Calpain functions in a caspase-independent manner to promote apoptosis-like events during platelet activation

Apoptosis and platelet activation share common morphological and biochemica l features. Because caspases are essential mediators of apoptosis, we exami ned whether platelets contain these proteinases and use them during platele t activation. Human platelets contained caspase-9, caspase-3, and the caspa se activators APAF-1 and cytochrome c as shown by sodium dodecyl sulfate-po lyacrylamide gel electrophoresis and Western blotting. Upon treatment with cytochrome c and dATP, platelet cytoplasmic extracts recapitulated apoptoti c events, including sequential activation of procaspase-9 and procaspase-3 and subsequent proteolysis of caspase substrates. Calcium ionophore-stimula ted platelets also recapitulated apoptotic events, including cell shrinkage , plasma membrane microvesiculation, phosphatidyl serine externalization, a nd proteolysis of procaspase-9, procaspase-3, gelsolin, and protein kinase C-delta. Strikingly, however, these events occurred without caspase activat ion or release of mitochondrial cytochrome c, suggesting a role for a nonca spase proteinase. Supporting this, inhibition of the calcium-dependent prot einase, calpain, prevented caspase proteolysis, 'apoptotic' substrate cleav age, and platelet microvesiculation. In vitro, purified calpain cleaved rec ombinant procaspase-9 and procaspase-3 without activating either caspase, c onfirming the inhibitor studies. These data implicate calpain as a potentia l regulator of caspases and suggest that calpain, not caspases, promotes ap optosis-like events during platelet activation. (C) 1999 by The American So ciety of Hematology.