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Role of protease-activated receptors in neutrophil degranulation
Medical Hypotheses (2002) 59(3), 266–267 ª 2002 Elsevier Science Ltd. All rights reserved. doi: 10.1016/S0306-9877(02)00214-1, available online at http://www.idealibrary.com
Role of protease-activated receptors in neutrophil degranulation S. Kannan Department of Physiology, School of Medicine, Temple University, Philadelphia, USA
Summary Inflammation is a process culminated by cellular components and soluble mediators act in concert to evolve a sustainable process to impart distress in vascularized tissue. The role of thrombin receptors in neutrophil degranulation is not fully understood. Thrombin-mediated signaling has been shown to occur at least in part by a family of G protein-coupled protease-activated receptors (PARs). Protease-activated receptors-1 (PAR1) is the prototype for understanding on the fundamental signaling mechanism and related characterization. The inference of thrombin receptors and various synthetic ligand formulations on neutrophil degranulation ascertained an enormous disparity in the response by human donors. Based on the previous reports and supposition in perpetuity, it is hypothesized that thrombin through its receptor, and its subtypes, may very improbably have any functional role in PMN degranulation. ª 2002 Elsevier Science Ltd. All rights reserved.
At the site of the vascular injury caused by one or more factor(s) (metabolic or mechanical, or infectious agent(s)) considerable amount of thrombin is generated. Thrombin, a coagulation protease (EC 3.4.21.5), is the best studied extracellular protease functioning like a hormone with confounding impact in myocardial infarction, stroke, and other thrombotic processes (1). Thrombin-mediated signaling has been shown to occur at least in part by a family of G-protein-coupled protease-activated receptors (PARs). Protease-activated receptor-1 (PAR1) is the prototype for understanding on the fundamental signaling mechanism and related characterization (2). Thrombin, recognizes the amino terminal exodomain of the G-protein-coupled thrombin receptor, PAR1 subsequently cleaves the peptide bond between Arg-41 and Ser-42. Thus, the newly formed amino terminus (SFLLRN) functions as a tethered peptide ligand, dock-
Received 23 August 2001 Accepted 19 December 2001 Correspondence to: S. Kannan, Department of Microbiology & Immunology, University of Texas Medical Branch, at Galveston, PO Box 25056, TX 77555, USA. Tel./Fax: +409-750-9060; E-mail: [email protected], [email protected]
266
ing intramoleculary to the PAR1 receptor to initiate the transmembrane signaling (2–4). Further, SFFLLRN represents the first six amino acids of unmasked PAR1 functions as an agonist independent of thrombin and proteolytic cleavage (5). To date, four PAR receptors are known namely. PAR1; PAR2; PAR3, and PAR4. All four subtype PARs have been characterized in both mouse and human. PAR1 plays a critical role in human platelet activation by thrombin, whereas PAR2 requires trypsin for its activation (6). While PAR3 is activated by thrombin and plays a pivotal role in mouse platelet activation (7), whereas PAR4 is activated by cathepsin G for neutrophil-dependent platelet activation (8). Thrombin-mediated effect occurs via (a) PAR1, a high affinity receptor requiring low concentration (sub-nanomolar) for platelet activation (2,3,9) and (b) PAR4 requires high concentrations of thrombin (low-affinity receptors) to mediate the events (10). A dual thrombin receptor model was proposed for platelet activation in mouse and human platelets. Where, PAR1 and PAR4 are required for human platelet activation in response to thrombin, PAR3 and PAR4 are corresponding receptor systems for thrombin-mediated mouse platelet activation (10,11). Thrombin has been shown to cause the liberation of arachidonic acid from endogenous phospholipid pool
Role of protease 267
in human neutrophil (12). Further, the phospholipase A2 (PLA2) isolated from human platelets has been shown to stimulate the production of leukotriene B4 from human polymorphonuclear leukocytes. It was suggested that soluble PLA2 released at the inflammation site might function as a facilitator in LTB4 generation in human neutrophils and further amplify the inflammatory processes involving degranulation (13). A direct evidence for thrombin-induced LTB4 generation and release by the neutrophil was observed in peripheral lymph (see Fig. 8 in (14)). LTB4 , an important leukotriene known to play a significant role in inflammatory diseases, is released into the extracellular milieu. LTB4 has been shown to act in an autocrine fashion via Gi coupled receptors on neutrophils and cause the degranulation. A systematic approach to evaluate the role of leukotrienes in PAR1-, PAR4-induced human neutrophil stimulation, and the differences in signaling events in the PAR and LTB4 -mediated neutrophil stimulation and degranulation were conflicting. The deficiency of the study could be concluded as follows: even though the localization of PAR-1 on neutrophils was successful PAR4 could not be localized (Tuluc and Kunapuli, pers. comm.). Neutrophils exposed to PAR1, TFLLRNPNDK, a PAR1-specific peptide (15) (not SFLLRN which activates PAR1 and PAR2) or PAR4 (GYPGKF) induced degranulation were not consistent among different donors (human). It should be pointed out that this evidence is concurrent with the accredited studies where neutrophils incubated with 14 C 5-HETE fail to cause the 5lipoxygenase activation (16). Furthermore, it is hypothesized that lack of 5-lipoxygenase activation in PAR1- or PAR4-stimulated human PMN may very likely be due to the lack of extracellular Ca2þ influx in to the cytosol. It is known that LTB4 receptor expression is unchanged in leukocytes from 5-lipoxygenase deficient mice compared to the wild-type mice (17). Failure to detect any significant difference between 5-lipoxygenase-deficient mice compare to the normal mice underscores the importance of thrombin receptors’ role in neutrophil degranulation. One plausible reason could be endogenous degradation and/or desensitization of thrombin receptors. Hypothetically, if the thrombin receptors are desensitized through an unknown mechanism, thrombin will be unresponsive in any of the signaling cascade leading to the neutrophil degranulation. Taken together, the published reports and also the lack of concurring experimental evidence on thrombin and its receptors, that it is very implausible for PAR to have any consequential role in PMN degranulation. ª 2002 Elsevier Science Ltd. All rights reserved.
