Involvement Of Calpain Research Articles

Cell‐associated activation of latent transforming growth factor‐β by calpain

Transforming growth factor-β (TGF-β) is normally secreted in a latent form, and plasmin-mediated proteolytic cleavage of latency-associated peptide (LAP), a component of latent TGF-β complex that makes the complex inactive, activates latent TGF-β. In the present study, we investigated the possible involvement of calpain, one of the cysteine proteases, in the activation of latent TGF-β. When recombinant latent TGF-β was incubated with calpain (1–10 u/ml) in a test tube, calpain cleaved LAP and released mature TGF-β from the latent complex. When calpain was applied to cultured bovine capillary endothelial (BCE) cells, a low concentration of calpain (0.05–0.1 u/ml) inhibited the migration and proliferation of the cells, and these inhibitory effects were abrogated by anti-TGF-β antibody as well as by calpain inhibitor peptide, but not by α2-antiplasmin, a specific inhibitor of plasmin. Active TGF-β was detected in the conditioned medium of BCE cells collected in the presence of calpain. Chemical cross-linking of 125I-calpain to BCE cells followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicated that calpain bound to the cell surface through chondroitinase ABC-sensitive proteoglycan. In addition, treatment of the BCE cells with chondroitinase ABC abrogated the inhibitory effect of calpain on the migration of these cells. Our data thus suggest that calpain is able to activate latent TGF-β through a mechanism independent of plasmin. This activation is efficient in the presence of cells, and calpain binds to the cell surface via proteoglycan and activates latent TGF-β, which is targeted to the same surface. J. Cell. Physiol. 174:186–193, 1998. © 1998 Wiley-Liss, Inc.

Rat brain contains multiple mRNAs for calpastatin

This work was undertaken to establish the forms of the calpain inhibitor, calpastatin, expressed in the brain tissue. Five cDNA clones were obtained and the corresponding amino acid sequences were deduced. Three of these proteins contain an N-terminal domain (domain L) and four inhibitory repeats typical of the calpastatin molecule. The other two are truncated forms, containing the domain L, free or associated with a single inhibitory repeat. Other differences, due to exon skipping, produce calpastatin forms with different susceptibility to posttranslational modifications. The more represented mRNA form corresponds to a calpastatin molecule containing the four inhibitory domains. These results may be useful to understand the involvement of calpain in the onset of acute and degenerative disorders of the central nervous system.

Exercise-induced muscle injury: a calpain hypothesis.

It is well established that periods of increased contractile activity result in significant changes in muscle structure and function. Such morphological changes as sarcomeric Z-line disruption and sarcoplasmic reticulum vacuolization are characteristic of exercise-induced muscle injury. While the precise mechanism(s) underlying the perturbations to muscle following exercise remains to be elucidated, it is clear that disturbances in Ca2+ homeostasis and changes in the rate of protein degradation occur. The resulting elevation in intracellular [Ca2+] activates the non-lysosomal cysteine protease, calpain. Because calpain cleaves a variety of protein substrates including cytoskeletal and myofibrillar proteins, calpain-mediated degradation is thought to contribute to the changes in muscle structure and function that occur immediately following exercise. In addition, calpain activation may trigger the adaptation response to muscle injury. The purpose of this paper is to: (i) review the chemistry of the calpain-calpastatin system; (ii) provide evidence for the involvement of the non-lysosomal, calcium-activated neutral protease (calpain) in the response of skeletal muscle protein breakdown to exercise (calpain hypothesis); and (iii) describe the possible involvement of calpain in the inflammatory and regeneration response to exercise.

