scholarly journals Neutrophil Cathepsin G Proteolysis of Protease Activated Receptor 4 Generates a Novel, Functional Tethered Ligand

2021 ◽  
Author(s):  
Michelle L. Stoller ◽  
Indranil Basak ◽  
Frederik Denorme ◽  
Jesse W Rowley ◽  
James Alsobrooks ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Synthetic Peptide ◽  
Cleavage Site ◽  
Serine Proteases ◽  
Cathepsin G ◽  
Mass Spectrometry Analysis ◽  
Calcium Flux ◽  
Spectrometry Analysis ◽  
Thrombin Cleavage ◽  
Tethered Ligand

Platelet-neutrophil interactions regulate ischemic vascular injury. Platelets are activated by serine proteases that cleave protease activated receptor (PAR) amino-termini, resulting in an activating tethered ligand. Neutrophils release cathepsin G (CatG) at sites of injury and inflammation, which activates PAR4 but not PAR1, although the molecular mechanism of CatG-induced PAR4 activation is unknown. We show that blockade of the canonical PAR4 thrombin cleavage site did not alter CatG-induced platelet aggregation, suggesting CatG cleaves a different site than thrombin. Mass spectrometry analysis using PAR4 N-terminus peptides revealed CatG cleavage at Ser67-Arg68. A synthetic peptide, RALLLGWVPTR, representing the tethered ligand resulting from CatG proteolyzed PAR4, induced PAR4-dependent calcium flux and greater platelet aggregation than the thrombin-generated GYPGQV peptide. Mutating PAR4 Ser67 or Arg68 reduced CatG-induced calcium flux without affecting thrombin-induced calcium flux. Dog platelets, which contain a conserved CatG PAR4 Ser-Arg cleavage site, aggregated in response to human CatG and RALLLGWVPTR, while mouse (Ser-Gln) and rat (Ser-Glu) platelets, were unresponsive. Thus, CatG amputates the PAR4 thrombin cleavage site by cleavage at Ser67-Arg68 and activates PAR4 by generating a new functional tethered ligand. These findings support PAR4 as an important CatG signaling receptor and suggest a novel therapeutic approach for blocking platelet-neutrophil-mediated pathophysiologies.

scholarly journals Immunologic analysis of the cloned platelet thrombin receptor activation mechanism: evidence supporting receptor cleavage, release of the N-terminal peptide, and insertion of the tethered ligand into a protected environment

Blood ◽  
1993 ◽  
Vol 82 (7) ◽  
pp. 2125-2136 ◽  
Author(s):  
KJ Norton ◽  
RM Scarborough ◽  
JL Kutok ◽  
MA Escobedo ◽  
L Nannizzi ◽  
...  
Keyword(s):  
Amino Acids ◽  
Platelet Aggregation ◽  
Platelet Activation ◽  
Cleavage Site ◽  
Receptor Activation ◽  
Activation Mechanism ◽  
Thrombin Receptor ◽  
Thrombin Cleavage ◽  
Thrombin Activation ◽  
Tethered Ligand

