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Biomedical subjects

M Chignard

Publications and source records attributed to M Chignard.

At least 19 recordsLinked to original sources
PubMed1996

Proteolysis of thrombospondin during cathepsin-G-induced platelet aggregation: functional role of the 165-kDa carboxy-terminal fragment.

The serine-proteinase cathepsin G (CG) is a potent agonist of platelet aggregation inducing the release and surface expression of alpha-granule adhesive proteins such as fibrinogen (Fg) and thrombospondin-1 (TSP-1). Because Fg and TSP-1 are potential substrates for the enzymatic activity of CG, we investigated the fate of these proteins during CG-induced platelet aggregation using an immunoblot technique. Only a small proportion of secreted Fg was proteolyzed by CG and platelet aggregation was efficiently inhibited by anti-fibrinogen Fab fragments. In contrast, TSP-1 was extensively proteolyzed on aggregated platelets releasing in the milieu a fragment with Mr approximately 28 000, corresponding to the amino-terminal heparin-binding domain (HBD). Several antibodies, directed against the cell-associated carboxy-terminal TSP-1f fragment (Mr approximately 165000) impaired the formation of stable macroaggregates, indicating that this fragment may contribute to platelet aggregation in the absence of the HBD.

Adult↗
PubMed1996

The phospholipase C/protein kinase C pathway is involved in cathepsin G-induced human platelet activation: comparison with thrombin.

Cathepsin G, an enzyme released by stimulated polymorphonuclear neutrophils, and thrombin are two human proteinases which potently trigger platelet activation. Unlike thrombin, the mechanisms by which cathepsin G initiates platelet activation have yet to be elucidated. The involvement of the phospholipase C (PLC)/protein kinase C (PKC) pathway in cathepsin G-induced activation was investigated and compared with stimulation by thrombin. Exposure of 5-[14C]hydroxytryptamine-labelled platelets to cathepsin G, in the presence of acetylsalicylic acid and phosphocreatine/creatine kinase, induced platelet aggregation and degranulation in a concentration-dependent manner (0.1-3.0 microM). Time-course studies (0-180 s) comparing equivalent concentrations of cathepsin G (3 microM) and thrombin (0.5 unit/ml) resulted in very similar transient hydrolysis of phosphatidylinositol 4,5-bisphosphate and steady accumulation of phosphatidic acid. In addition cathepsin G, like thrombin, initiated the production of inositol phosphates. The neutrophil-derived proteinase also induced phosphorylation of both the myosin light chain and pleckstrin, a substrate for PKC, to levels similar to those observed in platelets challenged with thrombin. Inhibition of PKC by GF 109203X, a specific inhibitor, suppressed platelet aggregation and degranulation to the same extent for both proteinases. Using fura 2-loaded platelets, the rise in the cytosolic free Ca2+ concentration induced by cathepsin G was shown to result, as for thrombin, from both mobilization of internal stores and Ca2+ entry across the plasma membrane. These findings provide evidence that cathepsin G stimulates the PLC/PKC pathway as potently as does thrombin, independently of thromboxane A2 formation and ADP release, and that this pathway is required for platelet functional responses.

Amino Acid Sequence↗
PubMed1996

Inhibition of neutrophil-endothelial cell adhension by a neutrophil product, cathepsin G.

In the present study we investigated the modulation of the polymorphonuclear neutrophil (PMN)-endothelial cell adhesion process by the two main proteinases released from activated PMN during their adhesion to endothelium. Our results showed that, in contrast with elastase, cathepsin G was a powerful inhibitor of PAIN adhesion to interleukin-1 (IL-1)-treated human umbilical vein endothelial cells. This inhibitory effect was linked to the enzymatic activity of the proteinase and was selectively directed against PMN. Because the viability and the reactivity of PMN were not modified by cathepsin G, we looked for a possible effect on adhesion molecules. L-selectin was not cleaved by cathepsin G, whereas it was by chymotrypsin, a closely related proteinase. Cathepsin G blocked PMN adhesion to activated endothelial cells, but also to serum- or fibrinogen-coated plates, three adhesion processes mediated by CD11b/CD18. However, by FACScan analysis or by immunoprecipitation, we failed to find evidence of modifications of CD11b/CD18 expression. Although the precise molecular target(s) of cathepsin G remain(s) to be defined, these data indicate that this proteinase, which is known as an inflammatory mediator, can also be considered as a potential down-regulator of adhesion reactions involved in the inflammatory process.

