Patricia Renesto

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 α‐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 M r ≈ 28 000, corresponding to the amino‐terminal heparin‐binding domain (HBD). Several antibodies, directed against the cell‐associated carboxy‐terminal TSP‐1f fragment (M r ≈ 165 000) impaired the formation of stable macroaggregates, indicating that this fragment may contribute to platelet aggregation in the absence of the HBD.

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.

Plasma antiproteinase screen and neutrophil-mediated platelet activation. A major role played by α1 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.

Proteinases and Cytokines in Neutrophil and Platelet Interactions In Vitro. Possible Relevance to the Adult Respiratory Distress Syndrome

While polymorphonuclear neutrophils are endowed with potent mechanisms for phagocytosis and the killing of microbes, and thus contribute to a host defense against microbicidal infections, they have also long been recognized as important effectors of inflammation. Indeed, when their migration in tissues is not regulated, their excessive accumulation could contribute to host tissue injury following release of two essential groups of toxic products: granular substances and toxic species of reduced oxygen.’ Lungs are particularly exposed to such insults and constitute a primary site of damage caused by host defense mechanisms. Indeed, if only a few neutrophils are normally present in the lung interstitium and alveolar spaces, these cells are susceptible to respond to chemotactic stimuli and accumulate in the lung. Thus, neutrophils have been specifically implicated in the adult respiratory distress syndrome (ARDS) and emphysema.’ After several years, the concept that neutrophils contribute to inflammation not only alone but also through interactions with platelets, has emerged. For example, a neutrophil-dependent platelet deposition was observed in different experimental models such as dermal inflammation,3 occlusion-reperfusion myocardial injury,4 or subendothelial immune complex glomerulonephritis.5 In a model of ARDS, it has been shown that not only neutrophils but also platelets contributed to lung injury.6 In this pathology, characterized by a loss of integrity of the alveolar-capillary wall, platelets have been described as potential contributors.’ I n uitro studies have revealed that such a cell-to-cell interaction was effective and was of extreme complexity. Different biochemical and biological pathways have thus been depicted: generation of oxygen derivatives, synthesis of arachidonate metabolites and platelet-activating factor (PAF), contact through the expression of P-selectin (GMP140 or CD62). formation of neutrophil-activating peptide 2 (NAP-2), and participation of p r o t e i n a s e ~ . ~ ~ ~ We have focused our interest on one of these mechanisms, that is, the neutroPhil-induced platelet activation mediated by proteinases and modulated by cyto-

Interference of anti-inflammatory and anti-asthmatic drugs with neutrophil-mediated platelet activation: singularity of azelastine

1 The capacity of various drugs (acetylsalicylic acid (ASA), ketoprofen, diclofenac, piroxicam, BW 755C, BW A4C, nedocromil sodium and azelastine) to inhibit human polymorphonuclear neutrophil (PMN)‐mediated platelet activation was investigated. In this model, stimulated PMN release cathepsin G (Cat G), a serine proteinase which, in turn, induces platelet activation. 2 Among the different tested drugs, azelastine (100 μm for 1 min) was the only one able to prevent platelet aggregation. The cyclo‐oxygenase inhibitors were all inactive, although used at effective concentrations as judged by inhibition of thromboxane B2 (TxB2) formation. Inhibition of platelet aggregation by azelastine was concentration‐dependent, the range of active concentrations being of 20–70 μm. Release from platelets of 5‐hydroxytryptamine was also inhibited at 30 μm and above, but never reached 100%. 3 The inhibition by azelastine is due to an effect on both cells. Indeed, β‐glucuronidase release from activated PMN and platelet activation by purified Cat G were both affected. 4 However, used at high concentrations (>100 μm) azelastine was toxic since it released significant amounts of lactate dehydrogenase (LDH) from PMN and platelets. 5 These results show the capacity of azelastine, an anti‐allergic and anti‐asthmatic compound, to inhibit the cell‐to‐cell communication between PMN and platelets, an effect which may be relevant for its therapeutic efficacy or for a new application in diseases in which PMN and platelets are involved.

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.

Combined activation of platelets by cathepsin G and platelet activating factor, two neutrophil-derived agonists.

Summary. In this paper we have studied the combined effects on platelet activation, of two polymorphonuclear neutrophil (PMN)‐derived agonists, namely platelet‐activating factor (PAF) and cathepsin G (Cat.G), used at threshold concentrations. Our results showed that the order of agonist addition was a determinant factor since the addition of Cat.G prior to PAF induced a full platelet activation while the reverse combination had no effects. The successive challenge of platelets by Cat.G and then PAF induced a strong aggregation accompanied by an enhancement of α and dense granule secretion. The observed phenomenon was also dependent on the time interval between agonist addition. It was significant at 30 s (P < 0·05) and plateaued over 1–2 min. Platelet activation resulting from the combination Cat.G‐PAF can be described as a function of PAF concentrations, the synergism being significant between 10 Nm and 1 μm. The mechanism by which Cat.G primes platelets remains to be elucidated. However, some points have been examined and have led us to conclude that an increase in expression and/or affinity of PAF receptors, [Ca2+]i movements, protein kinase C activation and phospholipase A2 pathway are not involved. Whatever the biochemical mechanism underlying this synergism which involved PMN and platelets, it may constitute a link between the inflammatory and haemostatic processes in response to tissue damage.

INHIBITION OF NEUTROPHIL-ENDOTHELIAL CELL ADHESION 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 PMN 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 GD11b/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.

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.

Protective effect of platelet activating factor antagonists on cultured endothelial cell lysis induced by elastase or activated neutrophils.

1 The mechanism(s) responsible for injury of endothelial cells induced by human leukocyte elastase (HLE) was investigated in an immortalized venous human endothelial cell line (IVEC). 2 First, the proteinase concentrations and incubation delays necessary to trigger a significant IVEC cytotoxicity were determined by chromium assays. Thus, exposure of IVEC for 6 h to 10 μ ml−1 HLE resulted in 22 ± 2.8% lysis and 36.4 ± 5.4% detachment (mean ± s.e. mean; n = 4; P < 0.05). 3 WEB 2086, a specific platelet‐activating factor (PAF) receptor antagonist, induced a significant concentration‐dependent decrease of such a lysis (39.6 ± 7.7% protection at 100 μ n = 4). This potential role for PAF was confirmed with two other antagonists of this lipid mediator, i.e., BN 52021 and RP 48740. 4 Finally, we demonstrated that pretreatment of IVEC with WEB 2086 protected significantly against cell lysis induced by stimulated human neutrophils, an experimental model in which HLE participates.