scholarly journals Hypochlorite-induced oxidation of proteins in plasma: formation of chloramines and nitrogen-centred radicals and their role in protein fragmentation

1999 ◽  
Vol 340 (2) ◽  
pp. 539-548 ◽  
Author(s):  
Clare L. HAWKINS ◽  
Michael J. DAVIES
Keyword(s):  
Plasma Protein ◽  
Plasma Proteins ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Nitrobenzoic Acid ◽  
Parent Protein ◽  
Sds Page ◽  
Amine Groups ◽  
Epr Spin Trapping ◽  
Protein Fragmentation

Activated phagocyte cells generate hypochlorite (HOCl) via the release of H2O2 and the enzyme myeloperoxidase. Plasma proteins are major targets for HOCl, although little information is available about the mechanism(s) of oxidation. In this study the reaction of HOCl (at least 50 μM) with diluted fresh human plasma has been shown to generate material that oxidizes 5-thio-2-nitrobenzoic acid; these oxidants are believed to be chloramines formed from the reaction of HOCl with protein amine groups. Chloramines have also been detected with isolated plasma proteins treated with HOCl. In both cases chloramine formation accounts for approx. 20-30% of the added HOCl. These chloramines decompose in a time-dependent manner when incubated at 20 or 37 °C but not at 4 °C. Ascorbate and urate remove these chloramines in a time- and concentration-dependent manner, with the former being more efficient. The reaction of fresh diluted plasma with HOCl also gives rise to protein-derived nitrogen-centred radicals in a time- and HOCl-concentration-dependent manner; these have been detected by EPR spin trapping. Identical radicals have been detected with isolated HOCl-treated plasma proteins. Radical formation was inhibited by excess methionine, implicating protein-derived chloramines (probably from lysine side chains) as the radical source. Plasma protein fragmentation occurs in a time- and HOCl-concentration-dependent manner, as evidenced by the increased mobility of the EPR spin adducts, the detection of further radical species believed to be intermediates in protein degradation and the loss of the parent protein bands on SDS/PAGE. Fragmentation can be inhibited by methionine and other agents (ascorbate, urate, Trolox C or GSH) capable of removing chloramines and reactive radicals. These results are consistent with protein-derived chloramines, and the radicals derived from them, as contributing agents in HOCl-induced plasma protein oxidation.

The Role of β2-Glycoprotein I in the Clearance of Platelet Microvesicles.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 145-145
Author(s):  
Hanan Abdel-Monem ◽  
Swapan Kumar Dasgupta ◽  
Anhquyen Le ◽  
Anthony Prakasam ◽  
Perumal Thiagarajan
Keyword(s):  
Plasma Proteins ◽  
Plasma Concentrations ◽  
Major Protein ◽  
Maximal Effect ◽  
Dependent Manner ◽  
Anionic Phospholipid ◽  
Concentration Dependent Manner ◽  
Glycoprotein I

