Calpain functions in a caspase-independent manner to promote apoptosis-like events during platelet activation.
Apoptosis and platelet activation share common morphological and biochemical features. Because caspases are essential mediators of apoptosis, we examined whether platelets contain these proteinases and use them during platelet activation. Human platelets contained caspase-9, caspase-3, and the caspase activators APAF-1 and cytochrome c as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Upon treatment with cytochrome c and dATP, platelet cytoplasmic extracts recapitulated apoptotic events, including sequential activation of procaspase-9 and procaspase-3 and subsequent proteolysis of caspase substrates. Calcium ionophore-stimulated platelets also recapitulated apoptotic events, including cell shrinkage, plasma membrane microvesiculation, phosphatidyl serine externalization, and proteolysis of procaspase-9, procaspase-3, gelsolin, and protein kinase C-delta. Strikingly, however, these events occurred without caspase activation or release of mitochondrial cytochrome c, suggesting a role for a noncaspase proteinase. Supporting this, inhibition of the calcium-dependent proteinase, calpain, prevented caspase proteolysis, 'apoptotic' substrate cleavage, and platelet microvesiculation. In vitro, purified calpain cleaved recombinant procaspase-9 and procaspase-3 without activating either caspase, confirming the inhibitor studies. These data implicate calpain as a potential regulator of caspases and suggest that calpain, not caspases, promotes apoptosis-like events during platelet activation.
Wolf
B B
BB
Division of Cellular Immunology, La Jolla Institute for Allergy and Immunology, San Diego, CA 92121, USA. 102251.1444@compuserve.com
Goldstein
J C
JC
Stennicke
H R
HR
Beere
H
H
Amarante-Mendes
G P
GP
Salvesen
G S
GS
Green
D R
DR
D000595:Amino Acid Sequence; D017209:Apoptosis / Q000502:physiology; D001792:Blood Platelets / Q000473:pathology / Q000502:physiology*; D002154:Calpain / Q000502:physiology*; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases / Q000502:physiology; D006801:Humans; D008969:Molecular Sequence Data; D015539:Platelet Activation; D015398:Signal Transduction
C505407:CASP3 protein, human; C506433:CASP9 protein, human; D002154:Calpain; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases