Identification of Protein Kinase Cα as an Essential, but Not Sufficient, Cytosolic Factor for Ca2+-induced α- and Dense-core Granule Secretion in Platelets

Akira Yoshioka,Ryutaro Shirakawa,Hiroaki Nishioka,Arata Tabuchi,Tomohito Higashi,Harunobu Ozaki,Akitsugu Yamamoto,Toru Kita,Hisanori Horiuchi

doi:10.1074/jbc.m102933200

Akira Yoshioka, Ryutaro Shirakawa + Show 7 more

Open Access

https://doi.org/10.1074/jbc.m102933200

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Abstract

Upon activation, platelets release many active substances. Here, we have analyzed the mechanism governing Ca(2+)-induced secretion of von Willebrand factor stored in alpha-granules and 5-hydroxytryptamine in dense-core granules in permeabilized human platelets. Both secretions were dependent on ATP and cytosol. An essential factor for both granule secretions was purified from rat brain cytosol and identified to be protein kinase Calpha (PKCalpha) by partial amino acid sequencing. Purified PKCalpha efficiently stimulated both secretions in the presence of cytosol, whereas PKCalpha alone did not support the secretion of either type of granules, suggesting that PKCalpha is not a sufficient factor. Finally, in human platelet cytosol fractionated by a gel filtration column, the stimulatory activity for dense-core granule secretion paralleled with the concentration of PKC, suggesting that PKC could also be such a stimulatory factor in platelet cytosol. Thus, we identified PKCalpha as an essential, but not sufficient, cytosolic factor for the Ca(2+)-induced secretions of both alpha- and dense-core granules in platelets.

Highlights

Certain types of cells contain specialized granules which are released in response to extracellular stimuli
An essential factor for both granule secretions was purified from rat brain cytosol and identified to be protein kinase C␣ (PKC␣) by partial amino acid sequencing
Purified Protein kinase C (PKC)␣ efficiently stimulated both secretions in the presence of cytosol, whereas PKC␣ alone did not support the secretion of either type of granules, suggesting that PKC␣ is not a sufficient factor

Summary

The abbreviations used are

5-HT, 5-hydroxytriptamine; vWF, von Willebrand factor; ␣-SNAP, ␣-soluble N-ethylmaleimide-sensitive factor-attachment protein; SNARE, SNAP receptor; SNAP 23, synaptosome-associated protein of 23 kDa; PKC, protein kinase C; SLO, streptolysin-O; GST, glutathione S-transferase; JAM, junctional adhesion molecule; PAGE, polyacrylamide gel electrophoresis. Ca2ϩ-induced [3H]5-HT secreting activity of all fractions was assayed under conditions in the presence of ATP and 0.5 mg of proteins/ml of rat brain cytosol. PKC inhibitors have been shown to inhibit platelet aggregation and granule release, and phorbol 12-myristate 13-acetate, a phorbol ester, have been shown to stimulate both functions [25,26,27]. PKC has been considered to be an important signaling molecule in platelet activation [7]. With reasons described above, further characterization of PKC function in platelet activation is required especially in the granule secretions. We have demonstrated that the Ca2ϩ-induced secretions of ␣- and dense-core granules in platelets are ATP- and cytosol-dependent and that PKC␣ is an essential, but not sufficient, cytosolic factor for both secretions

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION