Calpain I remains intact and intracellular during platelet activation. Immunochemical measurements with monoclonal and polyclonal antibodies.

As a step towards understanding the physiological function of calpain (Ca2+-activated neutral proteinase, EC 3.4.22.17) in blood platelets, and in view of some suggestions that calpain is transferred to the platelet external surface during platelet activation, the enzyme was studied with immunochemical methods in resting and thrombin-activated cells. (1) A mouse IgG1 monoclonal antibody was prepared which binds strongly only to the denatured large subunit of human calpain I, and weakly to that of human calpain II. A polyclonal antibody raised against rat calpain II was available which, apart from binding strongly to rat calpain II, binds to the large subunits of human calpain I and II about equally. (2) With these antibodies, it was found that calpain could be detected in fixed platelets in suspension only after permeabilization with 0.1% saponin, and could not be detected on the exterior surface of resting or of activated platelets, or in the supernatant media of these platelets. It was concluded that calpain is not significantly externalized during platelet activation. (3) Immunoblotting showed that conversion of the larger calpain I subunit from 80 kDa into 76-78 kDa occurred only when thrombin-activated platelets were stirred to permit aggregation, and did not occur during unstirred thrombin activation. Although an action of calpain in the 80 kDa form on possible platelet substrates such as cytoskeletal proteins cannot be excluded, calpain is certainly not present as the 76-78 kDa form, which is assumed to be its active form, until aggregation is initiated.     

Binding to erythrocyte membrane is the physiological mechanism for activation of Ca2+-dependent neutral proteinase

In the presence of micromolar concentrations of Ca2+ the catalytic 80 kDa subunit of human erythrocyte procalpain binds to the cytosolic surface of the erythrocyte membrane. Binding is rapid, highly specific and is reversed by the removal of Ca2+. In the bound form the 80 kDa catalytic subunit undergoes a rapid conversion to calpain, the active 75 kDa Ca2+-requiring proteinase. The activated proteinase produces extensive degradation of membrane components, particularly of band 4.1 and 2.1 proteins. Binding to membranes may represent an obligatory physiological mechanism for the conversion of procalpain to calpain.     

Activation by hemoglobin of the Ca2+-requiring neutral proteinase of human erythrocytes: structural requirements

The proenzyme form of the Ca2+-requiring neutral proteinase of human erythrocytes (procalpain) is converted to the active proteinase (calpain) by low concentrations of Ca2+ in the presence of appropriate substrates such as beta-hemoglobin or heme-free beta-globin chains. Modification of these substrates by limited proteolysis with calpain abolishes their ability to promote the conversion of procalpain. A similar requirement for the presence of unmodified beta-hemoglobin or heme-free beta-globin chains is observed for the autocatalytic inactivation of calpain. The conversion of procalpain to calpain is accompanied by a small decrease in the molecular mass of the catalytic subunit, from 80 kDa to 75 kDa; however, the activation is not accelerated by the addition of a small quantity of calpain. The autocatalytic inactivation of active CANP is related to the disappearance of the 75 kDa subunit and the formation of smaller peptide fragments.     

Localization of neutrophil adherence-inhibiting factor in guinea pig platelets

The effect of constituents of guinea pig platelets on neutrophil adherence was examined. The platelet sonicate supernatant contained adherence-inhibiting activity which strongly inhibited neutrophil adherence to glass. When the platelet sonicate supernatant was treated with neuraminidase or trypsin, the adherence-inhibiting activity was significantly inhibited, suggesting that the adherence-inhibiting factor (AIF) is a glycoprotein. The subcellular fractionation experiments indicated that the AIF activity was present at about 40% in both the cytosol and granule fractions. From the Sephadex G-200 gel filtration analysis, AIF of cytosol fraction and granule fraction proved to be different molecules, with molecular masses of about 230 and 12 kDa, respectively. When platelets were stimulated with thrombin, about 20% of total AIF was released extracellularly without the release of the cytoplasmic enzyme lactate dehydrogenase. These results suggest the possibility that a biologically active substance, AIF, is released from platelets in response to stimuli and regulates neutrophil functions through interference with neutrophil adherence.     