Abstract Abstract 145 The physiological function of β2-glycoprotein I is unclear and several studies suggest a role in the clearance of anionic phospholipid containing membranes. Anionic phospholipid containing liposomes are cleared rapidly from the circulation by the reticuloendothelial cells. In rats, uptake of liposomes by Kupffer cells requires that the liposomes bind to plasma proteins. In mice, the clearance of liposomes from the circulation is related to their ability to interact with plasma proteins. β2-glycoprotein I was identified as a major protein associated with rapid clearance of liposomes and pretreating the mice with antiβ2- glycoprotein I antibodies was found to significantly increase the half-life of the liposome. In vitro, β2-glycoprotein I was also shown to promote the phagocytosis of phosphatidylserine containing liposomes and apoptotic tumor cells. In conditions associated with increased microvesicles generation such as disseminated intravascular coagulation, plasma levels of β;2-glycoprotein I was reduced presumably due to its consumption. Antibodies to β2 glycoprotein I are frequently seen in patients with systemic lupus erythematosus and at times, in otherwise normal individuals. A subset of these antibodies prevents the assembly of the prothrombinase and the tenase complexes on phospholipid membrane, leading to the lupus anticoagulant effect. The presence of these antibodies is clinically very significant, as individuals harboring these antibodies are at risk for thromboembolic manifestations. We studied the role of β-glycoprotein I in the clearance of procoagulant platelet microvesicles and the effect of the auto antibodies in the phagocytosis of platelet microvesicles. We labeled β2-glycoprotein I with BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene)-hydrazide and β2-glycoprotein I incorporated 1.8 mole of BODIPY /mole. Labeling of β2-glycoprotein I with BODIPY did not change the binding efficacy of β2-glycoprotein I to cardiolipin as determined by Elisa assay. Binding of BODIPY-β2-glycoprotein I to platelet microvesicles was analyzed by flow cytometry. BODIPY- β2-glycoprotein I bound to phosphatidylserine-expressing platelet microvesicles in a concentration-dependent manner. Binding was inhibited by 50 fold molar excess of unlabeled β2-glycoprotein I, annexin A5 and the phosphatidylserine-binding C1C2 fragment of lactadherin. β2-glycoprotein I also promoted the phagocytosis of platelet microvesicles by THP-1 derived macrophages in vitro at physiological plasma concentrations with a half maximal effect at ∼10 ug/ml. β2-glycoprotein I-mediated phagocytosis was inhibited by annexin V and the C1C2 fragment of lactadherin. Furthermore, immunoaffinity purified β2-glycoprotein I-dependent antiphospholipid antibodies from 5 patients inhibited the phagocytosis in a concentration dependent manner. These studies suggest β2-glycoprotein I binding to phosphatidylserine-expressing procoagulant platelet microvesicles promotes their clearance by macrophages and autoantibodies to β2-glycoprotein I inhibit the process. The predictive value of antiβ-2 glycoprotein I for thrombosis is highly variable but the correlation is stronger in patients with lupus. In lupus, there is impaired clearance of procoagulant apoptotic cells. β2-glycoprotein I may have a significant role in their clearance and antibodies to β2-glycoprotein I may causally related to the thrombosis in these patients by inhibiting the clearance. Disclosures: No relevant conflicts of interest to declare.

Recombinant Plasmin from Lemna: Purification, Characterization and In Vitro Comparison with Human Plasma-Derived Plasmin.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4001-4001
Author(s):  
Gary L. Brookhart ◽  
Michael D. Tomalski ◽  
Karim C. Lounes ◽  
Piet Meijer ◽  
Cornelis Kluft ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Human Plasma ◽  
Lemna Minor ◽  
Tissue Type ◽  
Stable Complex ◽  
Dependent Manner ◽  
Dna Encoding ◽  
Concentration Dependent Manner ◽  
Western Blots ◽  
Sds Page

Abstract Background: The fibrinolytic enzyme plasmin has gained renewed interest as a direct thrombolytic agent. Local delivery of plasmin via catheter into thrombi, combined with the efficient neutralization of plasmin that may escape from the clot by its inhibitors α2-antiplasmin (α2-AP) and α2-macroglobulin may provide safe and effective removal of pathologic blood clots. Objective: To overcome the potential for pathogen contamination associated with plasmin prepared from pooled human plasma, we have generated recombinant plasmin (r-PLM) from transgenic Lemna plants and compared its biological properties with those of plasma-derived plasmin (pd-PLM). Results: Lemna minor, an aquatic plant, was transfected with the c-DNA encoding human plasminogen, and grown in media under controlled conditions. Lemna-derived plasminogen (r-PLG) was extracted from plant-tissue, separated on lysine-Sepharose and subsequently converted into plasmin (r-PLM), using urokinase-type plasminogen activator (u-PA). r-PLM was further purified to homogeneity using affinity chromatography on benzamidine-Sepharose. r-PLG and r-PLM co-migrate with pd-PLG and pd-PLM on non-reducing SDS-PAGE with the respective molecular weights of about 90,000 kDa and 82,000 kDa. When analyzed on reducing SDS-PAGE, r-PLM, just like pd-PLM, separates into a 56,000 kDa heavy chain and a 26,000 light chain. On Western blots r-PLG and r-PLM cross-react with polyclonal antibodies that were raised against pd-PLG, indicating immunologic identity of r-PLG/PLM and pd-PLG/PLM. All three plasminogen activators (PA), tissue-type PA, u-PA and streptokinase, activate r-PLG. Activity assays using the PLM-specific chromogenic substrate S-2403 show that catalytic rates of r-PLM are indistinguishable from pd-PLM, indicating that in this assay r-PLM and pd-PLM have comparable activity. Purified r-PLM and pd-PLM have similar specific activities. Like pd-PLM, r-PLM activity is inhibited by its physiological inhibitor α2-AP and Kunitz-type inhibitors SBTI and aprotinin. Inhibition of r-PLM by α2-AP results in the formation of a high-molecular weight SDS-stable complex, which migrates on SDS-PAGE with a molecular weight of 150,000 kDa, the cumulative molecular weight of r-PLM (82,000 kDa) and α2-AP (65,000 kDa). In microtiter-based fibrinolysis assays, both pd- and r-plasmin dissolve fibrin clots at the same rate and in a concentration-dependent manner. Conclusion: This work demonstrates that proteins of the fibrinolytic system can be expressed in Lemna. Lemna-derived r-PLM and pd-PLM have comparable biochemical properties, activity, and are equally sensitive to known plasmin inhibitors. Plant-derived r-PLM may be a valuable alternative to PLM preparations from pooled human plasma.

