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Cathepsin G and Thrombin: Evidence for Two Different Platelet Receptors

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Cathepsin G and Thrombin: Evidence for Two Different Platelet Receptors University of New Hampshire University of New Hampshire Scholars' Repository New Hampshire Agricultural Experiment Station Publications New Hampshire Agricultural Experiment Station 1-15-1994 Cathepsin G and thrombin: evidence for two different platelet receptors Mary A. Selak University of New Hampshire - Main Campus Follow this and additional works at: https://scholars.unh.edu/nhaes Part of the Biochemistry Commons Recommended Citation Selak, M.A. Cathepsin G and thrombin: evidence for two different platelet receptors. Biochemical Journal. 1994. V. 297 pp. 269-75 (Printed in Great Britain). This Article is brought to you for free and open access by the New Hampshire Agricultural Experiment Station at University of New Hampshire Scholars' Repository. It has been accepted for inclusion in New Hampshire Agricultural Experiment Station Publications by an authorized administrator of University of New Hampshire Scholars' Repository. For more information, please contact [email protected]. Biochem. J. (1994) 297, 269-275 (Printed in Great Britain) 269 Cathepsin G and thrombin: evidence for two different platelet receptors Mary A. SELAK Department of Biochemistry and Molecular Biology, University of New Hampshire, Durham, NH 03824, U.S.A. Neutrophil cathepsin G and thrombin, the only platelet agonists exhibiting an elevation in cytosolic Ca2+ concentration but did that are proteases, exhibit a mandatory requirement for catalytic not respond to cathepsin G; and (c) platelets pretreated with activity to induce platelet aggregation and signal transduction. neutrophil elastase failed to respond to thrombin but responded The thrombin receptor is a G-protein-coupled receptor which when rechallenged by cathepsin G. Thrombin and cathepsin G undergoes proteolysis to generate a tethered ligand that causes exhibit heterologous desensitization that is potentiated by self-activation. Since cathepsin G strongly resembles thrombin in okadaic acid and is attenuated by staurosporine, indicating that its ability to activate platelets, we have attempted to determine phosphorylation of serine/threonine residues is important for whether cathepsin G and thrombin function through the same or desensitization and that protein kinase C may be involved. Since different receptors. Evidence that thrombin and cathepsin G act catalytic activity of cathepsin G is required for platelet stimu- at different receptors was as follows: (a) an antibody directed lation, it is probable that platelet activation by cathepsin G against the thrombin receptor blocked thrombin-induced but not requires receptor proteolysis and that a tethered ligand mech- cathepsin G-induced platelet responses; (b) human fibroblasts anism is involved, suggesting that platelets may possess a family responded to thrombin and to a synthetic thrombin receptor of protease receptors. peptide (comprising residues 42-55 of the thrombin receptor) by INTRODUCTION by cathepsin G requires receptor proteolysis and that a tethered ligand mechanism is involved. While our previous results indicate We have previously demonstrated that neutrophil cathepsin G, that cathepsin G strongly resembles thrombin in its ability to like thrombin, is a strong platelet agonist which exhibits activate platelets [1], data presented here suggest that these two saturable, reversible binding to platelets, characteristic of the agonists function through distinct receptors rather than through involvement of a specific receptor [1,2]. In addition, cathepsin G a single receptor which is differentially cleaved to yield two and thrombin are the only platelet agonists that are proteases, different tethered ligands. Evidence indicating that platelets and most of the biological actions of both cathepsin G and possess different receptors for thrombin and cathepsin G suggests thrombin require the catalytic activity of the enzymes. In that platelets possess a family of protease receptors. particular, aggregation and stimulus-response coupling in plate- lets are abolished when the proteolytic activity ofeither cathepsin MATERIALS AND METHODS G or thrombin is blocked or neutralized [1-4]. The thrombin receptor has been cloned and sequenced and shown to possess Materials seven transmembrane domains characteristic of all members of Monoclonal antibody ATAP138, directed against the SFLLR- the family of G-protein-linked receptors [5]. However, unlike any NPNDKYEPF sequence of the thrombin receptor (Ser42-Phe55), other receptor yet described, proteolysis of the extracellular N- was a gift from Dr. Lawrence Brass (University of Pennsylvania, terminal receptor domain is required for activation. Cleavage of Philadelphia, PA, U.S.A.), and