Calpain Functions in a Caspase-Independent Manner to Promote Apoptosis-Like Events During Platelet Activation

Abstract

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-δ. 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.

The mechanism of apoptosis in human platelets during storage

“…This observation provides evidence that platelets, like NCs, do have a mechanism for apoptosis. During the preparation of this report, others 17,18 have reported the expression of caspases 3 and 9 in platelets.…”

“…Annexin V binding measures the surface expression of PS that increases with platelet activation and/or apoptosis. Third, Wolf et al 17 reported that, although the calcium‐dependent proteinase calpain was able to directly proteolyze procaspase 3 and procaspase 9 into smaller subunits during platelet activation, neither of their products was enzymatically active. These results therefore indicate that the increase in caspase enzymatic activity during platelet storage is related to the apoptotic mechanism and not platelet activation.…”

“…It is unlikely that this incomplete inhibition of gelsolin cleavage is due to a loss of the inhibitory effect of z‐VAD‐fmt on caspase activity, because no caspase 3 activity was detectable in platelets stored for up to 12 days in the presence of z‐VAD‐fmt (data not shown). It is more likely that gelsolin proteolysis is also occurring by a caspase‐ independent pathway such as that involving calpain 17 …”

High‐yield Platelet units revealed immediate pH decline and delayed mitochondrial dysfunction during storage in 100% plasma as compared with storage in SSP+

“…Some of the morphological and biochemical changes that characterize the PSL are reminiscent of cell death by apoptosis [3]. Platelets do contain all proteins required for apoptosis, including cytochrome C, procaspase 9, procaspase 8, procaspase 3 and the formation of active caspase 3 [4–6], illustrating that at least the intrinsic pathway of apoptosis seems to be functional [7].…”

Membrane array–based differential profiling of platelets during storage for 52 miRNAs associated with apoptosis

“…Platelets (PLTs) are enucleated cells, and during ex vivo storage, they undergo morphologic and physiologic changes also known collectively as storage lesions, which adversely affect PLT survival in vivo after transfusion 12‐15 . Apoptosis is a programmed process of cell death of nucleated cells rich in mitochondria and some artificially enucleated cells also manifest this phenomenon 16,17 . Several investigations have focused on studying PLTs in storage to understand the mechanisms underlying storage lesions and to find ways to improve PLT shelf life 12‐18 .…”