SNARE protein degradation upon platelet activation: calpain cleaves SNAP-23

In order to better understand the molecular mechanisms of platelet granule secretion, we evaluated the effect of activation-induced degranulation on three functional platelet SNARE proteins, SNAP-23, VAMP-3, and syntaxin 4. Initial studies showed that SNAP-23 is lost upon SFLLRN-induced platelet activation. Experiments with permeabilized platelets demonstrated that proteolysis of SNAP-23 was Ca(2+)-dependent. Ca(2+)-dependent proteolysis of SNAP-23 was inhibited by the cell-permeable calpain inhibitors, calpeptin and E-64d, as well as by the naturally occurring calpain inhibitor, calpastatin. In addition, purified calpain cleaved SNAP-23 in permeabilized platelets in a dose-dependent manner. In intact platelets, calpeptin prevented SFLLRN-induced degradation of SNAP-23. In contrast, calpeptin did not prevent SFLLRN-induced degradation of VAMP-3 and syntaxin 4 did not undergo substantial proteolysis following platelet activation. Calpain-induced cleavage of SNAP-23 was a late event occurring between 2.5 and 5 min following exposure of permeabilized platelets to Ca(2+). Experiments evaluating platelet alpha-granule secretion demonstrated that incubation of permeabilized platelets with 10 microM Ca(2+) prior to exposure to ATP inhibited ATP-dependent alpha-granule secretion from permeabilized platelets. SNAP-23 was cleaved under these conditions. Incubation of permeabilized platelets with either calpeptin or calpastatin prevented Ca(2+)-mediated degradation of SNAP-23 and reversed Ca(2+)-mediated inhibition of ATP-dependent alpha-granule secretion. Thus, activation of calpain prior to secretion results in loss of SNAP-23 and inhibits alpha-granule secretion. These studies suggest a mechanism whereby calpain activation serves to localize platelet secretion to areas of thrombus formation.     

Domain II of m-calpain is a Ca(2+)-dependent cysteine protease

Calpain, a Ca(2+)-dependent cytosolic cysteine protease, proteolytically modulates specific substrates involved in Ca(2+)-mediated intracellular events, such as signal transduction, cell cycle, differentiation, and apoptosis. The 3D structure of m-calpain, in the absence of Ca(2+), revealed that the two subdomains (domains IIa and IIb) of the protease domain (II) have an 'open' conformation, probably due to interactions with other domains. Although the presence of an EF-hand structure was once predicted in the protease domain, no explicit Ca(2+)-binding structure was identified in the 3D structure. Therefore, it is predicted that if the protease domain is excised from the calpain molecule, it will have a Ca(2+)-independent protease activity. In this study, we have characterized a truncated human m-calpain that consists of only the protease domain. Unexpectedly, the proteolytic activity was Ca(2+)-dependent, very weak, and not effectively inhibited by calpastatin, a calpain inhibitor. Ca(2+)-dependent modification of the protease domain by the cysteine protease inhibitor, E-64c, was clearly observed as a SDS-PAGE migration change, indicating that the conformational changes of this domain are a result of Ca(2+) binding. These results suggest that the Ca(2+) binding to domain II, as well as to domains III, IV, and VI, is critical in the process of complete activation of calpain.     

Association of calpastatin with inactive calpain: a novel mechanism to control the activation of the protease?

It is generally accepted that the Ca(2+)-dependent interaction of calpain with calpastatin is the most relevant mechanism involved in the regulation of Ca(2+)-induced proteolysis. We now report that a calpain-calpastatin association can occur also in the absence of Ca(2+) or at very low Ca(2+) concentrations, reflecting the physiological conditions under which calpain retains its inactive conformational state. The calpastatin binding region is localized in the non-inhibitory L-domain containing the amino acid sequences encoded by exons 4-7. This calpastatin region recognizes a calpain sequence located near the end of the DII-domain. Interaction of calpain with calpastatins lacking these sequences becomes strictly Ca(2+)-dependent because, under these conditions, the transition to an active state of the protease is an obligatory requirement. The occurrence of the molecular association between Ca(2+)-free calpain and various recombinant calpastatin forms has been demonstrated by the following experimental results. Addition of calpastatin protected calpain from trypsin digestion. Calpain was coprecipitated when calpastatin was immunoprecipitated. The calpastatin molecular size increased following exposure to calpain. The two proteins comigrated in zymogram analysis. Furthermore, calpain-calpastatin interaction was perturbed by protein kinase C phosphorylation occurring at sites located at the exons involved in the association. At a functional level, calpain-calpastatin interaction at a physiological concentration of Ca(2+) represents a novel mechanism for the control of the amount of the active form of the protease potentially generated in response to an intracellular Ca(2+) influx.     