REFERENCES 1. Coughlin S. R. How the protease thrombin talks to cells. Proc Natl Acad Sci USA 1999; 96: 11023–11027. 2. Vu T. K., Hung D. T., Wheaton V. I., Coughlin S. R. Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell 1991; 64: 1057–1068. 3. Vu T. K., Wheaton V. I., Hung D. T., Charo I., Coughlin S. R. Domains specifying thrombin-receptor interaction. Nature 1991; 353: 674–677. 4. Chen J. H., Karlberg K. E., Sylven C. Heparin and low molecular weight heparin but not hirudin stimulate platelet aggregation in whole blood from acetylsalicylic acid treated healthy volunteers. Thromb Res 1991; 63: 319–329. 5. Scarborough R. M., Naughton M. A., Teng W. et al. Tethered ligand agonist peptides. Structural requirements for thrombin receptor activation reveal mechanism of proteolytic unmasking of agonist function. J Biol Chem 1992; 267: 13146–13149. 6. Nystedt S., Emilsson K., Wahlestedt C., Sundelin J. Molecular cloning of a potential proteinase activated receptor. Proc Natl Acad Sci USA 1994; 91: 9208–9212. 7. Ishihara H., Connolly A. J., Zeng D., Kahn M. L., Zheng Y. W., Timmons C., Tram T., Coughlin S. R. Protease-activated receptor 3 is a second thrombin receptor in humans. Nature 1997; 386: 502–506. 8. Xu W. F., Andersen H., Whitmore T. E. et al. Cloning and characterization of human protease-activated receptor 4. Proc Natl Acad Sci USA 1998; 95: 6642–6646. 9. Rasmussen U. B., Vouret-Craviari V., Jallat S. et al. cDNA cloning and expression of a hamster alpha-thrombin receptor coupled to Ca2þ mobilization. FEBS Lett 1991; 288: 123–128. 10. Kahn M. L., Hammes S. R., Botka C., Coughlin S. R. Gene and locus structure and chromosomal localization of the protease-activated receptor gene family. J Biol Chem 1998; 273: 23290–23296. 11. Kahn M. L., Zheng Y. W., Huang W. et al. A dual thrombin receptor system for platelet activation. Nature 1998; 394: 690–694. 12. Ramesha C. S., Taylor L. A. Measurement of arachidonic acid release from human polymorphonuclear neutrophils and platelets: comparison between gas chromatographic and radiometric assays. Anal Biochem 1991; 192: 173–180. 13. Lam B. K., Gagnon L., Austen K. F., Soberman R. J. The mechanism of leukotriene B4 export from human polymorphonuclear leukocytes. J Biol Chem 1990; 265: 13438–13441. 14. Malik A. B., Lo S. K., Bizios R. Thrombin-induced alterations in endothelial permeability. Ann N Y Acad Sci 1986; 485: 293–309. 15. Damiano B. P., Cheung W. M., Santulli R. J. et al. Cardiovascular responses mediated by protease-activated receptor-2 (PAR-2) and thrombin receptor (PAR-1) are distinguished in mice deficient in PAR-2 or PAR-1. J Pharmacol Exp Ther 1999; 288: 671–678. 16. Baranes D., Matzner J., Razin E. Thrombin-induced calciumindependent degranulation of human neutrophils. Inflammation 1986; 10: 455–461. 17. Martin V., Ronde P., Unett D. et al. Leukotriene binding, signaling, and analysis of HIV coreceptor function in mouse and human leukotriene B4 receptor-transfected cells. J Biol Chem 1999; 274: 8597–8603.