scholarly journals Photochemical labeling of human erythrocyte membrane proteins with radioiodinated 4-azidosalicylic acid derivatives of G(M3), G(D3), G(M1), and FucG(M1) gangliosides.

1998 ◽  
Vol 45 (2) ◽  
pp. 509-521
Author(s):  
T Pacuszka ◽  
M Panasiewicz
Keyword(s):  
Specific Radioactivity ◽  
Band 3 ◽  
Uv Spectra ◽  
Dependent Manner ◽  
Protein Activity ◽  
Concentration Dependent Manner ◽  
High Specific Radioactivity ◽  
Sds Page ◽  
Erythrocyte Membrane Proteins ◽  
Derivatives Of

Photoreactive gangliosides of high specific radioactivity may prove useful for studies on glycosphingolipid functions. We prepared 4-azidosalicylic acid (ASA) acylated derivatives of GM3, GD3, GM1, and FucGM1 gangliosides (gangliosides-ASA). Gangliosides-ASA were characterized by their TLC mobility, UV spectra, carbohydrate composition, and digestion with leech endoceramidase. After radioiodination to about 200 Ci/mmole gangliosides-ASA were used for photochemical labeling of human erythrocytes. Radioiodinated gangliosides-ASA were incorporated into erythrocytes in a time and concentration dependent manner, the kinetics and extent of incorporation being similar for all the gangliosides-ASA used. Radioiodinated gangliosides-ASA incorporated into erythrocytes were resistant to trypsin digestion while treatment with 1% BSA removed about 90% of the label. Incubation with cholera toxin protected radioiodinated GM1-ASA and, to a lesser extent, FucGM1-ASA but not GM3-ASA and GD3-ASA, against removal with BSA. After photolysis about 40-50% of radioactivity was firmly bound to erythrocyte lipids and proteins. The ratio of lipid- to protein-bound radioactivity ranged from 2.2:1 to 3.2:1. Photolabeled proteins were analyzed by SDS/PAGE followed by autoradiography. Band 3 was the most extensively photolabeled protein with all the radioiodinated gangliosides-ASA used. DIDS, an inhibitor of band 3 protein activity, caused reduction in photolabeling of this protein by about 20%.

Comparative evaluation of plasma protein purification and 2-D gel electrophoresis protocols for analysis of HIV-1 infected plasma proteins (Preprint)

2021 ◽  
Author(s):  
Mr Sushanta Kumar Barik Sr ◽  
Deepika Varshney ◽  
Deepa Bisht Sr ◽  
Shripad A Patil Sr ◽  
Rananjaya Singh Sr ◽  
...  
Keyword(s):  
Protein Purification ◽  
Human Plasma ◽  
Plasma Protein ◽  
Gel Electrophoresis ◽  
Comparative Evaluation ◽  
Plasma Proteins ◽  
Human Plasma Protein ◽  
Sds Page ◽  
Hiv 1 ◽  
Abundance Proteins

BACKGROUND The focus of the study was the comparative evaluation of HIV-1 infected plasma protein purification and 2-D gel electrophoresis protocols. OBJECTIVE Human plasma protein purification is a risk task to perform 2-D gel electrophoresis. Human plasma proteins contain nearly 70% albumin and globulin. The removal of such high abundance high molecular weight proteins is very difficult to perform 2-D gel electrophoresis METHODS To the best of our knowledge, we searched several research papers, developed and adopted various organic and non-organic based protocols for HIV-1 infected human plasma protein purification and various isoelectrofocusing protocols in 2-D gel electrophoresis RESULTS After failure in 2-D gel-electrophoresis performance by these protocols, Aurum serum mini kit (Bio-Rad, USA) was adopted for plasma protein purification for performing 2-D gel electrophoresis. The low-abundance proteins were better resolved by 10% SDS-PAGE in 2-D gel-electrophoresis. Then, we extended the MALDI-TOF/TOF analysis of low-abundance proteins in human plasma by adopting the Aurum serum mini kit ( Bio-Rad, USA) for 2-D gel electrophoresis. CONCLUSIONS Thus, we concluded that, the Aurum serum mini kit (Bio-Rad, USA) is best to perform the 2-D gel electrophoresis of HIV-1 infected human plasma by depleting the high abundant proteins like albumin and globulin

scholarly journals Differentially expressed plasma protein fractions in workers of dyeing unit at a textile industry and comparable controls