Purification and characterization of a Ca(2+)-activated thiol protease from Drosophila melanogaster.

A Ca(2+)-activated thiol protease was purified from Drosophila melanogaster. The procedure involves Phenyl-Sepharose, Reactive Red-Agarose and Q-Sepharose fast flow (or MonoQ) chromatographic steps. The enzyme eluting from Q-Sepharose fast flow seems to be homogeneous as judged by silver staining on SDS-PAGE: it consists of a single polypeptide chain of M(r),app = 94K and pI = 5.46. The proteolytic activity of the purified enzyme is absolutely Ca(2+)-dependent, characterized by 0.6 mM free Ca2+ at half-maximal activity. Ca2+ ions cannot be replaced effectively by the divalent cations Mg2+, Mn2+, Zn2+, Ba2+, and Cd2+. The enzyme shows the inhibitor pattern of thiol proteases. Human recombinant calpastatin (domain I) completely inhibits the enzyme at a nearly 1:1 molar ratio. Several of these properties resemble those of vertebrate calpain II. However, various attempts to detect a small subunit of M(r) approximately 30K, common with vertebrate calpains, remained unsuccessful. We suggest that the Drosophila enzyme is a novel calpain II-like protease.     

Calpain-dependent proteolytic cleavage of the p35 cyclin-dependent kinase 5 activator to p25

Cyclin-dependent kinase 5 (CDK5) is a unique CDK, the activity of which can be detected in postmitotic neurons. To date, CDK5 purified from mammalian brains has always been associated with a truncated form of the 35-kDa major brain specific activator (p35, also known as nck5a) of CDK5, known as p25. In this study, we report that p35 can be cleaved to p25 both in vitro and in vivo by calpain. In a rat brain extract, p35 was cleaved to p25 by incubation with Ca(2+). This cleavage was inhibited by a calpain inhibitor peptide derived from calpastatin and was ablated by separating the p35.CDK5 from calpain by centrifugation. The p35 recovered in the pellet after centrifugation could then be cleaved to p25 by purified calpain. Cleavage of p35 was also induced in primary cultured neurons by treatment with a Ca(2+) ionophore and Ca(2+) and inhibited by calpain inhibitor I. The cleavage changed the solubility of the CDK5 active complex from the particulate fraction to the soluble fraction but did not affect the histone H1 kinase activity. Increased cleavage was detected in cultured neurons undergoing cell death, suggesting a role of the cleavage in neuronal cell death.     

Evidence that activation of platelet calpain is induced as a consequence of binding of adhesive ligand to the integrin, glycoprotein IIb-IIIa

Calpain (a Ca(2+)-dependent protease) is present in many cell types. Because it is present in the cytosol, the potential exists that it may regulate critical intracellular events by inducing crucial proteolytic cleavages. However, the concentrations of Ca2+ required to activate calpain are higher than those attained in the cytoplasm of most cells. Thus, the physiological importance of calpain and the mechanisms involved in its activation have remained elusive. In this study, we show that calpain rapidly moved to a peripheral location upon the addition of an agonist to suspensions of platelets, but it remained unactivated. We provide three lines of evidence that calpain was subsequently activated by a mechanism that required the binding of an adhesive ligand to the major platelet integrin, glycoprotein (GP) IIb- IIIa: calpain activation was prevented by RGDS, a tetrapeptide that inhibits the binding of adhesive ligand to GP IIb-IIIa; it was also prevented by monoclonal antibodies that inhibit adhesive ligand binding to GP IIb-IIIa; and its activation was markedly reduced in platelets from patients whose platelets have greatly reduced levels of functional GP IIb-IIIa. Thus, in platelets, binding of the extracellular domain of GP IIb-IIIa to its adhesive ligand can initiate a transmembrane signal that activates intracellular calpain. Because calpain is present in focal contacts of adherent cells, the interaction of integrins with adhesive ligands in the extracellular matrix may regulate activation of calpain in other cell types as well.     