a-thrombin was a generous gift the thrombin receptor at Arg4l exposes a new N-terminus which from Dr. John Fenton (Albany Medical College, Albany, NY, then functions as a tethered ligand to activate cells by binding to U.S.A.). Okadaic acid was obtained from Gibco or from Kamiya a site on the receptor [5,6]. The binding site for thrombin includes Biomedical Company, and staurosporine was purchased from a region of the N-terminus of the receptor distal to the cleavage Kamiya Biomedical Company. TRP42155 was purchased from site which bears a strong resemblance to hirudin and functions to Bacham Bioscience Inc., Philadelphia, PA, U.S.A. bind thrombin via its anion exosite [6-8]. A synthetic peptide composed of receptor residues Ser42-Phe55 (thrombin receptor Preparation and labelling of platelets peptide; TRP42155) has been shown to function as a full platelet agonist [5,9]. However, only the first six amino acids of this 14- Human blood was obtained from healthy volunteers and col- residue peptide are required for activity [10,11]. TRP42155 has also lected into acid-citrate dextose. The platelet-rich plasma, ob- been shown to activate human endothelial cells [12] and hamster tained by centrifugation at 180 g for 20 min at room temperature, fibroblasts [13], suggesting that endothelial cells, fibroblasts and was re-centrifuged at 800 g for 15 min at room temperature to platelets share a structurally similar thrombin receptor. obtain a platelet pellet. The platelet pellet was resuspended in Activation of the thrombin receptor by this novel intra- 10 ml of plasma and the platelets were incubated at 37 °C with molecular rearrangement raises the question of whether a family 3,M fura-2-acetoxymethyl ester for 45 min and with 1 ,uM 5- of similar G-protein-coupled protease receptors might exist. hydroxy[14C]tryptamine and 1 mM aspirin for 30 min (45 min Since the catalytic activity of cathepsin G is mandatory for total incubation time). Fura-2 acetoxymethyl ester and aspirin platelet stimulation, it is highly probable that platelet activation were dissolved in dimethyl sulphoxide and, when added to cells, Abbreviations used: TRP42155, thrombin receptor peptide (residues Ser42-Phe55 of the thrombin receptor); [Ca2+]i, cytosolic free Ca2+ concentration; PC/CK, phosphocreatine/creatine kinase; PAF, platelet-activating factor; GPlb, glycoprotein lb. 270 M. A. Selak the final dimethyl sulphoxide concentration never exceeded Tyrode's buffer containing 10 mM glucose and 5 mM EDTA 0.5 %. Platelets were gel-filtered using Sepharose CL-2B columns was added, and the cells were incubated for 10 min at 37 °C. equilibrated and eluted with calcium-free Tyrode's buffer supple- Cells were harvested mechanically with a cell scraper, dispersed mented with 0.20% fatty acid-free BSA and 1O mM glucose. by trituration, washed once and resuspended in Tyrode's buffer Imipramine (1,M) was added to 5-hydroxy['4C]tryptamine- containing 10 mM glucose and 1 mM CaCl2. Fibroblasts were labelled cells. handled as described above for platelets. Purification of neutrophil cathepsin G and elastase RESULTS Buffy coat leucocytes were used to isolate cathepsin G from Platelet responsiveness to cathepsin G and thrombin was neutrophil granules by the method of Baugh and Travis [14]. examined by measuring the extent of dense granule secretion Granule extracts were subjected to sequential aprotinin- and the amount of calcium discharged from internal stores, as Sepharose and CM-Sephadex chromatographies [15]. Protein estimated from the size of calcium transients in fura-2- and 5- concentration was determined using the BCA protein assay hydroxy[14C]tryptamine-loaded cells. To exclude the involvement (Pierce) with BSA as standard, as well as by absorbance at of endoperoxides/thromboxane A2 and ADP, all measurements 280 nm. The purity of cathepsin G and elastase was assessed by were performed using aspirinated platelets incubated in the specific substrate hydrolysis and by both SDS and non- presence of phosphocreatine/creatine kinase (PC/CK). Controls denaturing acid gel electrophoresis as described previously [1]. verified that the cells were unresponsive to ADP and arachidonic Based on these criteria, cathepsin G was totally devoid of acid. Platelet calcium and secretion responses were routinely neutrophil elastase and vice versa. Cathepsin G and elastase measured in the presence of EGTA to eliminate repletion of enzymic activities were measured spectrophotometrically at 37 °C internal pools due to agonist-induced calcium influx through the using N-succinyl-(Ala)2-Pro-Phe-p-nitroanilide and methoxy- plasma membrane. Though not always shown, ionomycin was succinyl-(Ala)2-Pro-Val-p-nitroanilide respectively as substrates, added to each sample to verify that depletion of intracellular according to the method of Nakajima et al. [16]. calcium was not responsible
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