Medical Hypotheses (2002) 59(3), 266–267
Role of protease-activated receptors in neutrophil degranulation S. Kannan Department of Physiology, School of Medicine, Temple University, Philadelphia, USA
Summary Inflammation is a process culminated by cellular components and soluble mediators act in concert to evolve a sustainable process to impart distress in vascularized tissue. The role of thrombin receptors in neutrophil degranulation is not fully understood. Thrombin-mediated signaling has been shown to occur at least in part by a family of G protein-coupled protease-activated receptors (PARs). Protease-activated receptors-1 (PAR1) is the prototype for understanding on the fundamental signaling mechanism and related characterization. The inference of thrombin receptors and various synthetic ligand formulations on neutrophil degranulation ascertained an enormous disparity in the response by human donors. Based on the previous reports and supposition in perpetuity, it is hypothesized that thrombin through its receptor, and its subtypes, may very improbably have any functional role in PMN degranulation. ª 2002 Elsevier Science Ltd. All rights reserved.
At the site of the vascular injury caused by one or more factor(s) (metabolic or mechanical, or infectious agent(s)) considerable amount of thrombin is generated. Thrombin, a coagulation protease (EC 3.4.21.5), is the best studied extracellular protease functioning like a hormone with confounding impact in myocardial infarction, stroke, and other thrombotic processes (1). Thrombin-mediated signaling has been shown to occur at least in part by a family of G-protein-coupled protease-activated receptors (PARs). Protease-activated receptor-1 (PAR1) is the prototype for understanding on the fundamental signaling mechanism and related characterization (2). Thrombin, recognizes the amino terminal exodomain of the G-protein-coupled thrombin receptor, PAR1 subsequently cleaves the peptide bond between Arg-41 and Ser-42. Thus, the newly formed amino terminus (SFLLRN) functions as a tethered peptide ligand, dock-
Received 23 August 2001 Accepted 19 December 2001 Correspondence to: S. Kannan, Department of Microbiology & Immunology, University of Texas Medical Branch, at Galveston, PO Box 25056, TX 77555, USA. Tel./Fax: +409-750-9060; E-mail: [email protected], [email protected]
266
ing intramoleculary to the PAR1 receptor to initiate the transmembrane signaling (2–4). Further, SFFLLRN represents the first six amino acids of unmasked PAR1 functions as an agonist independent of thrombin and proteolytic cleavage (5). To date, four PAR receptors are known namely. PAR1; PAR2; PAR3, and PAR4. All four subtype PARs have been characterized in both mouse and human. PAR1 plays a critical role in human platelet activation by thrombin, whereas PAR2 requires trypsin for its activation (6). While PAR3 is activated by thrombin and plays a pivotal role in mouse platelet activation (7), whereas PAR4 is activated by cathepsin G for neutrophil-dependent platelet activation (8). Thrombin-mediated effect occurs via (a) PAR1, a high affinity receptor requiring low concentration (sub-nanomolar) for platelet activation (2,3,9) and (b) PAR4 requires high concentrations of thrombin (low-affinity receptors) to mediate the events (10). A dual thrombin receptor model was proposed for platelet activation in mouse and human platelets. Where, PAR1 and PAR4 are required for human platelet activation in response to thrombin, PAR3 and PAR4 are corresponding receptor systems for thrombin-mediated mouse platelet activation (10,11). Thrombin has been shown to cause the liberation of arachidonic acid from endogenous phospholipid pool
Role of protease 267
in human neutrophil (12). Further, the phospholipase A2 (PLA2) isolated from human platelets has been shown to stimulate the production of leukotriene B4 from human polymorphonuclear leukocytes. It was suggested that soluble PLA2 released at the inflammation site might function as a facilitator in LTB4 generation in human neutrophils and further amplify the inflammatory processes involving degranulation (13). A direct evidence for thrombin-induced LTB4 generation and release by the neutrophil was observed in peripheral lymph (see Fig. 8 in (14)). LTB4 , an important leukotriene known to play a significant role in inflammatory diseases, is released into the extracellular milieu. LTB4 has been shown to act in an autocrine fashion via Gi coupled receptors on neutrophils and cause the degranulation. A systematic approach to evaluate the role of leukotrienes in PAR1-, PAR4-induced human neutrophil stimulation, and the differences in signaling events in the PAR and LTB4 -mediated neutrophil stimulation and degranulation were conflicting. The deficiency of the study could be concluded as follows: even though the localization of PAR-1 on neutrophils was successful PAR4 could not be localized (Tuluc and Kunapuli, pers. comm.). Neutrophils exposed to PAR1, TFLLRNPNDK, a PAR1-specific peptide (15) (not SFLLRN which activates PAR1 and PAR2) or PAR4 (GYPGKF) induced degranulation were not consistent among different donors (human). It should be pointed out that this evidence is concurrent with the accredited studies where neutrophils incubated with 14 C 5-HETE fail to cause the 5lipoxygenase activation (16). Furthermore, it is hypothesized that lack of 5-lipoxygenase activation in PAR1- or PAR4-stimulated human PMN may very likely be due to the lack of extracellular Ca2þ influx in to the cytosol. It is known that LTB4 receptor expression is unchanged in leukocytes from 5-lipoxygenase deficient mice compared to the wild-type mice (17). Failure to detect any significant difference between 5-lipoxygenase-deficient mice compare to the normal mice underscores the importance of thrombin receptors’ role in neutrophil degranulation. One plausible reason could be endogenous degradation and/or desensitization of thrombin receptors. Hypothetically, if the thrombin receptors are desensitized through an unknown mechanism, thrombin will be unresponsive in any of the signaling cascade leading to the neutrophil degranulation. Taken together, the published reports and also the lack of concurring experimental evidence on thrombin and its receptors, that it is very implausible for PAR to have any consequential role in PMN degranulation. ª 2002 Elsevier Science Ltd. All rights reserved.