CD18 Antigens↗
PubMed1996

Effect of cyclo-oxygenase inhibitors and modulators of cyclic AMP formation on lipopolysaccharide-induced neutrophil infiltration in mouse lung.

1. The adult respiratory distress syndrome (ARDS) is an acute lung inflammation developed after direct or indirect contact with pathogenic agents. In the present study, a mouse model was developed to mimic this condition using aerosolized bacterial lipopolysaccharide (LPS) and to investigate the mechanisms involved in the lung inflammatory response. 2. Inhalation of LPS led to a time and dose-dependent increase in tumour necrosis factor-alpha (TNF-alpha) production and neutrophil recruitment into the bronchoalveolar lavage fluid (BALF) of Balb/c mice. Under the same conditions, neutrophil infiltration was also found in the BALF of the LPS-sensitive mouse strain C3H/HeN, but was absent in the LPS-resistant strain C3H/HeJ. Intranasal administration of murine recombinant TNF-alpha also triggered neutrophil recruitment. 3. One hour after inhalation of LPS, half of the maximal level of TNF-alpha was measured in the BALF, but only a few neutrophils were detected at this time. The peak TNF-alpha concentration was reached at 3 h, when the neutrophil amount started to increase. At 24 h, maximal neutrophil number was found in the BALF and TNF-alpha was no longer present. 4. Pretreatment of mice under different experimental conditions demonstrated that: (a) cycloheximide almost completely blocks both neutrophil recruitment and TNF-alpha production; (b) anti TNF-alpha antibodies block neutrophil recruitment; (c) indomethacin or aspirin enhance by two fold neutrophil recruitment; (d) indomethacin significantly increases TNF-alpha production 1 h after inhalation of LPS; (e) dibutyryl cyclic AMP and prostaglandin E2 (PGE2) block both neutrophil recruitment and TNF-alpha production. 5. It is concluded that aerosolized LPS in mice triggers an acute lung inflammation which can be used as a potential model of inhalational ARDS and that, strategies leading to the elevation of cyclic AMP levels in vivo can be effective in modulating LPS-induced TNF-alpha synthesis and neutrophil recruitment.

Administration, Inhalation↗
PubMed1995

Neutrophil-mediated platelet activation: a key role for serine proteinases.

1. Neutrophils and platelets interact in vitro through multiple biochemical pathways in both directions, resulting in an inhibition or a potentiation of their reactivity, depending on the experimental conditions. 2. Under some conditions, a full stimulation of platelets (aggregation and degranulation) can be induced by neutrophils. The present review is focused on this aspect for which serine proteinases released from the azurophilic granules of neutrophils activate surrounding platelets. 3. The different facets of this process at the cellular and molecular levels, are presently depicted and their relevance to the in vivo situation suggested.

Animals↗
PubMed1994

Plasma antiproteinase screen and neutrophil-mediated platelet activation. A major role played by alpha 1 antitrypsin.