Nuclear cataract and light scattering in cultured lenses from guinea pig and rabbit

Purpose. To compare calcium ionophore-induced cataract formation and in vitro light scattering in cultured lenses from guinea pig and rabbit.Methods. Lenses from guinea pig and rabbit were cultured for 5 or 6 days with calcium ionophore A23187. To assess the involvement of calpain in cataract formation; SDS-PAGE, immunoblotting and calcium determinations were performed. For in vitro light scattering, lens soluble proteins from rabbit were hydrolyzed for 24 h by either endogenous lens calpain, or by addition of purified m-calpain and then further incubated for up to 10 days. Light scattering was measured daily at 405 nm.Results. Lenses from younger guinea pigs cultured in A23187 first developed outer cortical opacities followed by nuclear cataract. Total calcium was markedly increased by A23187 in lenses of all ages. Proteolysis of crystallins and a-spectrin were observed in nuclear cataract in younger guinea pigs. This was attenuated with age, in association with the attenuation of cataract formation with age. Calpain 80 kDa subunit in the lenses cultured with A23187 was also decreased. Co-culture with SJA6017 or E64d (reversible and irreversible inhibitors of calpain, respectively) reduced A23187-induced nuclear opacities, proteolysis of crystallins and a-spectrin, and loss of calpain without affecting increased total calcium. In contrast, rabbit lenses cultured in A23187 did not develop nuclear cataract, although biochemical changes in cultured rabbit lenses were similar to those in cultured guinea pig lenses. Furthermore, no appreciable in vitro light scattering occurred in soluble proteins from rabbit lenses after activation of endogenous m-calpain, or after addition of exogenous purified m-calpain, although crystallins were partially hydrolyzed by calpain.Conclusions. Both rabbit and guinea pig lenses undergo calpain-induced proteolysis upon elevation of lenticular calcium. However, factors in intact guinea pig lenses may promote light scattering and insolubilization after proteolysis by calpain, but these factors were not functional in rabbit lenses. Discovery of the factors promoting light scatter and insolubilization after proteolysis will help to explain the role of certain crystallin polypeptides in cataract formation. Curr. Eye Res. 17: 623–635, 1998.

Nuclear cataract and light scattering in cultured lenses from guinea pig and rabbit

To compare calcium ionophore-induced cataract formation and in vitro light scattering in cultured lenses from guinea pig and rabbit. Lenses from guinea pig and rabbit were cultured for 5 or 6 days with calcium ionophore A23187. To assess the involvement of calpain in cataract formation; SDS-PAGE, immunoblotting and calcium determinations were performed. For in vitro light scattering, lens soluble proteins from rabbit were hydrolyzed for 24 h by either endogenous lens calpain, or by addition of purified m-calpain and then further incubated for up to 10 days. Light scattering was measured daily at 405 nm. Lenses from younger guinea pigs cultured in A23187 first developed outer cortical opacities followed by nuclear cataract. Total calcium was markedly increased by A23187 in lenses of all ages. Proteolysis of crystallins and alpha-spectrin were observed in nuclear cataract in younger guinea pigs. This was attenuated with age, in association with the attenuation of cataract formation with age. Calpain 80 kDa subunit in the lenses cultured with A23187 was also decreased. Co-culture with SJA6017 or E64d (reversible and irreversible inhibitors of calpain, respectively) reduced A23187-induced nuclear opacities, proteolysis of crystallins and alpha-spectrin, and loss of calpain without affecting increased total calcium. In contrast, rabbit lenses cultured in A23187 did not develop nuclear cataract, although biochemical changes in cultured rabbit lenses were similar to those in cultured guinea pig lenses. Furthermore, no appreciable in vitro light scattering occurred in soluble proteins from rabbit lenses after activation of endogenous m-calpain, or after addition of exogenous purified m-calpain, although crystallins were partially hydrolyzed by calpain. Both rabbit and guinea pig lenses undergo calpain-induced proteolysis upon elevation of lenticular calcium. However, factors in intact guinea pig lenses may promote light scattering and insolubilization after proteolysis by calpain, but these factors were not functional in rabbit lenses. Discovery of the factors promoting light scatter and insolubilization after proteolysis will help to explain the role of certain crystallin polypeptides in cataract formation.