Evidence that agonist-induced activation of calpain causes the shedding of procoagulant-containing microvesicles from the membrane of aggregating platelets

One of the responses of platelets to stimulation is activation of intracellular calpain (the Ca(2+)-dependent protease). Previously, we have shown that activation of calpain in platelets is involved in the generation of platelet procoagulant activity. Because procoagulant activity is present on the microvesicles that are shed from activated platelets, in this study we examined whether calpain is involved in the shedding of microvesicles. Platelets were incubated with the physiological agonists collagen or thrombin. The extent of activation of calpain correlated positively with the amount of procoagulant-containing microvesicles that formed, and the shedding of procoagulant-containing microvesicles was inhibited by calpeptin, MDL, and EST (E-64-d), three membrane-penetrating inhibitors of calpain. The protein composition of the microvesicles shed from aggregating platelets was similar to that of microvesicles shed by platelets in which the association of the membrane skeleton with the plasma membrane had been disrupted by incubation of platelets with dibucaine or ionophore A23187. Furthermore, like microvesicles shed from dibucaine- or ionophore A23187-treated platelets, those shed from the aggregating platelets possessed procoagulant activity. These results are consistent with the possibility that activation of calpain in aggregating platelets causes the shedding of procoagulant-containing microvesicles. We suggest that the shedding of microvesicles results from the calpain-induced hydrolysis of the platelet membrane skeleton.     

A role for the intracellular protease calpain in the activation of store-operated calcium entry in human platelets

Here, we report a novel role for the cysteine protease calpain in store-operated calcium entry. Several structurally and mechanistically unrelated inhibitors of calpain inhibited Ca2+ entry activated in human platelets by thapsigargin-evoked Ca2+ store depletion or the physiological agonist thrombin, whereas inhibitors of other cysteine proteases were without effect. The use of the cell-permeable fluorogenic calpain substrate 7-amino-4-chloromethylcoumarin, t-BOC-l-leucyl-l-methionine amide revealed rapid activation of calpain which was closely temporally correlated with Ca2+ store depletion even in the absence of a rise in cytosolic [Ca2+]. Calpain inhibition prevented the tyrosine phosphorylation of several proteins upon Ca2+ store depletion, suggesting that calpain may lie upstream of protein tyrosine phosphorylation that is known to be required for the activation of store-operated Ca2+ entry in human platelets. Earlier studies using calpain inhibitors may need reinterpretation in the light of this finding that calpain plays a role in the activation of physiological Ca2+ entry pathways.     

Dissociation and aggregation of calpain in the presence of calcium

Calpain is a heterodimeric Ca(2+)-dependent cysteine protease consisting of a large (80 kDa) catalytic subunit and a small (28 kDa) regulatory subunit. The effects of Ca(2+) on the enzyme include activation, aggregation, and autolysis. They may also include subunit dissociation, which has been the subject of some debate. Using the inactive C105S-80k/21k form of calpain to eliminate autolysis, we have studied its disassociation and aggregation in the presence of Ca(2+) and the inhibition of its aggregation by means of crystallization, light scattering, and sedimentation. Aggregation, as assessed by light scattering, depended on the ionic strength and pH of the buffer, on the Ca(2+) concentration, and on the presence or absence of calpastatin. At low ionic strength, calpain aggregated rapidly in the presence of Ca(2+), but this was fully reversible by EDTA. With Ca(2+) in 0.2 m NaCl, no aggregation was visible but ultracentrifugation showed that a mixture of soluble high molecular weight complexes was present. Calpastatin prevented aggregation, leading instead to the formation of a calpastatin-calpain complex. Crystallization in the presence of Ca(2+) gave rise to crystals mixed with an amorphous precipitate. The crystals contained only the small subunit, thereby demonstrating subunit dissociation, and the precipitate was highly enriched in the large subunit. Reversible dissociation in the presence of Ca(2+) was also unequivocally demonstrated by the exchange of slightly different small subunits between mu-calpain and m-calpain. We conclude that subunit dissociation is a dynamic process and is not complete in most buffer conditions unless driven by factors such as crystal formation or autolysis of active enzymes. Exposure of the hydrophobic dimerization surface following subunit dissociation may be the main factor responsible for Ca(2+)-induced aggregation of calpain. It is likely that dissociation serves as an early step in calpain activation by releasing the constraints upon protease domain I.     