Upon activation, human polymorphonuclear neutrophils (PMN) release two serine proteinases, cathepsin G (Cat.G) and elastase (HLE), which in turn synergize to activate nearby platelets. We looked for the inhibitory effect of plasma and the involvement of alpha 1 antichymotrypsin (alpha 1 ACT) and alpha 1 antitrypsin (alpha 1 AT), on this cell-to-cell cooperation. It was observed that inhibition by plasma of PMN-mediated platelet activation was rather correlated with an effect on HLE (r = 0.95) than on Cat.G (r = 0.65) enzymatic activity. Purified alpha 1 AT suppressed in a concentration-dependent manner HLE activity present in the supernatant of activated PMN. When HLE was fully blocked, alpha 1 AT started to inhibit Cat.G activity. By contrast and as expected, purified alpha 1 ACT inhibited only Cat.G activity. Using specific blocking polyclonal antibodies against alpha 1 AT and alpha 1 ACT, it was demonstrated that the inhibitory effect of plasma vs. HLE was entirely mediated by alpha 1 AT. By contrast, blockade of Cat.G activity was only partly due to plasma alpha 1 ACT and around 50% was attributable to alpha 1 AT. When plasma from patients with an acute inflammatory state was used in place of plasma from normal subjects, the inhibitory effect was more pronounced, while plasma depleted in alpha 1 AT and alpha 1 ACT was less effective. These data indicate a predominant role of alpha 1 AT in the inhibition by plasma of the PMN-mediated platelet activation.

Amino Acid Sequence↗
PubMed1994

Neutrophil proteinase cathepsin G is proteolytically active on the human platelet glycoprotein Ib-IX receptor: characterization of the cleavage sites within the glycoprotein Ib alpha subunit.

The proteolytic activity of the neutrophil serine-proteinase cathepsin G (CG) on platelet adherence receptors, the glycoprotein (GP) Ib-IX complex and the integrin alpha IIb beta 3, has been investigated. In the range 50 to 200 nmol/l, CG is a potent platelet agonist which induces shape change, granule exocytosis and aggregation. Investigation of the proteolysis of the receptors' subunits during the course of platelet activation by CG was performed by immunoblot analysis of platelet proteins using a panel of specific antibodies. Exposure of platelets for 3 min at 37 degrees C to CG at a concentration that induces full cell activation resulted in an extensive cleavage of the N-terminal region of the extracellular domain of GPIb alpha, the largest (relative molecular mass, M(r), 143,000) of the three subunits constituting the GPIb-IX complex. In contrast, no detectable proteolytic modification of the two other subunits, GPIb beta and GPIX, was detected. Similarly, we observed that neither of the two subunits of the alpha IIb beta 3 receptor were proteolytically modified by CG. Cleavage of GPIb alpha by CG leaves a remnant of the polypeptide chain with M(r) approx. 106,000 in the plasma membrane, while releasing into the extracellular milieu the N-terminal domain with M(r) in the range 40,000 to 46,000. N-terminal sequencing of the CG-derived fragments of GPIb alpha indicated that the Leu275-Tyr276 peptide bond was the primary cleavage site for this proteinase. Proteolysis of GPIb alpha was already detectable at concentrations of CG as low as 25 nmol/l, while with 200 nmol/l the cleavage was detected as soon as 10 s after exposure of platelets to the proteinase. Comparison of the kinetics and concentration dependency for the proteolysis of GPIb alpha and for the activation of platelets by CG showed that cleavage of the GPIb-IX receptor is an early event that accompanies exocytosis and aggregation. Quantitative evaluation of the conversion of GPIb alpha into its membrane fragment indicated that, under optimal conditions, a maximum of approx. 50% of the total GPIb alpha can be affected by proteolysis. However, this proteolysis was > 90% complete when platelets were in the presence of the potent antagonist prostacyclin, suggesting that cellular redistribution of the GPIb-IX receptor may also occur during activation by CG. These results thus indicate that the very early phase of platelet activation by CG is accompanied by extensive modifications in the structure and expression of the GPIb-IX receptor, an effect that might be of functional significance for the interaction of platelets with the vessel wall.

Amino Acid Sequence↗
PubMed1994

Proteinase 3. A neutrophil proteinase with activity on platelets.