2015 ◽  
Vol 49 (2) ◽  
pp. 89-94
Author(s):  
N Roohi ◽  
A Shaukat ◽  
MA Iqbal ◽  
T Mumtaz
Keyword(s):  
Health Status ◽  
Adverse Effects ◽  
Plasma Protein ◽  
Textile Industry ◽  
Plasma Proteins ◽  
T Test ◽  
Protein Fractions ◽  
Significant Decrement ◽  
Textile Dyeing ◽  
Sds Page

Workers of textile industry are exposed to different hazardous chemicals that cause adverse effects on their health. As variations in plasma proteins are the best indicators of health status of the subjects, hence, we compared plasma protein fractions, resolved by SDS-PAGE of textile dyeing industry workers (n=30) with control subjects (n=30). Proteins were quantified by Total Lab Quant software and analyzed, statistically, by Student t-test. Thirteen protein fractions were detected ranging 250-17kDa. A significant increase in 132 and 53kDa fractions, whereas, a significant decrement in 87 and 66kDa fractions with a highly significant reduction in 112kDa fraction was observed. However, 250, 224, 43, 23, 19 and 17kDa protein fractions did not vary considerably. As some proteins circulate only during particular physiological or pathological circumstances and may serve as biomarkers of anomalies, the altered expressions of these biomolecules observed in textile dyeing industry workers may be strong predictors of different ailments. DOI: http://dx.doi.org/10.3329/bjsir.v49i2.22002 Bangladesh J. Sci. Ind. Res. 49(2), 89-94, 2014

scholarly journals Comparative evaluation of plasma protein purification and 2-D gel electrophoresis protocols for analysis of HIV-1 infected human plasma proteins

2021 ◽  
Author(s):  
Sushanta Kumar Barik ◽  
Deepika Varshney ◽  
Keshar Kunja Mohanty ◽  
Deepa Bisht ◽  
Shripad A. Patil ◽  
...  
Keyword(s):  
Protein Purification ◽  
Human Plasma ◽  
Plasma Protein ◽  
Gel Electrophoresis ◽  
Comparative Evaluation ◽  
Plasma Proteins ◽  
Sds Page ◽  
High Molecular Weight Proteins ◽  
Hiv 1 ◽  
Abundance Proteins

Abstract Purification of proteins from human plasma is a herculean task to perform 2-D gel electrophoresis. Human plasma contains nearly 70% albumin and globulin. The removal of such high abundance high molecular weight proteins is very difficult before performing 2-D gel electrophoresis. It becomes more difficult when we intent to investigate in infectious diseases like HIV/AIDS. We tried to the best of our efforts adopting various organic and non-organic based protocols based on various published papers. After failure of these protocols in results of 2-D gel-electrophoresis Aurum serum mini kit (Bio-Rad, USA) was adopted for plasma protein purification for performing 2-D gel electrophoresis. The low-abundance proteins were better resolved by 10% SDS-PAGE in 2-D gel-electrophoresis. Then,we extended the MALDI-TOF/TOF analysis of low-abundance proteins in human plasma by adopting the Aurum serum mini kit (Bio-Rad, USA) for 2-D gel electrophoresis. Thus, we concluded that, depletion of high abundant proteins like albumin and globulin, the use of the Aurum serum mini kit (Bio-Rad, USA) is the protocol of choice to perform the 2-D gel electrophoresis of HIV-1 infected human plasma.

