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SKELETON-ASSOCIATED AND CYTOSOLIC PROTEIN TYROSINE KINASE ACTIVITY IN STIMULATED PLATELETS

By Ferri A., Calza R. and Cannoletta R.

Abstract

Platelets contain high levels of non-receptor protein tyrosine kinases, of which the most abundant is p60c-src (about 0.4% of total platelets proteins) suggesting that it has one or more important roles in platelets functions. Reversible interactions of p60c-src with contractile proteins, including actin, occurs via its SH3 and/or SH2 domains but it is unknown how this interaction influences the organization of microfilaments. We compared platelets aggregation induced by an extracellular agonist (ristocetin supplemented with plasma von Willebrand factor (Rist.)) with that promoted by an intracellular, non physiological stimulus (vanadate(V), obtained by mixing equimolecular amounts of vanadyl(IV)acetylacetonate and hydrogen peroxide). Addition of either vanadate or ristocetin to washed platelets induced aggregation accompanied with increased phosphotyrosine levels in various proteins (20-40 Kd, 60 Kd, 70-100 Kd, 200 Kd). Among these, the 60 Kd band resulted to be the most affected in both cases.\ud Vanadate or ristocetin stimulated platelets were lysed and divided in three fractions: cytosol, cytoskeleton and membrane-associated skeleton; changes in actin and 60Kd protein distribution and changes in phosphotyrosine levels in the 60 Kd protein in these fractions were compared with those obtained after fractionation of resting platelets. Aggregation induced by vanadate was accompanied with increases in phosphotyrosine levels of either cytosolic and cytoskeleton-bound 60 Kd protein; the cytoskeletal 60 Kd phosphotyrosine level increase is effectively due to an increase in the 60 Kd protein amount, accompanied by a similar increase in cytoskeletal actin, whereas the increase in phosphotyrosine level in the cytosolic 60 Kd protein is due to an increase of a cytosolic kinase (or autokinase) activity, being it accompanied with a marked decrease in both actin and 60 Kd protein amounts in this fraction. No appreciable changes were observed in the membrane-associated skeleton. These results can be explained by supposing that vanadate induces phosphorylation (or autophosphorylation) in the cytosolic 60 Kd protein that becomes capable to act as nucleating center on which new actin filaments can grow from cytosolic G-actin. Results obtained by ristocetin-induced aggregation are consistent with this hypothesis. Ristocetin induced aggregation was accompanied with a decrease in both actin and 60 Kd protein amounts either in the cytoskeleton and in the membrane-associated skeleton (and consequently the amounts of these proteins increased in the cytosol). Also the 60 Kd phosphotyrosine levels resulted to be decreased in the skeletal fractions and increased in the cytosol. This can be consequence of a marked disassembly of microfilaments starting from agonist-induced membrane changes, accompanied with actin and 60 Kd protein migration to the cytosol in which the 60 Kd protein can undergo to phosphorylation (or autophosphorylation) and initiate contractile systems reorganization: The observed decrease in skeletal actin amount after ristocetin induced aggregation can be due to the fact that that this process is followed by wery weak secretion of granules content, therefore after massive depolymerization of microfilaments induced by the external signal, a strong repolymerization is not required

Publisher: IBST
Year: 1996
OAI identifier: oai:iris.unife.it:11392/1739308
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