Purified proteinase 3 (PR3) devoid of any elastase (HLE) and cathepsin G (Cat.G) contaminants, was prepared from azurophilic granules of human polymorphonuclear neutrophils by using a novel procedure. Although unable to induce platelet activation (up to 25 micrograms/ml) by itself, PR3 at a concentration as low as 2.5 micrograms/ml enhanced the platelet response to a concomitantly added threshold concentration of Cat.G, a recognized platelet agonist. In the presence of 10 micrograms/ml PR3, aggregation and degranulation of platelets induced by Cat.G were 43.2 +/- 5.9% and 27.1 +/- 1.9% as compared with 5.5 +/- 2.9% and 4.2 +/- 1.5% (n = 4) for Cat.G alone. This enhancing effect by PR3 was also observed with collagen and a cyclic endoperoxide analogue, and was inhibited by eglin C and elafin, two PR3 inhibitors. Associated with the removal of activity by a anti-PR3 mAb and the lack of effect of the secretory leukocyte proteinase inhibitor, these data demonstrated that the effect is specifically related to the enzymatic activity of PR3. It is hypothesized that this mechanism could play a role in the polymorphonuclear neutrophil-mediated platelet activation, an event already known to be dependent on Cat.G and HLE. This is supported by the fact that the association of PR3 and HLE, at concentrations ineffective by themselves, was able to potentiate Cat.G-induced platelet activation.

Blood Platelets↗
PubMed1994

Modulation by superoxide anions of neutrophil-mediated platelet activation.

When polymorphonuclear neutrophil-platelet suspensions were stimulated by 0.5 microM N-formyl-Met-Leu-Phe in the presence of 40 U/mL of superoxide dismutase, a significant reduction of platelet secretion was observed (51.4 +/- 6.3% vs 62.4 +/- 4.6% for control; mean +/- SEM; N = 6; P < 0.01). This was due to the superoxide anion scavenging property of superoxide dismutase since neutrophil degranulation, cathepsin G and elastase enzymatic activities (the two main mediators of this cell-to-cell interaction) and platelet reactivity were not affected. Involvement of superoxide anions was confirmed using leukotriene B4, a neutrophil agonist which induces degranulation with minimal superoxide anion production. Indeed, serotonin release induced by this agonist was unchanged whether superoxide dismutase was added or not.

Cathepsin G↗
PubMed1994

Regulation of the structure and activity of platelet adhesion receptors by leukocyte proteinases.

Two major membrane receptors implicated in the adhesive properties of blood platelets are the GPIb-IX complex, a receptor for subendothelial von Willebrand factor, and the alpha IIb beta 3 integrin, the receptor for plasma fibrinogen. We have evaluated how the biological activities of these receptors can be potentially modulated through limited proteolysis when platelets are exposed to the serine-proteinases secreted by activated polymorphonuclear neutrophils, i.e., leukocyte elastase (EL) and cathepsin G (CG). CG can activate the alpha IIb beta 3 integrin through intracellular metabolic pathways, but has no direct proteolytic activity on the receptor subunits. By contrast, EL does not activate the platelet metabolism, but specifically cleaves a short peptide sequence within the alpha IIb subunit, and this cleavage occurs in parallel with an up-regulation of the activity of the fibrinogen receptor. On another hand, both EL and CG cleave the amino-terminal portion of the GPIb alpha subunit of the GPIb-IX receptor, eliminating the binding site for von Willebrand factor and diminishing the capacity of platelets to interact with this adhesion protein. Thus, neutrophil proteinases have the potential to regulate the activity of platelet adhesion receptors, and such experimental observations may prove to be relevant in vivo in various pathological conditions.

Endopeptidases↗
PubMed1994

Progressive inactivation of cathepsin G and elastase released from activated neutrophils in vitro: lack of participation of the neutrophil alpha 1-proteinase inhibitor.