Inhibition of Recombinant Human Acetylcholinesterase Activity by Antipsychotics

Pharmacology ◽  
2019 ◽  
Vol 104 (1-2) ◽  
pp. 43-50 ◽  
Author(s):  
Keisuke Obara ◽  
Ayano Fujii ◽  
Chiaki Arie ◽  
Natsuki Harada ◽  
Fumiko Yamaki ◽  
...  
Keyword(s):  
Acetylcholinesterase Activity ◽  
Suppressive Effect ◽  
Blood Levels ◽  
Dependent Manner ◽  
Ache Inhibition ◽  
Inhibitory Effects ◽  
Concentration Dependent Manner ◽  
Nitrobenzoic Acid ◽  
Cholinergic Effects

Background/Aims: Extrapyramidal symptoms (EPS) are representative side effects of antipsychotics, caused by their inhibitory action on dopaminergic nerves in nigrostriatal pathways. EPS could be also caused by direct augmentation of cholinergic effects, for example, by acetylcholinesterase (AChE) inhibition. We investigated the potential inhibitory effects of 26 clinically available antipsychotics on the activity of recombinant human AChE (rhAChE) to predict the role of antipsychotic-induced AChE inhibition in EPS onset. Method: The degree of rhAChE activity inhibition was calculated using the 5,5′-dithio-bis-(2-nitrobenzoic acid) method. Results: At a concentration of 10–5 mol/L, haloperidol, bromperidol, timiperone, nemonapride, pimozide, risperidone, blonanserin, aripiprazole, and brexpiprazole inhibited rhAChE activity by >20%. Risperidone, aripiprazole, and brexpiprazole inhibited rhAChE activity in a concentration-dependent manner, and their effects were more potent than those of other antipsychotics. The inhibitory effects of these 3 drugs were evident from 10–6 mol/L, and their pIC50 values were 4.74 ± 0.04, 4.80 ± 0.04, and 4.93 ± 0.06, respectively. Notably, the concentration range in which aripiprazole inhibited rhAChE activity (≥10–6 mol/L) overlapped with its clinically achievable blood levels. Conclusion: Aripiprazole may cause EPS at clinical dosages by augmenting cholinergic effects via AChE inhibition, in addition to its suppressive effect on dopaminergic neurons.

scholarly journals Rapid Purification of a New P-I Class Metalloproteinase fromBothrops moojeniVenom with Antiplatelet Activity

2014 ◽  
Vol 2014 ◽  
pp. 1-12 ◽  
Author(s):  
Mayara R. de Queiroz ◽  
Carla C. Neves Mamede ◽  
Kelly C. Fonseca ◽  
Nadia C. G. de Morais ◽  
Bruna B. de Sousa ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Snake Venom ◽  
Biological Activities ◽  
Dependent Manner ◽  
Inhibitory Effects ◽  
Concentration Dependent Manner ◽  
Morphological Alterations ◽  
Sds Page ◽  
Fibrinogenolytic Activity ◽  
Rapid Purification

The present study aimed to evaluate the proteolytic and biological activities of a new metalloproteinase fromB. moojenivenom. The purification of BmooMPα-II was carried out through two chromatographic steps (ion-exchange and affinity). BmooMPα-II is a monomeric protein with an apparent molecular mass of 22.5 kDa on SDS-PAGE 14% under nonreducing conditions. The N-terminal sequence (FSPRYIELVVVADHGMFTKYKSNLN) revealed homology with other snake venom metalloproteinases, mainly among P-I class. BmooMPα-II cleaves Aα-chain of fibrinogen followed by Bβ-chain, and does not show any effect on theγ-chain. Its optimum temperature and pH for the fibrinogenolytic activity were 30–50°C and pH 8, respectively. The inhibitory effects of EDTA and 1,10-phenantroline on the fibrinogenolytic activity suggest that BmooMPα-II is a metalloproteinase. This proteinase was devoid of haemorrhagic, coagulant, or anticoagulant activities. BmooMPα-II caused morphological alterations in liver, lung, kidney, and muscle of Swiss mice. The enzymatically active protein yet inhibited collagen, ADP, and ristocetin-induced platelet aggregation in a concentration-dependent manner. Our results suggest that BmooMPα-II contributes to the toxic effect of the envenomation and that more investigations to elucidate the mechanisms of inhibition of platelet aggregation may contribute to the studies of snake venom on thrombotic disorders.

