Abstract
Sphingosine is a potent inhibitor of [3H]phorbol dibutyrate binding and protein kinase C activity in vitro and in human platelets (Hannun, Y., Loomis, C., Merrill, A., and Bell, R. (1986) J. Biol. Chem. 261, 12604-12609). Preincubation of platelets with sphingosine resulted in the inhibition of platelet secretion and second phase aggregation in response to ADP, gamma-thrombin, collagen, arachidonic acid, and platelet activating factor. Sphingosine did not affect the initial shape change of platelets or the first phase of aggregation in response to these agonists. Ristocetin-induced platelet agglutination was not affected by sphingosine. Sphingosine inhibition of secondary aggregation (secretion and second phase aggregation) was overcome by phorbol dibutyrate and by the cell-permeable protein kinase C activator, dioctanoylglycerol. Furthermore, platelet secretion and irreversible aggregation were induced by protein kinase C activators in platelets that had been "primed" to undergo initial shape change and first phase aggregation by low concentrations of agonists. These results suggest that protein kinase C activation is a necessary component in the signal transducing pathways that lead to platelet activation. Higher concentrations of agonists, however, induced irreversible aggregation and partial secretion in the presence of sphingosine, suggesting the existence of protein kinase C-independent pathways for platelet activation. These results demonstrate the utility of sphingosine as a pharmacologic tool in probing the role of protein kinase C in signal transduction.
Highlights
12609).Preincubation of platelets with sphingosine resulted in theinhibition of platelet secretion andsecond phase aggregation inresponse to ADP, y-thrombin, collagen, arachidonic acid, and platelet activatingfactor
Secretion and irreversible aggregation were induced In contrast,diCaand OAG, two cell-permeable protein kinase by protein kinase C activators in platelets that had C activators,fail to induce either aggregation or secretion (12, been “primed”to undergo initial shacpheange and first 13). These seemingly contradictory results may be a consephase aggregation by low concentrations of agonists. quence of phorbol ester action on targets other than protein. These results suggest that protein kinase C activation kinase C, or may result from the rapid metabolism ofDAG
Arachidonic acid and ristocetin were from Bio/Data (Hatboro, PA). [3H]PDBu (12.5 Ci/nmol) was from New England Nuclear
Summary
Materials-Sphingosine, PDBu, collagen, and ADP were from Sigma. Arachidonic acid and ristocetin were from Bio/Data (Hatboro, PA). [3H]PDBu (12.5 Ci/nmol) was from New England Nuclear. [3H]PDBu (12.5 Ci/nmol) was from New England Nuclear. Luciferin-luciferase reagent (Chromo-Lume) was from Chrono-Log Corp. N-Acetylsphingosine was prepared from sphingosine as described [20].y-Thrombin was a gift from John Fenton 11, Wadsworth Center for Laboratories, New York State Department of Health, Albany, NY. Preparation of Human Platelets-Platelet rich plasma (PRP) was prepared as described [23] and diluted to a final concentration of 3.5 X IORplatelets/ml. Washed platelets were prepared according to Siess et al [24]and suspended in modified Tyrode's buffer to a concentration of 3.5 X 10%. Platelet Aggregation and ATP Secretion-Aggregation of PRP and secretion of ATP were measured using a Chrono-Log Lumi-Aggregometer as described [25]. For quantitation of total ATP secretion, Chromo-Lume was added after thecompletion of aggregation. DAG Quantitation-Diacylglycerol mass in platelet extracts was quantitated using an assay employing Esherichiu coli DAG kinase [26]
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