Addition of platelets to activated human polymorphonuclear neutrophils (PMNs) led to their aggregation and degranulation, with these responses decreasing as a function of the time interval between PMN activation and platelet addition. Thus, for a 15-second interval platelet aggregation and serotonin release reached 51.3% +/- 6.7% (n = 11) and 64.3% +/- 4.9% (n = 8), respectively, but after a 5-minute interval they were totally absent. This effect was correlated with the decrease in enzymatic activities of elastase (HLE) and cathepsin G (CAT-G) that were released on PMN activation and responsible for the activation of nearby platelets (r = 0.86 and 0.90 for CAT-G and HLE, respectively; p < 0.05). Because it has been recently shown that PMNs express an alpha 1-proteinase inhibitor (alpha 1-Pl) gene and secrete this antiproteinase at their surface, we investigated whether the PMN alpha 1-Pl could regulate the biologic activities of both proteinases. Although superoxide anions oxidize alpha 1-Pl and reduce its affinity for CAT-G and HLE, maneuvers aimed at modifying their concentrations did not modify the loss of CAT-G and HLE. In fact, the progressive decrease of the two proteinase enzymatic activities followed the same pattern whether PMNs were present or not. It is concluded that PMN alpha 1-Pl is not involved in the time-dependent inactivation of CAT-G and HLE released from activated PMNs.

Blood Platelets↗
PubMed1993

Inhibition by human leukocyte elastase of neutrophil-mediated platelet activation.

When human polymorphonuclear neutrophils and platelets were incubated with human leukocyte elastase before N-formyl-Met-Leu-Phe (FMLP) challenge, a time- and concentration-dependent inhibition of the resulting platelet activation was observed. Thus, when the mixed cell suspension was preincubated for 6 min with 1 microM elastase before stimulation of neutrophils with 0.5 microM FMLP, resulting aggregations and serotonin releases were respectively only 4.4 +/- 4.1% (n = 4) and 1.6 +/- 2.4% (n = 4) as compared to 41.6 +/- 5.2% (n = 9) and 71.3 +/- 16.0 (n = 9) for controls. A direct inhibitory action of elastase on neutrophil activation was ruled out, as well as a breakdown of cathepsin G, a mediator involved in neutrophil-mediated platelet activation. In fact, we demonstrated that the target for the inhibitory effect of elastase in such a cell-to-cell cooperation system was the platelet. This phenomenon is likely to play a role under in vivo conditions in pathologies in which a significant granulocytic proteolytic activity has been detected in the plasma.

Blood Platelets↗
PubMed1993

Enhancement of cathepsin G-induced platelet activation by leukocyte elastase: consequence for the neutrophil-mediated platelet activation.

We have focused our interest on the platelet-activating properties of two polymorphonuclear neutrophil (PMN)-derived proteinases, namely elastase (HLE) and cathepsin G (Cat.G). First of all, we observed that whereas HLE was unable to trigger platelet activation by itself, it enhanced platelet activation induced by Cat.G when both proteinases were added simultaneously. It has been recently described that, upon stimulation, PMN released Cat.G, which in turn activated surrounding platelets. Thus, we looked for a combined effect of Cat.G and HLE during this cell-to-cell interaction. When PMN (5 x 10(6)/mL) were stimulated by 0.5 mumol/L N-formyl-Met-Leu-Phe, they released 237.9 +/- 49.1 nmol/L Cat.G and 381.7 +/- 28.0 nmol/L HLE. Such a concentration of purified Cat.G (240 nmol/L) induced only a moderate platelet activation when added to a PMN-platelet mixture. However, when Cat.G (240 nmol/L) and HLE (380 nmol/L) were added together, the resulting platelet activation was strictly comparable to that corresponding to the addition of N-formyl-Met-Leu-Phe (P > .05) in terms of aggregation, dense and alpha granule secretion, and thromboxane B2 production. In fact, Elafin, a specific HLE inhibitor, when added to the PMN-platelet cooperation system triggered by N-formyl-Met-Leu-Phe, prevented platelet activation within the same range of concentrations as for inhibition of HLE activity. In conclusion, we now show that not only Cat.G, but also HLE is involved in the PMN-mediated platelet activation.

Amino Acid Sequence↗
PubMed1993

Inhibition by recombinant SLPI and half-SLPI (Asn55-Ala107) of elastase and cathepsin G activities: consequence for neutrophil-platelet cooperation.

