Acid sphingomyelinase regulates platelet cell membrane scrambling, secretion, and thrombus formation.
OBJECTIVE
Platelet activation is essential for primary hemostasis and acute thrombotic vascular occlusions. On activation, platelets release their prothrombotic granules and expose phosphatidylserine, thus fostering thrombin generation and thrombus formation. In other cell types, both degranulation and phosphatidylserine exposure are modified by sphingomyelinase-dependent formation of ceramide. The present study thus explored whether acid sphingomyelinase participates in the regulation of platelet secretion, phosphatidylserine exposure, and thrombus formation.


RESULTS
Collagen-related peptide-induced or thrombin-induced ATP release and P-selectin exposure were significantly blunted in platelets from Asm-deficient mice (Smpd1(-/-)) when compared with platelets from wild-type mice (Smpd1(+/+)). Moreover, phosphatidylserine exposure and thrombin generation were significantly less pronounced in Smpd1(-/-) platelets than in Smpd1(+/+) platelets. In contrast, platelet integrin αIIbβ3 activation and aggregation, as well as activation-dependent Ca(2+) flux, were not significantly different between Smpd1(-/-) and Smpd1(+/+) platelets. In vitro thrombus formation at shear rates of 1700 s(-1) and in vivo thrombus formation after FeCl3 injury were significantly blunted in Smpd1(-/-) mice while bleeding time was unaffected. Asm-deficient platelets showed significantly reduced activation-dependent ceramide formation, whereas exogenous ceramide rescued diminished platelet secretion and thrombus formation caused by Asm deficiency. Treatment of Smpd1(+/+) platelets with bacterial sphingomyelinase (0.01 U/mL) increased, whereas treatment with functional acid sphingomyelinase-inhibitors, amitriptyline or fluoxetine (5 μmol/L), blunted activation-dependent platelet degranulation, phosphatidylserine exposure, and thrombus formation. Impaired degranulation and thrombus formation of Smpd1(-/-) platelets were again overcome by exogenous bacterial sphingomyelinase.


CONCLUSIONS
Acid sphingomyelinase is a completely novel element in the regulation of platelet plasma membrane properties, secretion, and thrombus formation.
From the Departments of Physiology (P.M., O.B., B.W., E.S., E.-M.S., S.S., S.T.T., C.L., F.L.), Cardiology and Cardiovascular Medicine (O.B., M.C., M.E., P.S., A.E.M., M.G.), and Dermatology (M.S.), University of Tübingen, Germany; Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands (M.A. H.F., J.M.E.M.C., J.W.M.H.); Bristol-Myers Squibb GmbH&CoKGaA, Munich, Germany (K.F.); Institute of Molecular Biology, University of Duisburg-Essen, Essen, Germany (E.G.); and Department of Clinical and Experimental Hemostasis, Heinrich Heine University Düsseldorf, Düsseldorf, Germany (M.E.).
Patrick
P
Münzer
Oliver
O
Borst
Britta
B
Walker
Evi
E
Schmid
Marion A H
MA
Feijge
Judith M E M
JM
Cosemans
Madhumita
M
Chatterjee
Eva-Maria
EM
Schmidt
Sebastian
S
Schmidt
Syeda T
ST
Towhid
Christina
C
Leibrock
Margitta
M
Elvers
Martin
M
Schaller
Peter
P
Seizer
Klaus
K
Ferlinz
Andreas E
AE
May
Erich
E
Gulbins
Johan W M
JW
Heemskerk
Meinrad
M
Gawaz
Florian
F
Lang
D000255:Adenosine Triphosphate / Q000097:blood; D000818:Animals; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology*; D002118:Calcium / Q000097:blood; D015550:Cell Degranulation / Q000187:drug effects; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology*; D002518:Ceramides / Q000097:blood; D002712:Chlorides; D004195:Disease Models, Animal; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D005290:Ferric Compounds; D005343:Fibrinolytic Agents / Q000494:pharmacology; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D019007:P-Selectin / Q000097:blood; D010718:Phosphatidylserines / Q000097:blood; D015539:Platelet Activation / Q000187:drug effects; D010974:Platelet Aggregation; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism; D013108:Sphingomyelin Phosphodiesterase / Q000037:antagonists & inhibitors / Q000097:blood* / Q000172:deficiency / Q000235:genetics; D013917:Thrombin / Q000378:metabolism; D013927:Thrombosis / Q000097:blood / Q000139:chemically induced / Q000201:enzymology* / Q000235:genetics / Q000517:prevention & control; D013997:Time Factors
D002518:Ceramides; D002712:Chlorides; D004791:Enzyme Inhibitors; D005290:Ferric Compounds; D005343:Fibrinolytic Agents; D019007:P-Selectin; D010718:Phosphatidylserines; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D000255:Adenosine Triphosphate; C577975:ASMase, mouse; D013108:Sphingomyelin Phosphodiesterase; D013917:Thrombin; D002118:Calcium; C024555:ferric chloride
acid sphingomyelinase; amitriptyline; granule secretion; phosphatidylserine exposure; platelets; thrombin generation; thrombus formation