Localization of neutrophil adherence-inhibiting factor in guinea pig platelets

The effect of constituents of guinea pig platelets on neutrophil adherence was examined. The platelet sonicate supernatant contained adherence-inhibiting activity which strongly inhibited neutrophil adherence to glass. When the platelet sonicate supernatant was treated with neuraminidase or trypsin, the adherence-inhibiting activity was significantly inhibited, suggesting that the adherence-inhibiting factor (AIF) is a glycoprotein. The subcellular fractionation experiments indicated that the AIF activity was present at about 40% in both the cytosol and granule fractions. From the Sephadex G-200 gel filtration analysis, AIF of cytosol fraction and granule fraction proved to be different molecules, with molecular masses of about 230 and 12 kDa, respectively. When platelets were stimulated with thrombin, about 20% of total AIF was released extracellularly without the release of the cytoplasmic enzyme lactate dehydrogenase. These results suggest the possibility that a biologically active substance, AIF, is released from platelets in response to stimuli and regulates neutrophil functions through interference with neutrophil adherence.     

Role of phospholipids in the activation of the Ca2+-dependent neutral proteinase of human erythrocytes

Activation of the Ca2+-dependent neutral proteinase of human erythrocytes in the presence of Ca2+ and a digestible substrate (Pontremoli, S., Sparatore, B., Melloni, E., Michetti, M. and Horecker, B.L. 1984, Biochem. Biophys. Res. Communs. 123, 331-337) is promoted by phospholipids such as phosphatidylcholine, phosphatidylinositol and phosphatidylserine. The presence of at least one unsaturated fatty acid chain is essential and metabolic derivatives such as dioleylglycerol, phosphorylserine and free fatty acids are ineffective. The most effective promoter was a freshly prepared mixture of phospholipids from human erythrocyte membranes. Activation involves conversion of the 80 kDa proenzyme (procalpain) subunit to the 75 kDa active proteinase and is irreversible. Phospholipids act by producing a large decrease in the concentration of Ca2+ required for the conversion of procalpain to active calpain.     

Procalpain is activated on the plasma membrane and the calpain acts on the membrane

The mechanism of activation of human erythrocyte calpain was investigated using the immunoblotting technique with anticalpain monoclonal antibody. The purified calpain underwent a Ca2+-induced fragmentation of the 80 kDa subunit to 76 kDa and 36 kDa fragments. The behavior of the 76 kDa fragment in electrophoresis corresponded to the proteinase activity of calpain, whereas the behavior of the 80 kDa subunit and the 36 kDa fragment did not. When inside-out membrane vesicles were added to the reaction mixture of calpain and Ca2+ and the vesicles were separated from the supernatant solution by centrifugation, the 80 kDa subunit and 76 kDa fragment were found in the vesicle fraction. No other fragments were found in this fraction. On the other hand, the 80 kDa subunit and 36 kDa fragment were found in the supernatant fraction. When right-side-out membrane vesicles were added to the reaction mixture and the vesicles were separated from the supernatant fraction, no fragment was found in the vesicle fraction, while only the 36 kDa fragment was found in the supernatant fraction. These results indicate that the 80 kDa subunit of procalpain was bound in a Ca2+-dependent manner to the cytosolic surface of the plasma membrane and then underwent fragmentation to produce the 76 kDa fragment (active form) and that it expressed its proteinase activity at the surface of the membrane.     

Following association to the membrane, human erythrocyte procalpain is converted and released as fully active calpain

When exposed to inside-out human erythrocyte vesicles, in the presence of micromolar Ca2+, the 80 kDa catalytic subunit of procalpain is processed through three successive and sequential steps. These include binding to the cytosolic surface of the membrane, followed by a very rapid conversion into the 75 kDa active subunit, and ultimately by spontaneous and complete release of this active proteinase form. Binding to the membranes is competitively inhibited by the endogenous natural inhibitor through the formation of the proteinase-inhibitor complex, in which form the 80 kDa subunit can no longer be associated to the membranes. Calcium ions and the natural endogenous inhibitor appear to be crucially involved in the modulation of this novel membrane-bound mediated activation of human red cell procalpain.     

Annexin V relocates to the periphery of activated platelets following thrombin activation: an ultrastructural immunohistochemical approach

We have previously shown biochemically that the physiological agonist thrombin can cause translocation of endogenous annexin V to a fraction containing all platelet membranes. This paper reports ultrastructural immunohistochemical data revealing that annexin V molecules localize with plasma membranes of blood platelets following thrombin activation. When ultrathin sections of resting platelets were examined by immunogold staining, annexin V was found to be cytosolic, having a generalized distribution throughout the platelet. After thrombin activation, annexin V became peripheral in location and plasmalemma association increased. Morphometric analysis of gold particles shows that annexin V relocates specifically to the plasma membrane and its underlying cytoskeleton following treatment with thrombin. In control platelets 6.1% +/- 0.78 of annexin V is present at the plasma membrane and 15.0% +/- 0.82 in the region corresponding to the membrane cytoskeleton (10-80 nm); after stimulation with 0.5 unit/ml thrombin for 2 min this increased to 16.7% +/- 0.22 and 40.4% +/- 0.53, respectively.     