REFERENCES 1. Coughlin S. R. How the protease thrombin talks to cells. Proc Natl Acad Sci USA 1999; 96: 11023–11027. 2. Vu T. K., Hung D. T., Wheaton V. I., Coughlin S. R. Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell 1991; 64: 1057–1068. 3. Vu T. K., Wheaton V. I., Hung D. T., Charo I., Coughlin S. R. Domains specifying thrombin-receptor interaction. Nature 1991; 353: 674–677. 4. Chen J. H., Karlberg K. E., Sylven C. Heparin and low molecular weight heparin but not hirudin stimulate platelet aggregation in whole blood from acetylsalicylic acid treated healthy volunteers. Thromb Res 1991; 63: 319–329. 5. Scarborough R. M., Naughton M. A., Teng W. et al. Tethered ligand agonist peptides. Structural requirements for thrombin receptor activation reveal mechanism of proteolytic unmasking of agonist function. J Biol Chem 1992; 267: 13146–13149. 6. Nystedt S., Emilsson K., Wahlestedt C., Sundelin J. Molecular cloning of a potential proteinase activated receptor. Proc Natl Acad Sci USA 1994; 91: 9208–9212. 7. Ishihara H., Connolly A. J., Zeng D., Kahn M. L., Zheng Y. W., Timmons C., Tram T., Coughlin S. R. Protease-activated receptor 3 is a second thrombin receptor in humans. Nature 1997; 386: 502–506. 8. Xu W. F., Andersen H., Whitmore T. E. et al. Cloning and characterization of human protease-activated receptor 4. Proc Natl Acad Sci USA 1998; 95: 6642–6646. 9. Rasmussen U. B., Vouret-Craviari V., Jallat S. et al. cDNA cloning and expression of a hamster alpha-thrombin receptor coupled to Ca2þ mobilization. FEBS Lett 1991; 288: 123–128. 10. Kahn M. L., Hammes S. R., Botka C., Coughlin S. R. Gene and locus structure and chromosomal localization of the protease-activated receptor gene family. J Biol Chem 1998; 273: 23290–23296. 11. Kahn M. L., Zheng Y. W., Huang W. et al. A dual thrombin receptor system for platelet activation. Nature 1998; 394: 690–694. 12. Ramesha C. S., Taylor L. A. Measurement of arachidonic acid release from human polymorphonuclear neutrophils and platelets: comparison between gas chromatographic and radiometric assays. Anal Biochem 1991; 192: 173–180. 13. Lam B. K., Gagnon L., Austen K. F., Soberman R. J. The mechanism of leukotriene B4 export from human polymorphonuclear leukocytes. J Biol Chem 1990; 265: 13438–13441. 14. Malik A. B., Lo S. K., Bizios R. Thrombin-induced alterations in endothelial permeability. Ann N Y Acad Sci 1986; 485: 293–309. 15. Damiano B. P., Cheung W. M., Santulli R. J. et al. Cardiovascular responses mediated by protease-activated receptor-2 (PAR-2) and thrombin receptor (PAR-1) are distinguished in mice deficient in PAR-2 or PAR-1. J Pharmacol Exp Ther 1999; 288: 671–678. 16. Baranes D., Matzner J., Razin E. Thrombin-induced calciumindependent degranulation of human neutrophils. Inflammation 1986; 10: 455–461. 17. Martin V., Ronde P., Unett D. et al. Leukotriene binding, signaling, and analysis of HIV coreceptor function in mouse and human leukotriene B4 receptor-transfected cells. J Biol Chem 1999; 274: 8597–8603.
Medical Hypotheses (2002) 59(3), 266–267