The relative contributions of apoptosis and necrosis in brain injury have been a matter of much debate. Caspase-3 has been identified as a key protease in the execution of apoptosis, whereas calpains have mainly been implicated in excitotoxic neuronal injury. In a model of unilateral hypoxia-ischemia in 7-day-old rats, caspase-3-like activity increased 16-fold 24 h postinsult, coinciding with cleavage of the caspase-3 proenzyme and endogenous caspase-3 substrates. This activation was significantly decreased by pharmacological calpain inhibition, using CX295, a calpain inhibitor that did not inhibit purified caspase-3 in vitro. Activation of caspase-3 by m-calpain, but not -calpain, was facilitated in a dose-dependent manner in vitro by incubating cytosolic fractions, containing caspase-3 proform, with calpains. This facilitation required the presence of some active caspase-3 and could be abolished by including the specific calpain inhibitor calpastatin. This indicates that initial cleavage of caspase-3 by m-calpain, producing a 29-kDa fragment, facilitates the subsequent cleavage into active forms. This is the first report to our knowledge suggesting a direct link between the early, excitotoxic, calcium-mediated activation of calpain after cerebral hypoxia-ischemia and the subsequent activation of caspase-3, thus representing a tentative pathway of "pathological apoptosis."The relative contributions of necrosis and apoptosis to the injury that develops after cerebral hypoxia-ischemia (HI) 1 has been a matter of much debate (1). Recent studies suggest that cell death after HI is different from developmentally regulated cell death in most cases and cannot appropriately be described as apoptotic (2-5). Nevertheless, HI cell death shares important morphological and biochemical features with apoptotic cell death, such as activation of caspases and nucleosomal DNA fragmentation (6 -17). Caspases, a family of cysteine proteases with an unusual substrate specificity, requiring an aspartate residue in the P1 position, have been identified as key executors of apoptosis (18). Calpains, another family of cysteine proteases, are calcium-activated and are proposed to participate in the turnover of cytoskeletal proteins and regulation of kinases, transcription factors, and receptors (19,20). Calpains have mainly been implicated in excitotoxic neuronal injury and necrosis (21-23). Pharmacological inhibitors of calpains and caspases exert cerebroprotective effects (9, 14 -16, 24 -26). A growing body of literature has emerged, demonstrating functional connections between calpains and caspases (27). Common substrate proteins have been identified, such as fodrin (28 -31), calpastatin (32, 33), actin (34), PARP (35), and tau (36). There are reports demonstrating calpain-mediated cleavage of caspase-3 (35, 37) and caspase-7 (38, 39) as well as caspase-8 and -9 (39). Furthermore, the proapoptotic protein Bax was cleaved by calpain during drug-induced apoptosis of HL-60 cells (40), and calpain may be responsible for cleaving the loop r...