Evidence for the involvement of calpain in cataractogenesis in Shumiya cataract rat (SCR)

The Shumiya cataract rat (SCR) is a hereditary cataract model in which lens opacity appears spontaneously in the nuclear and perinuclear portions at 11–12 weeks of age. It was found that the proteolysis of some crystallins and cytoskeletal proteins is significantly enhanced in cataractous SCR lenses. The calcium concentrations in cataractous lenses rise markedly with age as compared with control lenses and the autolytic product of calpain is also detected in cataractous lenses. In order to provide direct evidence for the involvement of calpain in the proteolytic modification of lens proteins, we developed antibodies exclusively specific to the proteolytic products of some lens proteins produced by the action of calpain and analyzed their degradation during cataractogenesis in SCR by Western blotting and immunohistochemical staining. The results demonstrate that calpain participates in the proteolytic modification of lens proteins, at least α-crystallin (A and B chain), βB1-crystallin, and α-fodrin. The proteolytic products formed by the action of calpain on these proteins are detected in cataractous lenses of SCR as young as 8 weeks of age and accumulate with age. It was also found that βB1-crystallin, originally a soluble protein, is converted to an insoluble form by limited calpain proteolysis. The chaperon-like activity of α-crystallin from control lens is markedly reduced by calpain proteolysis in vitro, and α-crystallin in opaque lens that has already undergone proteolysis by calpain shows significantly reduced chaperon-like activity. Immunohistochemical studies reveal that the area where the calpain-mediated α-crystallin proteolysis is in progress coincides well with the area developing and destined to develop the opacification. These results strongly suggest that calpain may contribute to lens opacification during cataract formation in SCR.

Distribution of ankyrin isoforms and their proteolysis after ischemia and reperfusion in rat brain.

The distribution of brain-type ankyrin (ankyrinB, 212 kDa) and erythrocyte-type ankyrin (ankyrinR, 239 kDa) was investigated in the subcellular fractions of rat forebrain (P1, 1,000 g pellet; P2, 15,000 g pellet; P3, 100,000 g pellet; S, 100,000 g supernatant) by immunoblotting using specific antibodies. The P2 fraction contained approximately 40% of the 212- and 163-kDa isoforms of ankyrinB and the 239-kDa isoform of ankyrinR. Further subfractionation of the P2 by Percoll gradient centrifugation followed by separation of myelin showed association of the three ankyrin isoforms with the synaptosome-rich fraction but not with the myelin-rich fraction. The plasma membrane-rich P3 fraction contained a concentration of ankyrin isoforms similar to that in the P2 fraction. In vitro proteolysis of ankyrin in the P2 fraction with calpain showed that the 212-kDa ankyrinB was more susceptible to calpain than was ankyrinR. In the two-vessel occlusion model, ischemia for 30 min generated the 160-kDa fragment of ankyrinR, and reperfusion for 60 min after 30 min of ischemia remarkably increased the 160-kDa fragment. The reperfusion also significantly decreased the 212-kDa isoform of ankyrinB. Both ischemia-reperfusion and in vitro proteolysis with calpain generated the 160-kDa fragment of ankyrinR, suggesting the involvement of calpain.

On the involvement of calpains in the degradation of the tumor suppressor protein p53

A crude fraction that contains ubiquitin–protein ligases contains also a proteolytic activity of ∼100 kDa that cleaves p53 to several fragments. The protease does not require ATP and is inhibited in the crude extract by an endogenous ∼250 kDa inhibitor. The proteinase can be inhibited by chelating the Ca 2+ ions, by specific cysteine proteinase inhibitors and by peptide aldehyde derivatives that inhibit calpains. Purified calpain demonstrates an identical activity that can be inhibited by calpastatin, the specific protein inhibitor of the enzyme. Thus, it appears that the activity we have identified in the extract is catalyzed by calpain. The calpain in the extract degrades also N-myc, c-Fos and c-Jun, but not lysozyme. In crude extract, the calpain activity can be demonstrated only when the molar ratio of the calpain exceeds that of its native inhibitor. Recent experimental evidence implicates both the ubiquitin proteasome pathway and calpain in the degradation of the tumor suppressor, and it was proposed that the two pathways may play a role in targeting the protein under various conditions. The potential role of the two systems in this important metabolic process is discussed.