The recently cloned functional thrombin receptor is thought to be activated by thrombin cleavage of the bond between R41 and S42, followed by the insertion of the new N-terminal region (“tethered ligand”) into an unknown site in the receptor. Antibodies to peptides at or near the cleavage site have been reported to inhibit thrombin- induced platelet activation to varying extents, but the precise mechanism(s) of their inhibition is unknown. We have produced: (1) a polyclonal antibody in rabbits to a peptide containing amino acids 34 to 52 (anti-TR34–52); enzyme-linked immunosorbent assays (ELISA) indicate that anti-TR34–52 contains antibodies to regions on both sides of the thrombin cleavage site; (2) two murine monoclonal antibodies (MoAbs) to a peptide containing amino acids 29 to 68; one antibody reacts primarily with residues N-terminal to the thrombin cleavage site, and the other reacts primarily with residues C-terminal to the cleavage site; and (3) a polyclonal rabbit antibody to a peptide containing amino acids 83 to 94 (anti-TR83–94). Anti-TR34–52 binds to platelets as judged by flow cytometry, and pretreating platelets with a thrombin receptor peptide ligand does not lead to loss of antibody reactivity, suggesting that platelet activation does not initiate redistribution or internalization of surface thrombin receptors. In contrast, pretreating platelets with thrombin leads to complete loss of anti-TR34–52 binding. Similarly, the binding of both MoAbs to platelets is dramatically reduced by pretreatment with thrombin. However, the binding of anti-TR83–94 is not decreased by thrombin activation, confirming that the receptor is not internalized. Anti-TR34–52 profoundly inhibits low dose thrombin-induced platelet shape change and aggregation, but the inhibition can be overcome with higher thrombin doses. However, anti-TR34–52 does not inhibit platelet aggregation induced by tethered ligand peptides. The TR34–52 peptide is a thrombin substrate, with cleavage occurring at the R41-S42 bond as judged by high performance liquid chromatography (HPLC) and platelet aggregation analysis. Anti-TR34–52 prevented cleavage of the TR34–52 peptide, suggesting that the antibody prevents platelet activation, at least in part, by preventing cleavage of the thrombin receptor. These data, although indirect, provide additional support for a thrombin activation mechanism involving thrombin cleavage of the receptor; in addition, they provide new evidence indicating that receptor cleavage is followed by loss of the N-terminal peptide, and insertion of the tethered ligand into a protected domain.

scholarly journals Immunologic analysis of the cloned platelet thrombin receptor activation mechanism: evidence supporting receptor cleavage, release of the N-terminal peptide, and insertion of the tethered ligand into a protected environment

Blood ◽  
1993 ◽  
Vol 82 (7) ◽  
pp. 2125-2136 ◽  
Author(s):  
KJ Norton ◽  
RM Scarborough ◽  
JL Kutok ◽  
MA Escobedo ◽  
L Nannizzi ◽  
...  
Keyword(s):  
Amino Acids ◽  
Platelet Aggregation ◽  
Platelet Activation ◽  
Cleavage Site ◽  
Receptor Activation ◽  
Activation Mechanism ◽  
Thrombin Receptor ◽  
Thrombin Cleavage ◽  
Thrombin Activation ◽  
Tethered Ligand

Abstract The recently cloned functional thrombin receptor is thought to be activated by thrombin cleavage of the bond between R41 and S42, followed by the insertion of the new N-terminal region (“tethered ligand”) into an unknown site in the receptor. Antibodies to peptides at or near the cleavage site have been reported to inhibit thrombin- induced platelet activation to varying extents, but the precise mechanism(s) of their inhibition is unknown. We have produced: (1) a polyclonal antibody in rabbits to a peptide containing amino acids 34 to 52 (anti-TR34–52); enzyme-linked immunosorbent assays (ELISA) indicate that anti-TR34–52 contains antibodies to regions on both sides of the thrombin cleavage site; (2) two murine monoclonal antibodies (MoAbs) to a peptide containing amino acids 29 to 68; one antibody reacts primarily with residues N-terminal to the thrombin cleavage site, and the other reacts primarily with residues C-terminal to the cleavage site; and (3) a polyclonal rabbit antibody to a peptide containing amino acids 83 to 94 (anti-TR83–94). Anti-TR34–52 binds to platelets as judged by flow cytometry, and pretreating platelets with a thrombin receptor peptide ligand does not lead to loss of antibody reactivity, suggesting that platelet activation does not initiate redistribution or internalization of surface thrombin receptors. In contrast, pretreating platelets with thrombin leads to complete loss of anti-TR34–52 binding. Similarly, the binding of both MoAbs to platelets is dramatically reduced by pretreatment with thrombin. However, the binding of anti-TR83–94 is not decreased by thrombin activation, confirming that the receptor is not internalized. Anti-TR34–52 profoundly inhibits low dose thrombin-induced platelet shape change and aggregation, but the inhibition can be overcome with higher thrombin doses. However, anti-TR34–52 does not inhibit platelet aggregation induced by tethered ligand peptides. The TR34–52 peptide is a thrombin substrate, with cleavage occurring at the R41-S42 bond as judged by high performance liquid chromatography (HPLC) and platelet aggregation analysis. Anti-TR34–52 prevented cleavage of the TR34–52 peptide, suggesting that the antibody prevents platelet activation, at least in part, by preventing cleavage of the thrombin receptor. These data, although indirect, provide additional support for a thrombin activation mechanism involving thrombin cleavage of the receptor; in addition, they provide new evidence indicating that receptor cleavage is followed by loss of the N-terminal peptide, and insertion of the tethered ligand into a protected domain.

