Conventional calpains are ubiquitous calcium-regulated cysteine proteases that have been implicated in cytoskeletal organization, cell proliferation, apoptosis, cell motility, and hemostasis. There are two forms of conventional calpains: the -calpain, or calpain I, which requires micromolar calcium for half-maximal activation, and the m-calpain, or calpain II, which functions at millimolar calcium concentrations. We evaluated the functional role of the 80-kDa catalytic subunit of -calpain by genetic inactivation using homologous recombination in embryonic stem cells. The -calpain-deficient mice are viable and fertile. The complete deficiency of -calpain causes significant reduction in platelet aggregation and clot retraction but surprisingly the mutant mice display normal bleeding times. No detectable differences were observed in the cleavage pattern and kinetics of calpain substrates such as the ␤3 subunit of ␣IIb␤3 integrin, talin, and ABP-280 (filamin). However, -calpain null platelets exhibit impaired tyrosine phosphorylation of several proteins including the ␤3 subunit of ␣IIb␤3 integrin, correlating with the agonist-induced reduction in platelet aggregation. These results provide the first direct evidence that -calpain is essential for normal platelet function, not by affecting the cleavage of cytoskeletal proteins but by potentially regulating the state of tyrosine phosphorylation of the platelet proteins.The calpains are a family of calcium-dependent neutral cysteine proteases present in essentially all tissues of higher animals (8,34,37). Calpain homologues distantly related to the catalytic subunits of conventional calpains are also found in lower organisms such as parasites, insects, nematodes, fungi, and yeast (34). They are believed to play functionally important roles in diverse biological processes such as reorganization of cortical cytoskeleton, cell motility, cell proliferation, apoptosis, and hemostasis (9,27,31,39). Calpains are divided into two broad classes, ubiquitous and tissue specific. Calpain I (also referred to as -calpain) and calpain II (also referred to as m-calpain) are expressed in all tissues in varying amounts and share ϳ61% sequence identity (20). Both the -and m-calpains contain an 80-kDa catalytic subunit that forms a heterodimer with the regulatory 30-kDa subunit (34). The 80-kDa catalytic subunits of the -and m-calpains are products of separate but closely related genes (referred to as Capn1 and Capn2, respectively), while the 30-kDa subunit (encoded by the Capn4 gene) is common to both (34). The -calpain is fully active in micromolar concentrations of calcium, while the mcalpain requires millimolar calcium concentrations for full activation. Larger tissue-specific calpains have been cloned from stomach and smooth muscle tissues (35, 37). Mutations of the muscle-specific Capn3 (calpain 3 gene) have been shown to cause one form of limb-girdle muscular dystrophy type 2A (30). More recently, several groups have identified CAPN10 (calpain 10) as the target gene for mutations in...

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Dematin is an actin-binding and bundling protein of the erythrocyte membrane skeleton. Dematin is localized to the spectrin-actin junctions, and its actin-bundling activity is regulated by phosphorylation of cAMP-dependent protein kinase. The carboxyl terminus of dematin is homologous to the ''headpiece'' domain of villin, an actin-bundling protein of the microvillus cytoskeleton. The headpiece domain contains an actin-binding site, a cAMP-kinase phosphorylation site, plays an essential role in dematin self-assembly, and bundles F-actin in vitro. By using homologous recombination in mouse embryonic stem cells, the headpiece domain of dematin was deleted to evaluate its function in vivo. Dematin headpiece null mice were viable and born at the expected Mendelian ratio. Hematological evaluation revealed evidence of compensated anemia and spherocytosis in the dematin headpiece null mice. The headpiece null erythrocytes were osmotically fragile, and ektacytometry͞micropore filtration measurements demonstrated reduced deformability and filterability. In vitro membrane stability measurements indicated significantly greater membrane fragmentation of the dematin headpiece null erythrocytes. Finally, biochemical characterization, including the vesicle͞cytoskel-eton dissociation, spectrin self-association, and chemical crosslinking measurements, revealed a weakened membrane skeleton evidenced by reduced association of spectrin and actin to the plasma membrane. Together, these results provide evidence for the physiological significance of dematin and demonstrate a role for the headpiece domain in the maintenance of structural integrity and mechanical properties of erythrocytes in vivo.T he membrane bilayer and the network of membrane-associated proteins together regulate the characteristic shape and elastic properties of red blood cells (1, 2). When membrane skeletons are prepared in the presence of a high concentration of monovalent salt, the core of the membrane skeleton consists of spectrin, actin, protein 4.1, and dematin (3). Although the functions of spectrin, actin, and protein 4.1 have been extensively characterized (4, 5), virtually nothing is known about the physiological function in mature erythrocytes of dematin, a three-subunit protein that migrates in the protein 4.9 region during electrophoresis. The earliest evidence suggesting a membrane stabilizing role for dematin came from Holdstock and Ralston (6). They demonstrated that charged sulfhydryl compounds such as p-chloromercuribenzene sulfonateS preferentially attach to dematin and cause the disruption of erythrocyte cytoskeletons. Dematin is a substrate for multiple protein kinases, and phosphorylation of dematin by the cAMP-dependent protein kinase is known to regulate dematin's actin-bundling activity in vitro (7-9). The major phosphorylation site of the cAMPdependent protein kinase is located within the headpiece domain of dematin (10), but the physiological significance of dematin phosphorylation is not known.Siegel and Branton (11) conducted the first ...