1. The capacity of recombinant human secretory leukocyte proteinase inhibitor (SLPI) to inhibit human leukocyte elastase (HLE) and cathepsin G (Cat G) was investigated and compared with a recombinant truncated form (carboxyl-terminal domain, Asn55-Ala107) called 1/2 SLPI. 2. Both compounds were efficient when tested against enzymatic activities of purified HLE and Cat G indicating that the HLE- and Cat G-inhibitory sites were preserved in the truncated form. SLPI and 1/2 SLPI also affected platelet activation induced by 0.2 microM Cat G (IC50 = 112 +/- 13 nM for SLPI and 280 +/- 12 nM for 1/2 SLPI). 3. The effects of SLPI and 1/2 SLPI were then tested against polymorphonuclear neutrophil (PMN)-mediated platelet activation, a cell-to-cell interaction mediated by HLE and Cat G released from PMN. In this experimental system, addition of SLPI or 1/2 SLPI before N-formyl-Met-Leu-Phe (fMLP) led to the inhibition of the resulting platelet activation. As was the case for Cat G enzymatic activity and Cat G-induced platelet activation, SLPI was more efficient than 1/2 SLPI (IC50 = 676 +/- 69 nM vs 1121 +/- 150 nM). 4. The ratio of the IC50 against PMN-mediated platelet activation compared to purified Cat G-mediated platelet activation was 6.03 for SLPI and 4.32 for 1/2 SLPI. This difference may be due to the smaller size of the truncated form which could allow this molecule to diffuse more easily between PMN and platelets. 5. In conclusion, 1/2 SLPI could be a promising candidate in the treatment of pathological states linked to inflammation in which participation of HLE and Cat G has been evoked.

Amino Acid Sequence↗
PubMed1993

Leucocyte elastase-mediated release of von Willebrand factor from cultured endothelial cells.

The aim of this study was to investigate a possible activation of human endothelial cells in monolayer culture by a purified neutrophil-derived proteinase, i.e. elastase. Cells were isolated from human umbilical cord veins, and incubated either in primary culture or after two passages, in the presence of various concentrations of this proteinase. Although a lack of prostacyclin formation was noted, elastase induced a large release of von Willebrand factor (vWf) in a concentration-dependent manner. Thus, upon incubation with 1 microgram.ml-1 elastase for 30 min, 70 mU.ml-1 vWf were detected in the incubation medium, as compared to 5 mU.ml-1 for control. Using cells in primary culture, a fivefold higher concentration of vWf was recovered following incubation with 10 micrograms.ml-1 elastase than with 0.5 IU.ml-1 thrombin. This effect was linked to the enzymatic activity of elastase and not to its cationic charge, as deduced from the inhibition by eglin C, and the lack of effect of the phenylmethylsulphonyl fluoride (PMSF) treated proteinase. We conclude that vWf release was not due to cell activation, since cytoplasmic calcium mobilization was absent, and inhibition of protein kinase C did not modify the response. In fact, this release was the consequence of cell damage, since concentrations of vWf recovered correlated with cell lysis. These results support the hypothesis that the high level of plasma vWf in patients with sepsis or adult respiratory distress syndrome could result from damage to the endothelial cells by elastase released from activated neutrophils.

Calcium↗
PubMed1992

Activation and damage of cultured airway epithelial cells by human elastase and cathepsin G.

Accumulation of polymorphonuclear neutrophils (PMN) and epithelium damage have often been described during airway inflammation. We studied the effects of two PMN-derived proteinases, namely elastase and cathepsin G, on guinea-pig tracheal epithelial cells in culture. Both proteinases activated tracheal epithelial cells in terms of prostaglandin (PG) E2 production. A concentration- and time-dependent effect was observed with 10 micrograms/ml and 6 h as the optimal conditions for both enzymes. Optical microscopic studies confirmed an effect on tracheal epithelial cells as intercellular gaps were observed upon incubation of the monolayers with proteinases. A small cytotoxic effect was observed after 1 h incubation but remained stable up to 6 h. This cytotoxic effect, more pronounced with elastase than with cathepsin G, was dissociated from PGE2 formation.

Animals↗