Platelet senescence and phosphatidylserine exposure.
BACKGROUND
The exposure of phosphatidylserine occurs during platelet (PLT) activation and during in vitro storage. Phosphatidylserine exposure also occurs during apoptosis after the release of mitochondrial cytochrome c. We have examined the role of cytochrome c release, mitochondrial membrane potential (ΔΨm), and cyclophilin D (CypD) in phosphatidylserine exposure due to activation and storage.


METHODS
The exposure of phosphatidylserine and the loss of ΔΨm were determined in a flow cytometer using fluorescein isothiocyanate-lactadherin and JC-1, a lipophilic cationic reporter dye. The role of CypD was determined with cyclosporin A and CypD-deficient murine PLTs. Cytochrome c-induced caspase-3 and Rho-associated kinase I (ROCK1) activation were determined by immunoblotting and using their inhibitors.


RESULTS
Collagen- and thrombin-induced exposure of phosphatidylserine was accompanied by a decrease in ΔΨm. Cyclosporin A inhibited the phosphatidylserine exposure and the loss of ΔΨm. CypD(-/-) mice had decreased loss of ΔΨm and impaired phosphatidylserine exposure. Collagen and thrombin did not induce the release of cytochrome c nor the activation of caspase-3 and ROCK1. In contrast, in PLTs stored for more than 5 days, the phosphatidylserine exposure was associated with cytochrome c-induced caspase-3 and ROCK1 activation. ABT737, a BH3 mimetic that induces mitochondrial pathway of apoptosis, induced cytochrome c release and activation of caspase-3 and ROCK1 and phosphatidylserine exposure independent of CypD.


CONCLUSIONS
These results show that in stored PLTs cytochrome c release and the subsequent activation of caspase-3 and ROCK1 mediate phosphatidylserine exposure and it is distinct from activation-induced phosphatidylserine exposure.
Dasgupta
Swapan Kumar
SK
Michael E. DeBakey Veterans Affairs Medical Center, Department of Pathology, Baylor College of Medicine.
Argaiz
Eduardo Rios
ER
Mercado
Jose Emmanel Chedid
JE
Maul
Hector Omar Elizondo
HO
Garza
Jorge
J
Enriquez
Ana Bety
AB
Abdel-Monem
Hanan
H
Prakasam
Anthony
A
Andreeff
Michael
M
Thiagarajan
Perumal
P
D000818:Animals; D001792:Blood Platelets / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D016922:Cellular Senescence / Q000187:drug effects* / Q000235:genetics; D000081406:Peptidyl-Prolyl Isomerase F; D021983:Cyclophilins / Q000235:genetics; D045304:Cytochromes c / Q000378:metabolism; D005434:Flow Cytometry; D051379:Mice; D018345:Mice, Knockout; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D010718:Phosphatidylserines / Q000494:pharmacology*; D015539:Platelet Activation / Q000187:drug effects* / Q000235:genetics
D000081406:Peptidyl-Prolyl Isomerase F; C000631446:PPIF protein, mouse; D010718:Phosphatidylserines; D045304:Cytochromes c; D021983:Cyclophilins
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