Crystal structure of calpain reveals the structural basis for Ca(2+)-dependent protease activity and a novel mode of enzyme activation

The combination of thiol protease activity and calmodulin-like EF-hands is a feature unique to the calpains. The regulatory mechanisms governing calpain activity are complex, and the nature of the Ca(2+)-induced switch between inactive and active forms has remained elusive in the absence of structural information. We describe here the 2.6 A crystal structure of m-calpain in the Ca(2+)-free form, which illustrates the structural basis for the inactivity of calpain in the absence of Ca(2+). It also reveals an unusual thiol protease fold, which is associated with Ca(2+)-binding domains through heterodimerization and a C(2)-like beta-sandwich domain. Strikingly, the structure shows that the catalytic triad is not assembled, indicating that Ca(2+)-binding must induce conformational changes that re-orient the protease domains to form a functional active site. The alpha-helical N-terminal anchor of the catalytic subunit does not occupy the active site but inhibits its assembly and regulates Ca(2+)-sensitivity through association with the regulatory subunit. This Ca(2+)-dependent activation mechanism is clearly distinct from those of classical proteases.     

Sidekicks: synaptic adhesion molecules that promote lamina-specific connectivity in the retina

Ca(2+) signaling by calpains leads to controlled proteolysis during processes ranging from cytoskeleton remodeling in mammals to sex determination in nematodes. Deregulated Ca(2+) levels result in aberrant proteolysis by calpains, which contributes to tissue damage in heart and brain ischemias as well as neurodegeneration in Alzheimer's disease. Here we show that activation of the protease core of mu calpain requires cooperative binding of two Ca(2+) atoms at two non-EF-hand sites revealed in the 2.1 A crystal structure. Conservation of the Ca(2+) binding residues defines an ancestral general mechanism of activation for most calpain isoforms, including some that lack EF-hand domains. The protease region is not affected by the endogenous inhibitor, calpastatin, and may contribute to calpain-mediated pathologies when the core is released by autoproteolysis.     

Effect of extremely low frequency magnetic fields on calpain activation

The effects of low intensity, low frequency magnetic fields (MFs) on catalytic activity of the calcium dependent protease calpain was determined following the enzyme activation both in "in vitro" and "in vivo" conditions. We have observed that a 0.3 mT MF induces a significant increase in the requirement of the protease for this metal ion. This change is detectable at low [Ca(2+)] and disappears when the level of Ca(2+) is raised to saturating amounts. The observed effects are not due to transient MF(-) induced conformational changes occurring in calpain, but to direct effects of the MF on Ca(2+) ions, which become less available for the binding sites present in calpain. Altogether, these results indicate that exposure to low intensity, low frequency MFs alters the intracellular Ca(2+) "availability," thereby modifying the related cell response.     

Calcium-induced calpain mediates apoptosis via caspase-3 in a mouse photoreceptor cell line

The rd mouse, an accepted animal model for photoreceptor degeneration in retinitis pigmentosa, has a recessive mutation for the gene encoding the beta-subunit of the cGMP phosphodiesterase. This mutation results in high levels of cGMP, which leaves an increased number of the cGMP-gated channels in the open state, thus allowing intracellular calcium (Ca(2+)) to rise to toxic levels, and rapid photoreceptor degeneration follows. To delineate the events in rd photoreceptor degeneration, we demonstrated an increase in calpain and caspase-3 activity, hypothesizing that Ca(2+)-mediated apoptosis in photoreceptors is mediated by calpain, involving mitochondrial depolarization and caspase-3 activation. To examine this hypothesis further, a murine photoreceptor-derived cell line (661W) was treated with the Ca(2+) ionophore A23187, cGMP-gated channel agonist 8-bromo-cGMP, or phosphodiesterase inhibitor isobutylmethylxanthine to mimic the increased Ca(2+) influx seen in the rd photoreceptors. Ca(2+)-induced cell death in 661W cells was found to be mediated by calpain and caspase-3 and could be completely inhibited by the calpain inhibitor SJA6017, implicating both calpain and caspases in the apoptotic process. The apoptotic events correlated in an SJA6017-inhibitable manner with bid cleavage, mitochondrial depolarization, cytochrome c release, and caspase-3 and -9 activation. We concluded that Ca(2+) influx in the rd model of photoreceptor degeneration leads to the activation of the cysteine protease calpain, which executes apoptosis via modulation of caspase-3 activity.     

