CAPTURE BY CHEMICAL CROSS-LINKERS PROVIDES EVIDENCE THAT INTEGRIN ALPHA-IIB-BETA-3 FORMS A COMPLEX WITH PROTEIN-TYROSINE KINASES IN INTACT PLATELETS

Platelet activation is accompanied by a cascade of kinase reactions in which numerous specific proteins are phosphorylated on tyrosine. Thes e events are strictly dependent upon functional activation of an integ rin receptor, generally alpha IIb beta 3 (also known as glycoprotein I Ib-IIIa). It is not known how alpha IIb beta 3 regulates protein tyros ine kinase activation and, in particular, neither this nor any other i ntegrin has been shown to associate with a protein tyrosine kinase. We employed chemical crosslinking of intact platelets with the bifunctio nal reagents dithiobis(succinimidyl propionate) (DSP) (lipid-soluble) and dithiobis(sulpho succinimidyl propionate) (DTSSP) (lipid-insoluble ) followed by in vitro kinase assays of immunoprecipitated proteins to identify kinase activity associated with alpha IIb beta 3 in intact p latelets. It was found that DSP but not DTSSP crosslinked kinase activ ity to alpha IIb beta 3, suggesting an internal association. In these immunoprecipitates from DSP-crosslinked platelets, the in vitro kinase reaction revealed a complex of several phosphoproteins in association with alpha IIb beta 3. This association was not seen when the resting platelets were lysed before crosslinking, indicating the specificity of the reaction in crosslinking only molecules in preformed spatial as sociation. The beta 3 subunit of alpha IIb beta 3 was identified as on e of the phosphoproteins in the complex obtained after subjecting anti -beta 3 immunoprecipitates from lysates of DSP-treated platelets to an in vitro kinase reaction and SDS/PAGE analysis. Phosphorylation of th is subunit is shown to be predominantly on tyrosine. Affinity purifica tion of the crosslinked phosphoprotein complex with anti-beta 3 follow ed by elution and re-precipitation identified pp60(c-src) and pp54/58( c-lyn) as two protein tyrosine kinases associating with the integrin. These results suggest that, upon platelet activation, alpha IIb beta 3 may provide a transmembrane focus for proteins involved in signal tra nsduction.