The Anti-Atherogenic Properties of Sesamin are Mediated via Improved Macrophage Cholesterol Efflux Through PPARγ1-LXRα and MAPK Signaling

2014 ◽  
Vol 84 (1-2) ◽  
pp. 79-91 ◽  
Author(s):  
Amin F. Majdalawieh ◽  
Hyo-Sung Ro
Keyword(s):  
Cholesterol Efflux ◽  
Transcriptional Activity ◽  
Molecular Mechanisms ◽  
Foam Cell ◽  
Mapk Signaling ◽  
Luciferase Reporter ◽  
Foam Cell Formation ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Macrophage Cholesterol Efflux

Background: Foam cell formation resulting from disrupted macrophage cholesterol efflux, which is triggered by PPARγ1 and LXRα, is a hallmark of atherosclerosis. Sesamin and sesame oil exert anti-atherogenic effects in vivo. However, the exact molecular mechanisms underlying such effects are not fully understood. Aim: This study examines the potential effects of sesamin (0, 25, 50, 75, 100 μM) on PPARγ1 and LXRα expression and transcriptional activity as well as macrophage cholesterol efflux. Methods: PPARγ1 and LXRα expression and transcriptional activity are assessed by luciferase reporter assays. Macrophage cholesterol efflux is evaluated by ApoAI-specific cholesterol efflux assays. Results: The 50 μM, 75 μM, and 100 μM concentrations of sesamin up-regulated the expression of PPARγ1 (p< 0.001, p < 0.001, p < 0.001, respectively) and LXRα (p = 0.002, p < 0.001, p < 0.001, respectively) in a concentration-dependent manner. Moreover, 75 μM and 100 μM concentrations of sesamin led to 5.2-fold (p < 0.001) and 6.0-fold (p<0.001) increases in PPAR transcriptional activity and 3.9-fold (p< 0.001) and 4.2-fold (p < 0.001) increases in LXR transcriptional activity, respectively, in a concentration- and time-dependent manner via MAPK signaling. Consistently, 50 μM, 75 μM, and 100 μM concentrations of sesamin improved macrophage cholesterol efflux by 2.7-fold (p < 0.001), 4.2-fold (p < 0.001), and 4.2-fold (p < 0.001), respectively, via MAPK signaling. Conclusion: Our findings shed light on the molecular mechanism(s) underlying sesamin’s anti-atherogenic effects, which seem to be due, at least in part, to its ability to up-regulate PPARγ1 and LXRα expression and transcriptional activity, improving macrophage cholesterol efflux. We anticipate that sesamin may be used as a therapeutic agent for treating atherosclerosis.

Neutrophil Elastase Potentiates Cathepsin G-lnduced Platelet Activation

1992 ◽  
Vol 68 (05) ◽  
pp. 570-576 ◽  
Author(s):  
Mary A Selak
Keyword(s):  
Neutrophil Elastase ◽  
Cell Activation ◽  
Thromboxane A2 ◽  
Creatine Phosphate ◽  
Dense Granule ◽  
Cathepsin G ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Low Concentrations ◽  
High Concentrations

SummaryWe have previously demonstrated that human neutrophil cathepsin G is a strong platelet agonist that binds to a specific receptor. This work describes the effect of neutrophil elastase on cathepsin G-induced platelet responses. While platelets were not activated by high concentrations of neutrophil elastase by itself, elastase enhanced aggregation, secretion and calcium mobilization induced by low concentrations of cathepsin G. Platelet aggregation and secretion were potentiated in a concentration-dependent manner by neutrophil elastase with maximal responses observable at 200 nM. Enhancement was observed when elastase was preincubated with platelets for time intervals of 10–60 s prior to addition of a low concentration of cathepsin G and required catalytically-active elastase since phenylmethanesulphonyl fluoride-inhibited enzyme failed to potentiate cell activation. Neutrophil elastase potentiation of platelet responses induced by low concentrations of cathepsin G was markedly inhibited by creatine phosphate/creatine phosphokinase and/or indomethacin, indicating that the synergism between elastase and cathepsin G required the participation of ADP and thromboxane A2. On the other hand, platelet responses were not attenuated by the PAF antagonist BN 52021, signifying that PAF-acether did not play a role in elastase potentiation. At higher concentrations porcine pancreatic elastase exhibits similar effects to neutrophil elastase, demonstrating that the effect of elastase was not unique to the neutrophil protease. While neutrophil elastase failed to alter the ability of cathepsin G to hydrolyze a synthetic chromogenic substrate, preincubation of platelets with elastase increased the apparent affinity of cathepsin G binding to platelets. In contrast to their effect on cathepsin G-induced platelet responses, neither neutrophil nor pancreatic elasatse potentiated aggregation or dense granule release initiated by ADP, PAF-acether, arachidonic acid or U46619, a thromboxane A2 mimetic. Moreover, unlike its effect on cathepsin G, neutrophil elastase inhibited thrombin-induced responses. The current observations demonstrate that elastase can potentiate platelet responses mediated by low concentrations of cathepsin G, suggesting that both enzymes may function synergistically to activate platelets under conditions where neutrophil degranulation occurs.

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