Calpains are involved in phosphatidylinositol 3′,4′- bisphosphate synthesis dependent on the α IIbβ 3 integrin engagement in thrombin-stimulated platelets

In thrombin-stimulated platelets α IIbβ 3 integrin engagement triggers both phosphatidylinositol 3′,4′- bisphosphate synthesis and calpain activation. We checked the possible involvement of calpains in phosphatidylinositol 3-kinase signalling pathway using a cell permeant specific inhibitor of calpains, calpeptin. In conditions where thrombin-induced platelet aggregation and secretion were not impaired, we found a dose-dependent inhibition of phosphatidylinositol 3,4- bisphosphate synthesis by calpeptin from 50 μg/ml. Moreover, pretreatment of platelets by both calpeptin and the peptide RGDS, an inhibitor of fibrinogen binding to activated α IIbβ 3 integrin, did not induce additive effects on phosphatidylinositol 3,4- bisphosphate inhibition. Finally, the p85 regulatory subunit of phosphatidylinositol 3-kinase was still translocated to the cytoskeleton in calpeptin-treated platelets. These data indicate that calpains are involved in the regulation of α IIbβ 3 integrin-dependent phosphatidylinositol 3-kinase signalling pathway. © 1997 Federation of European Biochemical Societies.

Involvement of calpain in postmortem proteolysis in the rat brain

Calpain, a Ca 2+-dependent neutral protease was examined to investigate its involvement in postmortem proteolysis in the rat brain. Western blotting analysis showed that the 240 kDa α-subunit of fodrin, a well-known substrate for calpain, was degraded to generate 150 kDa and 145 kDa fragments in the postmortem interval (0–24 h) at 25 ± 3 °C. Postmortem proteolysis was dependent on ambient temperature. In in vitro experiments, the 150 kDa and 145 kDa fragments appeared in the homogenate with addition of Ca 2+ (1 μM–1 mM) or in the microsomal fraction by incubation with purified calpain. Both calpain inhibitor-1 and leupeptin suppressed in vitro proteolysis. During the initial 0–24 h postmortem, the activity of m-calpain in the brain remained unaltered, while that of its endogenous inhibitor, calpastatin, decreased with the postmortem interval. These results indicate that calpain is involved in fodrin proteolysis in the postmortem rat brain. The ratio of the amount of the 150 kDa proteolytic product to that of the 240 kDa fodrin α-subunit was correlated significantly with the postmortem interval (0–16 h; r = 0.745).

Involvement of Calpain in Integrin-Mediated Signal Transduction

An antibody specific to the calpain cleavage site in talin, a cytoskeletal protein, was produced. This antibody selectively recognizes the C-terminal 200-kDa fragment generated when talin is digested by calpain and does not react at all with intact talin or the N-terminal 47-kDa fragment. To assess the involvement of calpain in the integrin-mediated signaling pathway, the effect of limited proteolysis of talin by calpain on platelet activation and aggregation was analyzed using this antibody. It was revealed that thrombin-stimulated platelet aggregation accompanies the autolytic activation of μ-calpain and the accumulation of the μ-calpain-generated 200-kDa fragment of talin. These changes were blocked by RGDS peptide which inhibits the binding of fibrinogen, an adhesive ligand, to the major integrin in platelets, αIIbβ3, while RGES peptide, which has no fibrinogen-binding-inhibitory activity, had no effect. Membrane-permeable calpain inhibitors calpeptin and E-64d inhibited platelet aggregation, μ-calpain activation, and the limited proteolysis of talin. These results strongly suggest that calpain is involved in the integrin-mediated signal transduction pathway.

