Phosphorylation Of Platelet Proteins In Response To Activation By Phorbol 12-Myristate 13-Acetate

Abstract

We have investigated the 32P-1abelling of platelet proteins in response to 5uM to l0uM phorbol 12-myristate 13- acetate (PMA) which triggers pseudopod formation and aggregation but an atypical release without granule centralization by a contractile gel. Total platelet protein samples resolved on polyacrylamide-sodium dodecyl sulfate gels showed greater than 3 fold increases sustained over a 15 minute time course in the 32p-abelling of 260,000; 40,000; and 20,000 apparent molecular weight peptides. While similar increases in 32p-labelling are observed with other activators, such as thrombin, arachidonate, and A23187, peak phosphorylation routinely occurs between 30 to 60 seconds followed by an aggregation dependent dephosphorylation to less than 50% of peak levels between 3 to 5 minutes. The cytoskeletal cores which remain after 1% Triton X-100 extraction of platelets activated by typical stimuli contain mostly actin, myosin, and actin-binding protein. The presence in this cytoskeletal core most of the 32p-label associated with the 260,000 and 20,000 molecular weight peptides suggests that these phosphopeptide are the 260,000 molecular weight actin binding protein, and the 20,000 molecular weight myosin light chain subunits. Cytoskeletal cores prepared from PMA activated platelets still contain greater than 90% of the 32p-labelled 260,000 molecular weight peptide but less than 20% of the 32p-labelled 20,000 molecular weight peptide, most of which is found in the solubilized fraction. These results suggest that the lack of granule centralization by a contractile gel is due to a disruption of actin-myosin interaction even though the myosin light chain is phosphorylated. This effect seems to be specific in that actin-binding protein - actin interaction believed to be responsible for pseudopod formation is still present in PMA activated platelets.