The Neutrophil Proteases, Elastase and Cathepsin G, May Modulate the Activity of Factor VIII.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1716-1716
Author(s):  
Andrew J. Gale ◽  
Diana Rozenshteyn ◽  
Justin Riceberg
Keyword(s):  
Factor Viii ◽  
Human Neutrophil ◽  
Cleavage Site ◽  
Maximum Level ◽  
Cathepsin G ◽  
Human Neutrophil Elastase ◽  
Factor V ◽  
Cleavage Sites ◽  
Thrombin Cleavage ◽  
A1 Domain

Abstract Neutrophils and monocytes express cathepsin G and elastase and also can bind to activated platelets, thus they can be localized to the site of active coagulation. Early studies suggested that cathepsin G and elastase inactivated factor VIII (FVIII) and were thus anticoagulant. But other studies have suggested procoagulant functions for cathepsin G and elastase in activation of factor V or activation of platelets among other possible mechanisms. Therefore, we investigated the effects of human neutrophil elastase and human neutrophil cathepsin G on FVIII/VIIIa. Elastase does inactivate both FVIII and FVIIIa but cathepsin G activates FVIII while having very little effect on FVIIIa. Cathepsin G activation of FVIII is enhanced by phospholipid vesicles, apparently due to enhanced rate of cleavage and stabilization of the resulting molecule. The maximum level of activation is less than that of thrombin, but it is still four-fold as measured in an APTT assay. Cleavage sites for both proteases in FVIII were identified by Edman degradation and gel analysis. FVIII cleavages are limited to a few specific sites that are mostly located near known activating and inactivating cleavage sites. A notable exception is a cleavage site for elastase after valine 26 in the A1 domain. Cathepsin G cleavage sites near to thrombin cleavage sites likely contribute to the partial activation of FVIII. The unique elastase cleavage site at valine 26 likely contributes to the inactivation of FVIII and FVIIIa. Therefore, it is possible that neutrophils and monocytes may provide some pro-coagulant effect by activating FVIII and may also provide negative feedback by inactivating FVIIIa as well.

scholarly journals A tick salivary protein targets cathepsin G and chymase and inhibits host inflammation and platelet aggregation

Blood ◽  
2011 ◽  
Vol 117 (2) ◽  
pp. 736-744 ◽  
Author(s):  
Jindrich Chmelar ◽  
Carlo J. Oliveira ◽  
Pavlina Rezacova ◽  
Ivo M. B. Francischetti ◽  
Zuzana Kovarova ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Serine Proteases ◽  
Acute Inflammation ◽  
Biological Activities ◽  
Salivary Protein ◽  
Cathepsin G ◽  
Skin Injury ◽  
Edema Formation ◽  
Protein Targets ◽  
First Time