Three distinct phases of fodrin proteolysis induced in postischemic hippocampus. Involvement of calpain and unidentified protease.

Fodrin, a neuronal cytoskeleton protein, is proteolyzed by calpain after ischemic insult. We examined proteolysis of fodrin induced by global forebrain ischemia in gerbil hippocampus in spatial terms by using the antibody specific to the calpain-proteolyzed form of fodrin. In gerbils, a 10-minute forebrain ischemia was produced by occlusion of both carotid arteries. After recirculation, the hippocampus was processed for immunohistochemical and immunoblot study with the antibody against the calpain-proteolyzed form of fodrin. Additionally, short-term ischemia was studied to find the threshold of fodrin proteolysis. Three phases of fodrin proteolysis distinct in chronology and distribution arose: (1) an early predegeneration phase in the molecular layer and stratum oriens of the CA1 and CA3 sectors within the first 15 minutes, which lasted up to 4 hours; (2) a late predegeneration phase in the whole CA1 sector, except for the pyramidal cells, between 12 hours and 2 days; and (3) a postdegeneration phase in the cytoplasm of the CA1 neurons, which arose in 3 to 7 days. A 4-minute (not a 3-minute) forebrain ischemia induced the late predegeneration phase of fodrin proteolysis and delayed neuronal death in CA1. Immunoblotting showed that the primary product of calpain action was further proteolyzed by an unidentified protease. Calpain induced proteolysis of fodrin in ischemic hippocampus, and the late predegeneration phase of the proteolysis was closely associated with the delayed neuronal death in the CA1 sector. Calpain and another protease may play a role in the development of neuronal death after transient forebrain ischemia.

The Role of the Calpain-Calpastatin System in Thyrotropin-releasing Hormone-induced Selective Down-regulation of a Protein Kinase C Isozyme, nPKCε, in Rat Pituitary GH4C1 Cells

We have examined the mechanism for the selective down-regulation of protein kinase C epsilon (nPKC epsilon) in rat pituitary GH4C1 cells responding to thyrotropin-releasing hormone (TRH) stimulation. Among various low molecular weight protease inhibitors examined, only a cysteine protease inhibitor (calpain inhibitor I, N-acetyl-Leu-Leu-norleucinal) blocked the down-regulation of nPKC epsilon. Furthermore, the introduction of a synthetic calpastatin peptide, an exclusively specific inhibitor of calpain, into the cells also reduced the down-regulation, suggesting the involvement of calpain among all the intracellular cysteine proteases in this process. In accordance, we observed TRH-induced translocation of m-calpain from the cytosol to the membrane and the concomitant up-regulation of calpastatin isoforms; presumably, the former represents activation of the protease initiating the kinase degradation, while the latter constitutes a negative feedback system protecting the cells from activated calpain. These results suggest that in GH4C1 cells, TRH mobilizes both protease (m-calpain) and inhibitor (calpastatin) as a strictly regulating system for the nPKC epsilon pathway mediating TRH signals.

Action of calpastatin in prevention of cardiac L-type Ca2+ channel run-down cannot be mimicked by synthetic calpain inhibitors.

