Platelet Activation Mediated through Membrane Glycoproteins: Involvement of Tyrosine Kinases

ABSTRACT

Fc γ RII cross-linking and anti-CD9 mAbs incuded tyrosine phosphorylation of Fc γ RII, Syk, and Lyn associated with Fc γ RII in Fc γ RII cross-linking but not in anti-CD9 mAb-induced platelet activation. We prepared various GST fusion proteins expressing one or two SH2 domains of Syk and evaluated the association between these GST fusion proteins with Fc γ RII. Based on the results obtained from these experiments, we suggest that only one tyrosine residue in ITAM of Fc γ RII is phosphorylated with anti-CD9 mAb and that both are phosphorylated with Fc γ RII cross-linking. Platelet activation mediated by GPIb, the receptor for vWF, is also related with tyrosine phosphorylation. Botrocetin and vWF induced Syk activation. Shc was also rapidly and heavily tyrosine phosphorylated. Sre and Lyn, a 54-kDa tyrosine kinase, was associated with cytoskeletal proteins. When GPIb was immunoprecipitated with nonfunctional anti-GPIb mAbs after platelets were activated with vWF and botrocetin, an in vitro kinase assay revealed the transient association of a kinase activity with GPIb after platelet activation. Phosphoamino acid analysis of phosphorylated proteins in this assay demonstrated that only tyrosine residues but not serine or threonine were phosphorylated, suggesting that the kinase was indeed a tyrosine kinase.

KEYWORD

Tyrosine kinases - platelet activation - GPIb - botrocetin - Fc γ - RII

REFERENCES

• 1 Hunter T, Sefton B M. Transforming gene product of Rous sarcoma virus phosphorylates tyrosine.  Proc Natl Acad Sci USA . 1980;  77 1311-1315
• 2 Druker B J, Mamon H, Robert T M. Oncogenes, growth factors, and signal transduction.  N Engl J Med . 1989;  321 1383-1391
• 3 Ullrich A, Schlessinger J. Signal transduction by receptors with tyrosine kinase activity.  Cell . 1990;  61 203-212
• 4 Horvath A R, Kiss Z S, Anand R, Kellie S. Association of pp60c-src with the cytoskeleton upon platelet activation.  Biochem Soc Trans . 1991;  19 1150-1154
• 5 Songyang Z, Schoelson S E, Chaudhuri M. SH2 domains recognize specific phosphopeptide sequences.  Cell . 1993;  72 767-778
• 6 Weiss A, Littman D R. Signal transduction by lymphocyte antigen receptors.  Cell . 1994;  76 263-274
• 7 Chacko G, Brandt J T, Coggeshall K M, Anderson C L. Phosphoinositide 3-kinase and p72^syk noncovalently associate with the low affinity Fcγ receptor on human platelets through an immunoreceptor tyrosine-based activation motif.  J Biol Chem . 1996;  271 10775-10781
• 8 Worthington R E, Carrol R C, Boucheix C. Platelet activation by CD9 monoclonal antibodies is mediated by the Fc γ II receptor.  Br J Haematol . 1990;  74 216-222
• 9 Kuroda K, Ozaki Y, Qi R M. Fc γ II receptor-mediated platelet activation induced by anti-CD9 monoclonal antibody opens Ca²⁺ channels which are distinct from those associated with Ca²⁺ store depletion.  J Immunol . 1995;  155 4427-4436
• 10 Qi R, Ozaki Y, Kuroda K. Differential activation of human platelets induced by Fc γ II cross-linking and by anti-CD9 monoclonal antibody.  J Immunol . 1996;  157 5638-5645
• 11 Qi R, Ozaki Y, Asazuma N. Fc γ RII tyrosine phosphorylation differs between Fc γ RII cross-linking and platelet-activating anti-platelet monoclonal antibodies.  Biochim Biophys Acta . 1999;  1451 353-363
• 12 Kroll M H, Hellums J D, Guo Z. Protein kinase C is activated in platelets subjected to pathological shear stress.  J Biol Chem . 1993;  268 3520-3508
• 13 Jackson S P, Schoenwaelder S M, Yuan Y. Adhesion receptor activation of phosphatidylinositol 3-kinase. Von Willebrand factor stimulates the cytoskeletal association and activation of phosphatidylinositol 3-kinase and pp60^c-src in human platelets.  J Biol Chem . 1994;  269 27093-27099
• 14 Ozaki Y, Satoh K, Yatomi Y. Protein tyrosine phosphorylation in human platelets induced by interaction between glycoprotein Ib and von Willebrand factor.  Biochim Biophys Acta . 1995;  1243 482-488
• 15 Yanabu M, Ozaki Y, Nomura S. Tyrosine phosphorylation and p72syk activation by an anti-glycoprotein Ib monoclonal antibody.  Blood . 1997;  89 1590-1598
• 16 Asazuma N, Ozaki Y, Satoh K. Glycoprotein Ib-von Willebrand factor interactions activate tyrosine kinases in human platelets.  Blood . 1997;  90 4789-4798
• 17 Du X, Fox J E, Pei S. Identification of a binding sequence for the 14-3-3 protein within the cytoplasmic domain of the adhesion receptor, platelet glycoprotein Ibα.  J Biol Chem . 1996;  271 7362-7367
• 18 Aakhus A M, Wilkinson J M, Solum N O. Binding of human platelet glycoprotein Ib and actin to fragments of actin-binding protein.  Thromb Haemost . 1992;  67 252-257