Characteristics of mitochondrial calpains

Calpains are considered to be cytoplasmic enzymes, although several studies have shown that calpain-like protease activities also exist in mitochondria. We partially purified mitochondrial calpain from swine liver mitochondria and characterized. Only one type of mitochondrial calpain was detected by the column chromatographies. The mitochondrial calpain was stained with anti-mu-calpain and calpain small subunit antibodies. The susceptibility of mitochondrial calpain to calpain inhibitors and the optimum pH differ from those of cytosolic mu- and m-calpains. The Ca(2+)-dependency of mitochondrial calpain was similar to that of cytosolic mu-calpain. Therefore, we named the protease mitochondrial mu-like calpain. In zymogram analysis, two types of caseinolytic enzymes existed in mitochondria and showed different mobilities from cytosolic mu- and m-calpains. The upper major band was stained with anti-mu-calpain and calpain small subunit antibodies (mitochondrial calpain I, mitochondrial mu-like calpain). The lower band was stained only with anti-calpain small subunit antibody (mitochondrial calpain II, unknown mitochondrial calpain). Calpastatin was not detected in mitochondrial compartments. The mitochondrial calpain processed apoptosis-inducing factor (AIF) to truncated AIF (tAIF), releasing tAIF into the intermembrane space. These results indicate that mitochondrial calpain, which differs from mu- and m-calpains, seems to be a ubiquitous calpain and may play a role in mitochondrial apoptotic signalling.     

The importance of conserved amino acid residues in p94 protease sub-domain IIb and the IS2 region for constitutive autolysis

p94/calpain 3, a skeletal muscle-specific member of calpain protease family, is characterized by apparent Ca(2+)-independence during exhaustive autolysis and concomitant proteolysis of non-self substrates. The purpose of our study was to comprehensively profile the structural basis of p94 enabling activation in the cytosol without an extra Ca(2+). Ca(2+)-dependent p94 mutants were screened using "p94-trapping", which is an application of yeast genetic reporter system called "proteinase-trapping". Several amino acids were revealed as critical for apparent Ca(2+)-independent p94 activity. These results highlight the importance of conserved amino acids in domain IIb as well as in the p94-specific IS2 region.     

Thrombin-induced platelet aggregation involves an indirect proteolytic cleavage of aggregin by calpain

5'-p-Fluorosulfonylbenzoyl adenosine (FSBA), a nucleotide analog of ADP, has been shown to inhibit ADP-induced shape change, aggregation and exposure of fibrinogen binding sites concomitant with covalent modification of a single surface membrane polypeptide of Mr 100,000 (aggregin). Since thrombin can aggregate platelets which have been modified by FSBA and are refractory to ADP, we tested the hypothesis that thrombin-induced platelet aggregation might involve cleavage of aggregin. At a low concentration of thrombin (0.05 U/ml), platelet aggregation, exposure of fibrinogen receptors and cleavage of aggregin in FSBA-modified platelets did not occur, indicating ADP dependence. In contrast, incubation of [3H]FSBA-labeled intact platelets with a higher concentration of thrombin (0.2 U/ml) resulted in cleavage of radiolabeled aggregin, aggregation, and exposure of fibrinogen binding sites. Under identical conditions, aggregin in membranes isolated from [3H]FSBA-labeled platelets was not cleaved by thrombin. Thrombin-induced platelet aggregation and cleavage of aggregin were concomitantly inhibited by a mixture of 2-deoxy-D-glucose, D-gluconic acid 1,5-lactone, and antimycin A. These results suggest that thrombin cleaves aggregin indirectly by activating an endogeneous protease. Thrombin is known to elevate intracellular Ca2+ concentration and thereby activates intracellular calcium dependent thiol proteases (calpains). In contrast to serine protease inhibitors, calpain inhibitors including leupeptin, antipain, and ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (chelator of Ca2+) inhibited platelet aggregation and cleavage of aggregin in [3H]FSBA-labeled platelets. Leupeptin, at a concentration of 10-20 microM, used in these experiments, did not inhibit the amidolytic activity of thrombin, thrombin-induced platelet shape change, or the rise in intracellular Ca2+. Purified platelet calpain II caused aggregation of unmodified and FSBA-modified platelets and cleaved aggregin in [3H]FSBA-labeled platelets as well as in isolated membranes. The latter is in marked contrast to the action of thrombin on [3H]FSBA-labeled membranes. Thus, thrombin-induced platelet aggregation may involve intracellular activation of calpain which proteolytically cleaves aggregin thus unmasking latent fibrinogen receptors, a necessary prerequisite for platelet aggregation.     