Activity of L-type Ca2+ channels in a membrane patch disappears rapidly when the patch is excised from the cell into an artificial solution. This channel run-down observed in isolated membrane patches can however, be prevented by application of calpastatin, an endogenous protease inhibitor, and ATP. The high specificity of calpastatin for the protease calpain would clearly point to a participation of calpain activity in the run-down of Ca2+ channels. In an attempt to examine a possible involvement of calpain, three synthetic and rather specific calpain inhibitors were substituted for calpastatin. One of these inhibitors chosen for its membrane permeability in addition allowed calpain activity to be inhibited even before patch excision. The potency of these compounds in inhibiting calpain, specifically mu- and m-calpain, was first determined in a biochemical assay and then compared with their efficacy in preventing Ca2+ channel run-down. Surprisingly, calpastatin was least effective in calpain inhibition but by far the most potent in prevention of Ca2+ channel run-down. In addition run-down of Ca2+ channel activity was examined for its reversibility, which would not be expected upon involvement of a proteolytic process. However, Ca2+ channel activity clearly recovered after run-down by application of calpastatin. In contrast, synthetic calpain inhibitors were unable to reverse Ca2+ channel run-down. These results indicate that proteolysis might only be partially responsible for channel run-down and suggest an as yet unidentified function for calpastatin beyond its inhibitory action on calpain in the regulation of Ca2+ channel activity.

Unphosphorylated and tyrosine-phosphorylated forms of a focal adhesion protein, paxillin, are substrates for calpain II in vitro: Implications for the possible involvement of calpain II in mitosis-specific degradation of paxillin

Cell-to-substratum adhesion becomes weakened during mitosis of the cell cycle in fibroblasts. The level of one focal adhesion protein, paxillin, is greatly reduced in mitotic-arrested cells. We show here the possible involvement of calpain II, known to be localized in focal adhesion plaques, in the degradation of paxillin. Paxillin is tyrosine-phosphorylated during interphase of the cell cycle by protein tyrosine kinases (PTK) such as c-Src and Csk, and becomes dephosphorylated during mitosis. Our data, however, indicate that tyrosine phosphorylation of paxillin does not affect the rate of paxillin degradation by calpain in vitro.

Calpain-mediated regulation of AMPA receptors in adult rat brain.

Changes in AMPA receptors have been proposed to underlie changes in synaptic efficacy in hippocampus and other brain structures. Activation of calpain has also been discussed as a potential mechanism to produce lasting modifications of synaptic structure and function. We report here that preincubation of thin (10 microns) frozen rat brain sections with calcium at physiological temperature changes the immunological properties of AMPA receptors, an effect totally blocked by calpain inhibitors. Immunocytochemistry indicates that in situ calpain activation produces a decreased immunoreactivity for GluR1, and to a lesser extent for GluR2/3, in the neuropil throughout the brain and an increased immunoreactivity in cell bodies, particularly in hippocampus. Western blots of calcium-treated sections suggest that the decreased immunoreactivity for GluR subunits is due to partial proteolysis. These results strongly suggest the involvement of calpain in the regulation of glutamatergic synapses.

Ca 2+-dependent proteinases (calpains) and muscle cell differentiation

The chronology of appearance of calpain I and calpain II was analyzed during myogenesis of embryonic myoblasts in culture. The influence of the hormones insulin and corticosterone, and insulin growth factor-1 (IGF-1) and transforming growth factor-β (TGF-β) on the modulation of calpain-calpastatin levels during myogenesis was also analyzed. Immunodetection assays using specific antibodies and enzymic activities showed that during muscle cell differentiation in vitro, calpain II is present from the beginning of myoblast fusion (2nd day) increasing until the 6th day and then reaching a plateau. These observations were confirmed by an analysis of the expression of total calpain mRNAs which followed the same time profile, thereby providing evidence for a transcriptional regulation in the expression of calpains. Even if an increase in calpain II activity occurs at approximately the same time as an increase of fusion, calpain II activity and rate of fusion are not closely correlated. The involvement of calpain II in some event that follows myoblast fusion is suggested. On the other hand, calpain I and calpastatin were detected only on the 6th day of cell culture growth; these results enable us to argue that if calpain I has any biological role (which remains to be established), this role occurs during the final stages of muscle cell differentiation. The presence of exogenous factors which are known to affect muscle cell differentiation by altering either the rate of protein synthesis, or degradation or both, significantly affects the modulation of calpain-calpastatin levels. Such a regulation at the transcriptional level suggests that calpains do not act as housekeeping enzymes during myogenesis.