Altered phospholipid composition of plasma membranes from thrombin-stimulated human platelets

The individual phospholipid concentrations, and their respective fatty acid distributions, in whole platelet lysates and plasma membranes derived from unstimulated and thrombin-stimulated intact human platelets were studied. This was of interest, since previous work had led to the suggestion that altered phospholipid concentrations in plasma membranes of intact stimulated cells may be of importance in mediating cellular responses. The concentrations (nmol/mg protein) of phosphatidylinositol in whole platelet lysates and plasma membranes derived from thrombin-activated platelets decreased by 37 and 45%, respectively, a compared to their corresponding controls. As well, concentrations of plasma membrane phosphatidylcholine and phosphatidylethanolamine in thrombin-stimulated platelets decreased by 20 and 9%, respectively, when compared with their control values. The amounts of phosphatidylserine and sphingomyelin in whole platelet lysates and plasma membranes were unchanged by exposure to thrombin. Fatty acid analyses revealed that thrombin stimulation of intact human platelets induced a decrease in the arachidonate content (from 37.7 to 33.1 wt.% of total fatty acid) of plasma membrane phosphatidylinositol. Similar shifts in the wt% of arachidonic acid in plasma membrane phosphatidylcholine were found. These results indicate that thrombin stimulation of intact human platelets produces a significant decrease in the mass of phosphatidylinositol in plasma membranes and raises the suggestion that the preferential depletion of the plasma membrane in arachidonoyl-containing phosphatidylinositol may be of importance in mediating cellular responses to external stimuli.     

Regulation of membrane-associated and cytosolic phospholipase C activities in human platelets by guanosine triphosphate

The effects of guanine nucleotides, thrombin, and platelet cytosol (100,000 X g supernatant) on the hydrolysis of polyphosphoinositides by phospholipase C was examined in isolated platelet membranes labeled with [3H]inositol. Guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) (10 microM) caused a 2-fold stimulation of polyphosphoinositide hydrolysis, compared to background. GTP gamma S (10 microM) plus thrombin (1 unit/ml) stimulated the release of inositol triphosphate, inositol diphosphate, and inositol phosphate 500, 300, and 250%, respectively, compared to GTP gamma S alone. Cytosol prepared from unlabeled platelets slightly increased the release of inositol phosphates from [3H]inositol-labeled membranes. Addition of cytosol plus GTP gamma S (10 microM), however, resulted in a 300% enhancement of the release of inositol phosphates compared to membranes incubated with thrombin and GTP gamma S. The stimulatory effects of cytosol and GTP gamma S on polyphosphoinositide hydrolysis were also observed when membranes were replaced by sonicated lipid vesicles prepared from a total platelet lipid extract. These data suggest that PIP2 hydrolysis in platelets is catalyzed by a soluble phospholipase C which is regulated by a GTP-binding regulatory protein.     

Copper deficiency alters protein kinase C mediation of thrombin-induced dense granule secretion from rat platelets

Experiments were conducted to determine if copper deficiency enhances the rate of thrombin-induced dense granule secretion by modifying the major signal transduction pathways of rat platelets. Platelets were obtained from male, weanling Sprague-Dawley rats fed diets containing either deficient ( < 0.5 μg/g diet) or adequate (5.5 μg/g diet) copper for 5 weeks. Following stimulation with thrombin (0.1 U/mL), the rate of dense granule secretion as measured by ATP release was 160% higher in platelets from copper-deficient than from control rats. Inhibition of the rate of thrombin-induced ATP release by (6-aminohexyl)-1-naphthalene-sulfonamide, a calmodulin antagonist was independent of copper status. However, 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine, a protein kinase C inhibitor, inhibited the rate of ATP release only in platelets from copper-deficient rats. Aspirin had no effect on ATP release from platelets obtained from either copper-deficient or control rats. This suggests that copper deficiency alters the role of protein kinase C in regulating dense granule secretion. Analysis of autoradiographs showing [³²P]-labeled platelet proteins indicated that the phosphorylation of a 40 kDa protein, a known substrate for protein kinase C in platelets, was significantly less following thrombin stimulation in platelets from copper-deficient than from control rats. When protein kinase C was activated by phorbol 12-myristate 13-acetate prior to thrombin stimulation, ATP release was attenuated regardless of copper status. These findings suggest that protein kinase C can still function as a feedback inhibitor of platelet dense granule secretion in copper deficiency, but impaired activation of this enzyme following thrombin stimulation may prevent it from achieving full regulatory capacity.     

