pierpaoli
S
S
Guidotti
L
L
Capitani
S
S
D000199:Actins / Q000096:biosynthesis; D000730:Androstadienes / Q000494:pharmacology; D018952:Antigens, CD34 / Q000494:pharmacology; D001777:Blood Coagulation; D001792:Blood Platelets / Q000187:drug effects / Q000502:physiology; D002448:Cell Adhesion / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D005337:Fibrin; D005340:Fibrinogen / Q000187:drug effects / Q000378:metabolism*; D005353:Fibronectins / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D004915:Leukemia, Erythroblastic, Acute; D008533:Megakaryocytes / Q000187:drug effects* / Q000201:enzymology / Q000502:physiology*; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000096:biosynthesis / Q000187:drug effects / Q000502:physiology*; D011485:Protein Binding / Q000187:drug effects; D013926:Thrombopoietin / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D015854:Up-Regulation; D000077191:Wortmannin
D000199:Actins; D000730:Androstadienes; D018952:Antigens, CD34; D005353:Fibronectins; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D005337:Fibrin; D005340:Fibrinogen; D013926:Thrombopoietin; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D000077191:Wortmannin