Characterization of sphingomyelinase activity released by thrombin-stimulated platelets.
In this study we report that human platelets display neutral (nSMase) and acid sphingomyelinase (aSMase) as well as acid ceramidase (aCerase) activity. Cell activation by thrombin resulted in a marked decrease of intracellular aSMase activity, accompanied by the release of enzyme into the medium. In contrast, thrombin treatment did not affect aCerase activity. Two major protein bands of 73 and 70 kDa were recognized by aSMase antibodies in resting platelet lysates and in the medium of stimulated cells. Phorbol esters together with the calcium ionophore A23187 fully reproduced thrombin action on aSMase release. The secreted enzymatic activity was insensitive to digestion with endoglycosidase H but it was stimulated by Zn2+, although to a limited extent compared to aSMase constitutively released by murine endothelial cells. Taken together, these data suggest that secreted aSMase does not originate from the lysosomal compartment but rather from other platelet vesicles.
Romiti
E
E
Dipartimento di Scienze Biochimiche, Università di Firenze, Florence, Italy.
Vasta
V
V
Meacci
E
E
Farnararo
M
M
Linke
T
T
Ferlinz
K
K
Sandhoff
K
K
Bruni
P
P
D000818:Animals; D001792:Blood Platelets / Q000187:drug effects* / Q000201:enzymology*; D015153:Blotting, Western; D000001:Calcimycin / Q000494:pharmacology; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D004727:Endothelium / Q000378:metabolism; D002517:Galactosylgalactosylglucosylceramidase / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000494:pharmacology; D006801:Humans; D007476:Ionophores / Q000494:pharmacology; D051379:Mice; D010703:Phorbol Esters / Q000494:pharmacology; D015539:Platelet Activation; D011233:Precipitin Tests; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism*; D013917:Thrombin / Q000494:pharmacology*; D015032:Zinc / Q000378:metabolism
D007476:Ionophores; D010703:Phorbol Esters; D000001:Calcimycin; D013108:Sphingomyelin Phosphodiesterase; D006026:Glycoside Hydrolases; D002517:Galactosylgalactosylglucosylceramidase; D013917:Thrombin; D015032:Zinc