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.     

Dynamics of intracellular granules with CD63-GFP in rat basophilic leukemia cells

CD63 is located on the basophilic granule membranes in resting basophils, mast cells, and platelets, and is also located on the plasma membranes of the cells. We constructed a CD63-GFP (green fluorescent protein) plasmid and introduced it into rat basophilic leukemia (RBL-2H3) cells to observe the movements of CD63 on degranulation. The movements of CD63-GFP were studied in living RBL cells by confocal laser scanning microscopy (CLSM). CD63-GFP, in which GFP was conjugated to the C-terminus of CD63, was located on both the granule membranes and the plasma membranes of RBL cells. The diameter of the fluorescent granules in the cytoplasm varied from 0.5 to 1.5 microm. Before antigen stimulation most granules with CD63-GFP hardly moved in RBL cells. However, after antigen stimulation the plasma membranes ruffled violently and the granules moved dramatically. They reached the plasma membranes in a few minutes and fused with them instantaneously. Analysis of the movement of each granule provided a new insight into the elementary process of degranulation. The velocity of the granule movement toward the plasma membranes on antigen stimulation was calculated to be 0.1+/-0.02 microm/s. This shows that the granules are able to reach the plasma membranes in 2 or 3 min if the diameter of the cells is 20 microm.     

Evidence for membrane-associated calpain I in human erythrocytes. Detection by an immunoelectrophoretic blotting method using monospecific antibody

Low and high Ca2+-requiring forms of Ca2+-dependent cysteine proteinase are known as calpain I and calpain II, respectively. We have obtained, for the first time, monospecific antibodies for calpain I and for calpain II. Using these antibodies and an electrophoretic blotting method, we have found that a small, but reproducible, amount of calpain I was associated with human erythrocyte membranes while the bulk of the protease was contained in the cytosol. Most of membrane-associated calpain I was extractable with 1% Triton X-100, but not with 0.1% detergent. In the presence of 0.1 mM Ca2+ and 5 mM cysteine, membrane-associated calpain I degraded the membrane protein band 4.1 preferentially and band 3 protein only slowly. The Ca2+-induced autodigestion of the membrane preparation was inhibited by leupeptin but not by a cytosolic calpain inhibitor, calpastatin, added to the incubation medium. No calpain II was detected in either erythrocyte cytosol or membranes when anti-calpain II antibody was used under the same conditions as those for the detection of calpain I.     

Altered membrane fluidity and signal transduction in the platelets from patients of thrombotic stroke

Several earlier studies have implicated platelet activation with the pathogenesis of thrombotic stroke. In this report we have studied the changes in membrane physical microenvironment and signal transduction in the platelets obtained from the patients with thrombotic stroke. Aggregation induced by the synthetic agonist thrombin receptor-activating peptide was significantly enhanced (p < 0.001) in the platelets obtained from the patients. Steady-state fluorescence anisotropy measurements using diphenylhexatriene reflected a significant increase in membrane microviscosity from 3.315 (± 0.103) in the control to 4.600 (± 0.119) in the stroke. Proteins of relative mobilities of 131, 100, 47 and 38 kDa were found to remain phosphorylated on tyrosine in the resting platelets obtained from thrombotic stroke patients while they were not phosphorylated in the control counterparts. Besides, calpain, a calcium dependent thiol protease present in the platelets, was found to remain active in this disease as reflected from the proteolysis of calpain substrates. Taken together, these data indicated abnormal circulating platelets in the patients of thrombotic stroke, which could contribute to the etiopathogenesis of this disease.     

Subcellular localization and some properties of lipoxygenase activity in human blood platelets.