Calpain: new perspectives in molecular diversity and physiological‐pathological involvement

Calpain, calcium-activated neutral protease, stands as a unique receptor for calcium signals in biological systems; its activation leads to irreversible proteolytic processing of substrate proteins, modifying cellular situations in a manner distinct from that of reversible processes including the phosphorylation-dephosphorylation reactions. Because the enzyme participates not only in normal intracellular signal transduction cascades but also in various pathological states including ischemia, calpain research has attracted tremendous interest in wide areas of life sciences in both basic and clinical terms. This review will address the new perspectives evoked by recent discoveries since 1990. Molecular biological studies have established that calpain in fact constitutes a large family of distinct isozymes differing in structure and distribution, whereas an increasing number of reports describe physiological-pathological involvement of calpain. Another major accomplishment is the technical breakthrough allowing spatial resolution of calpain action presenting a clearer in vivo picture of how calpain acts in cells and tissues.

Involvement of calpain in myonephropathic metabolic syndrome (MNMS)

Myonephropathic metabolic syndrome (MNMS) is a serious muscle reperfusion injury associated with acute renal failure. The exact pathogenesis of MNMS has not been fully elucidated, nor effective treatment, through the renal failure is thought to be a consequence of rhabdomyolysis. In the present study, the possible involvement of calpain in the lysis was investigated in a MNMS animal model employing a cell permeable calpain antagonist calpeptin. Male rabbits were subjected to bilateral hind leg ischaemia for 5 hours by clamping the distal aorta, followed by reperfusion for 3 hours. Blood pressure, plasma N-acethyl-beta-D-glucosaminidase (NAG) and the presence of myoglobinuria were serially determined. Blood pressure remained constant during the ischemic period but dropped by about 25% immediately after reperfusion. This was significantly attenuated by intraaortic administration of calpeptin. NAG gradually increased during ischemia and during reperfusion and this was also significantly reduced by calpeptin. Myoglobinuria appeared immediately after reperfusion, and was also attenuated by calpeptin. Calpeptin prevented lytic and degenerative changes of the hind leg muscles, determined by light and electron microscopy. Thus it is concluded that activation of calpain in skeletal muscle is an important etiologic factor of MNMS and that the occurrence of MNMS may be prevented by administration of a calpain antagonist.

The effects of calpeptin (a calpain specific inhibitor) on agonist induced microparticle formation from the platelet plasma membrane

Platelets activated by various agonists produce formation of vesicles shed from the plasma membrane (microparticles). However, the mechanism of microparticle (MP) formation has not been clarified yet. The aim of the present study was to determine the possibility of involvement of calpain (a Ca 2+-dependent thiol protease) in MP formation. Washed platelets preincubated with calpeptin, a cell permeable calpain specific inhibitor, or with a vehicle were activated by thrombin plus collagen or by calcium ionophore A23187. Flow cytometry was used to detect the amount of microparticle formation by using murine monoclonal antibodies against GP IIb-IIIa or GP IIb and fluorescein 5-isothiocyanate labeled goat anti-mouse IgG. MP formation stimulated either by thrombin plus collagen or by A23187 was inhibited by calpeptin in a dose dependent manner. The microparticle formation from platelets activated by A23187 reached a plateau in approximately 5 min after activation, whereas that from platelets activated by thrombin plus collagen reached a plateau at 30 min following the stimulation. These time sequences corresponded well with those of degradation of actin-binding protein (ABP), a well known substrate of calpain, of platelets activated by these two stimulations. However, the inhibition of MP formation by calpeptin was more marked in the early stage (within 10 min) than in the late stage (after 30 min) of platelet activation. At 30 min after platelet activation by either two stimulations, a significant amount of microparticle formation was observed in the presence of 30 μM calpeptin, which inhibited hydrolysis of ABP almost completely. Our data suggest the involvement of calpain in the early stage (especially within 10 min) of microparticle formation.