title: Platelet microparticle suppressing antibody against GP Ibalpha acts independently of the filamin cleavage and increases protein tyrosine phosphorylation.
abstract: Our aim was to investigate the role of filamin cleavage and protein tyrosine phosphorylation in shear-stress-induced platelet microparticle formation and of its suppression by the monoclonal antibody (mAb) Ib-23 directed against GPIbalpha. PPACK-anticoagulated or EDTA-anticoagulated platelet-rich plasma or washed platelets were exposed to high shear stress (5000 s-1 for 5 min) in the presence of antagonists of GP Ibalpha (mAb Ib-23), of GP IIb/IIIa (abciximab) and their combination. We assessed the generation of microparticle by flow cytometry, the filamin cleavage and the protein tyrosine phosphorylation by western blotting. Microparticle formation decreased by more than 50% with mAb Ib-23 alone (P < 0.01, n = 23) but filamin cleavage was not inhibited. In contrast, abciximab did not change microparticle numbers nor filamin cleavage under the same conditions. However, when combined, the inhibitors against GP Ibalpha and GP IIb/IIIa decreased microparticle by 70% and the filamin cleavage by 20% (P < 0.05). Focal adhesion kinase and p72Syk phosphorylation was enhanced by mAb Ib-23, whereas treatment with abciximab reduced it. mAb Ib-23 inhibits platelet microparticle induced by high shear stress. The mechanism seems not to depend on filamin cleavage: abciximab allowed for full microparticle formation at similar levels of cleaved filamin, whereas the combined inhibitors reduced it. The effect of mAb Ib-23 on protein tyrosine phosphorylation supports previous data, which correlates microparticle formation with the extent of protein tyrosine dephosphorylation and mirrors the dephosphorylation by protein tyrosine phosphatase (PTP-1B) in platelets of calpain-/- knockout mice.
authors.affiliation: Department of Medicine, University Hospital of Bern, Switzerland.
authors.forename: Marc
authors.initials: M
authors.lastname: Forestier
authors.forename: Julio C
authors.initials: JC
authors.lastname: Reséndiz
authors.forename: Luca
authors.initials: L
authors.lastname: Pontiggia
authors.forename: Riitta
authors.initials: R
authors.lastname: Lassila
authors.forename: Juerg H
authors.initials: JH
authors.lastname: Beer
mesh_terms: D000077284:Abciximab; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001792:Blood Platelets / Q000378:metabolism* / Q000648:ultrastructure*; D055252:Cell-Derived Microparticles / Q000187:drug effects*; D003285:Contractile Proteins / Q000378:metabolism* / Q000502:physiology; D064448:Filamins; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000378:metabolism; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D008840:Microfilament Proteins / Q000378:metabolism* / Q000502:physiology; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000276:immunology*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D013314:Stress, Mechanical; D000072377:Syk Kinase; D014443:Tyrosine / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D003285:Contractile Proteins; D064448:Filamins; D007140:Immunoglobulin Fab Fragments; D047908:Intracellular Signaling Peptides and Proteins; D008840:Microfilament Proteins; D019038:Platelet Glycoprotein GPIb-IX Complex; D011506:Proteins; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D051416:Focal Adhesion Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase; C000606897:Syk protein, mouse; D000077284:Abciximab