Lipoxygenase activity was measured in human platelet subcellular fractions. From a sonicated platelet preparation, a granule fraction, mixed membranes (surface and intracellular) and cytosol fractions were separated by differential centrifugation. With respect to activities in the sonicated preparation, the lipoxygenase was slightly enriched in both the cytosol and mixed-membrane fractions and consistently de-enriched in the granule fractions. Approx. 65% and 20% of the total cell enzyme activity were found in the cytosol and mixed membranes respectively, with only 8% present in the granule fraction. Additionally we measured the lipoxygenase activity in purified surface- and intracellular-membrane subfractions prepared from the mixed membranes by free-flow electrophoresis. There was a slight enrichment in activity in the intracellular membrane fraction compared with that in the mixed membranes, and a depletion of activity in the surface membranes. Characterization of the enzyme activity, i.e. time course, pH-dependence, Ca2+-dependence, Vmax. and Km for arachidonic acid, and the carbon-position specificity for this acid, failed to reveal any significant differences between the membrane-bound and soluble forms of the lipoxygenase. These findings suggest that in human platelets the same lipoxygenase is associated with the membranes as in the cytosol and that the membrane-bound activity predominates in intracellular membrane elements.     

Association of gelsolin with actin filaments and cell membranes of macrophages and platelets

Recent evidence that polyphosphoinositides regulate the function of the actin-modulating protein gelsolin in vitro raises the possibility that gelsolin interacts with cell membranes. This paper reports ultrastructural immunohistochemical data revealing that gelsolin molecules localize with plasma and intracellular membranes, including rough endoplasmic reticulum, cortical vesicles and mitochondria of macrophages, and blood platelets. Anti-gelsolin gold also labeled the surface and interior of secondary lysosomes presumably representing plasma gelsolin ingested by these cells from the lung surface by endocytosis. Gelsolin molecules, visualized with colloidal gold in replicas of the cytoplasmic side of the substrate-adherent plasma membrane of mechanically unroofed and rapidly frozen and freeze-dried macrophages, associated with the ends of short actin filaments sitting on the cytoplasmic membrane surface. A generalized distribution of gelsolin molecules in thin sections of resting platelets rapidly became peripheral, and plasmalemma association increased following thrombin stimulation. At later times the distribution reverted to the cytoplasmic distribution of resting cells. These findings provide the first evidence for gelsolin binding to actin filament ends in cells and indicate that gelsolin functions in both cytoplasmic and membrane domains.     

Identification of calpain II in porcine sperm

The role that proteolytic enzymes may play in membrane-associated phenomena of sperm has been the subject of extensive investigation. In the present study, we have examined the possibility that a Ca2+-activated, neutral protease, calpain II, may be associated with sperm membranes. Using indirect immunofluorescence with primary antibodies, which are polyclonal and monoclonal antibodies directed against the 80 kDa subunit of calpain II, we have established the presence of this antigen in porcine sperm. Staining by anticalpain II (80 kDa subunit) of the apical segment of the acrosomal cap and basal body (centriolar) region was seen consistently. Variable staining of the sperm tail was also observed. These observations, combined with our positive identification of a 80 kDa protein in acrosomal membranes (via immunoblot), document the association of this protease with sperm membranes. The proximity of calpain II to the acrosome suggests a potential role for the protease in the Ca2+-mediation of the acrosome reaction.     

The effects of platelet secretion inhibitors on protein phosphorylation

Protein phosphorylation was investigated in human platelets after stimulation to secretion by thrombin. After stimulation by thrombin at 4 degrees C (in which secretion is inhibited), phosphorylations of the 80, 56, and 38 kDa polypeptides and dephosphorylation of the 67 kDa phosphopeptide eventually occurred. The phosphorylations of the 27 and 20 kDa polypeptides remained inhibited until the temperature was increased to 37 degree C, which also resulted in secretion. Various stimulants and inhibitors of platelet function were used to characterize individual protein phosphorylations. The divalent-cation ionophore, A23187, induced the phosphorylations (or dephosphorylation) of the same proteins as thrombin with the exception of the 80 kDa protein, which remained incompletely phosphorylated. The intracellular calcium antagonist, TMB-8, inhibited thrombin-stimulated secretion and phosphorylation of all the polypeptides except the 80 kDa protein. The dephosphorylation of the 67 kDa phosphoprotein was not affected by TMB-8. Incubation of platelets with prostaglandin E1 and isobutylmethylxanthine inhibited thrombin-stimulated secretion and the phosphorylation of the 38 and 20 kDa protein and increased the phosphorylation of the 67 and 27 kDa phosphoproteins. These observations may be used to correlate protein phosphorylation with secretion, suggesting a possible sequence of intracellular events that mediate thrombin-stimulated secretion.