Functional role of HSP90 complexes with endothelial nitric-oxide synthase (eNOS) and calpain on nitric oxide generation in endothelial cells

Although several reports have indicated that eNOS is a highly sensitive calpain substrate, the occurrence of a concomitant Ca(2+)-dependent activation of the synthase and of the protease has never been analyzed in specific direct experiments. In this study, we have explored in vivo how eNOS can undergo Ca(2+)-dependent translocation and activation, protected against degradation by activated calpain. Here we demonstrate that following a brief exposure to Ca(2+)-loading, the cytosolic eNOS-HSP90 complex recruits calpain in a form in which the chaperone and the synthase are almost completely resistant to digestion by the protease. Furthermore, in the presence of the HSP90 inhibitor geldanamycin, a significant decrease in NO production and an extensive degradation of eNOS protein occurs, indicating that dissociation from membranes and association with the chaperone is correlated to the protection of the synthase. Experiments with isolated membrane preparations confirm the primary role of HSP90 in dissociation of eNOS from caveolae. Prolonged exposure of cells to Ca(2+)-loading resulted in an extensive degradation of both eNOS and HSP90, accompanied by a large suppression of NO production. We propose that the protective effect exerted by HSP90 on eNOS degradation mediated by calpain represents a novel and critical mechanism that assures the reversibility of the intracellular trafficking and activation of the synthase.     

Action of calpain on the basic estrogen receptor molecule of porcine uterus

Basic estrogen receptor (ER) molecule (vero-ER) of the cytosol of porcine uterus was purified 1,200-fold after successive chromatographies on phenyl-Sepharose, hydroxylapatite, and DEAE-cellulose, followed by Sephadex G-150 gel filtration. The purified vero-ER was completely free from endogenous protease and ER-binding factor. The action of Ca2+-dependent cysteine proteinase (calpain) on vero-ER was studied by utilizing the purified receptor and calpains from porcine uterus (endogenous calpain), porcine kidney, and human erythrocytes. Proteolysis of vero-ER was followed by monitoring the disappearance of the binding capability of vero-ER with "8S" ER-forming factor. Vero-ER was proteolyzed by both the endogenous and the exogenous calpains in the presence of Ca2+. The calpains did not attack vero-ER in the absence of Ca2+. The results indicated the absolute requirement by calpain for Ca2+ for the limited hydrolysis of vero-ER. Uterine cytosol was shown to contain, in parallel with calpain, a protease which does not require Ca2+ for the limited proteolysis of vero-ER. The strongly hydrophobic domain of vero-ER, recently shown to be indispensable for the nuclear translocation of vero-ER (Murayama, A. & Fukai, F. (1983) FEBS Lett. 158, 255), was preferentially destroyed by both the Ca2+-requiring and -nonrequiring enzymes. It was assumed that calpain might intervene in the estrogen action by diminishing irreversibly the amount of the cytoplasmic ER capable of translocating into the nucleus.     

Inhibition of calpain blocks platelet secretion, aggregation, and spreading

Previous studies have indicated that the Ca(2+)-dependent protease, calpain, is activated in platelets within 30-60 s of thrombin stimulation, but specific roles of calpain in platelets remain to be identified. To directly test the functions of calpain during platelet activation, a novel strategy was developed for introducing calpain's specific biological inhibitor, calpastatin, into platelets prior to activation. This method involves treatment of platelets with a fusion peptide, calpastat, consisting of the cell-penetrating signal sequence from Kaposi's fibroblast growth factor connected to a calpain-inhibiting consensus sequence derived from calpastatin. Calpastat specifically inhibits thrombin peptide (SFLLR)-induced alpha-granule secretion (IC(50) = 20 microM) during the first 30 s of activation, thrombin-induced platelet aggregation (IC(50) = 50 microM), and platelet spreading on glass surfaces (IC(50) = 34 microM). Calpastat-Ala, a mutant peptide in which alanine is substituted at conserved calpastatin residues, lacks calpain inhibitory activity and fails to inhibit secretion, aggregation, or spreading. The peptidyl calpain inhibitors calpeptin, MDL 28,170 (MDL) and E64d also inhibit secretion, aggregation and spreading, but require 3-10-fold higher concentrations than calpastat for biological activity. Together, these findings demonstrate that calpain regulates platelet secretion, aggregation, and spreading and indicate that calpain plays an earlier role in platelet activation following thrombin receptor stimulation than had been previously detected.