Abstract Platelet aggregation and acute inflammation are key processes in vertebrate defense to a skin injury. Recent studies uncovered the mediation of 2 serine proteases, cathepsin G and chymase, in both mechanisms. Working with a mouse model of acute inflammation, we revealed that an exogenous salivary protein of Ixodes ricinus, the vector of Lyme disease pathogens in Europe, extensively inhibits edema formation and influx of neutrophils in the inflamed tissue. We named this tick salivary gland secreted effector as I ricinus serpin-2 (IRS-2), and we show that it primarily inhibits cathepsin G and chymase, while in higher molar excess, it affects thrombin activity as well. The inhibitory specificity was explained using the crystal structure, determined at a resolution of 1.8 Å. Moreover, we disclosed the ability of IRS-2 to inhibit cathepsin G-induced and thrombin-induced platelet aggregation. For the first time, an ectoparasite protein is shown to exhibit such pharmacological effects and target specificity. The stringent specificity and biological activities of IRS-2 combined with the knowledge of its structure can be the basis for the development of future pharmaceutical applications.

scholarly journals Molecular Basis for G-protein-Coupled Receptor (GPCR) Activation and Biased Signalling at the Platelet Thrombin Receptor Proteinase Activated Receptor-4 (PAR4)

2019 ◽  
Author(s):  
Pierre E. Thibeault ◽  
Jordan C. LeSarge ◽  
D’Arcy Arends ◽  
Michaela Fernandes ◽  
Peter Chidiac ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
G Protein ◽  
Molecular Basis ◽  
Calcium Signalling ◽  
Cathepsin G ◽  
Mitogen Activated Protein Kinase ◽  
Proteolytic Activation ◽  
Gpcr Activation ◽  
Tethered Ligand ◽  
G Protein Coupled

AbstractProteinase Activated Receptor-4 (PAR4) is a member of the proteolytically-activated PAR family of G-Protein-coupled Receptors (GPCRs). PARs are activated following proteolytic cleavage of the receptor N-terminus by enzymes such as thrombin, trypsin, and cathepsin-G to reveal the receptor-activating motif termed the tethered ligand. The tethered ligand binds intramolecularly to the receptor and triggers receptor signalling and cellular responses. In spite of this unusual mechanism of activation, PARs are fundamentally peptide receptors and can also be activated by exogenous application of short synthetic peptides derived from the tethered ligand sequence. In order to gain a better understanding of the molecular basis for PAR4-dependent signalling, we examined signalling responses to a library of peptides derived from the canonical PAR4 activating peptide (PAR4-AP), AYPGKF-NH2. We examined peptide residues involved in activation of the Gαq/11-coupled calcium signalling pathway, β-arrestin recruitment, and mitogen-activated protein kinase pathway activation. The peptide N-methyl-alanine-YPGKF-NH2 was identified as a compound that is a poor activator of PAR4-dependent calcium signalling but was fully competent in recruiting β-arrestin-1 and -2. In order to gain a better understanding of the ligand-binding pocket, we used in silico docking to identify key residues involved in PAR4 interaction with AYPGKF-NH2. The predicted interactions were verified by site-directed mutagenesis and analysis of calcium signalling and β-arrestin-1/-2 recruitment following proteolytic activation (with thrombin) or activation with the synthetic agonist peptide (AYPGKF-NH2). We determined that a key extracellular loop-2 aspartic acid residue (Asp230) is critical for signalling following both proteolytic and peptide activation of PAR4. Finally, we investigated platelet aggregation in response to AyPGKF-NH2 (a peptide with D-tyrosine in position two) which is unable to activate calcium signalling, and AYPGRF-NH2 a peptide that is equipotent to the parental peptide AYPGKF-NH2 for calcium signalling but is more potent at recruiting β-arrestins. We found that AyPGKF-NH2 fails to activate platelets while AYPGRF-NH2 causes a platelet aggregation response that is greater than that seen with the parental peptide and is comparable to that seen with thrombin stimulation. Overall, these studies uncover molecular determinants for agonist binding and signalling through a non-canonically activated GPCR and provide a template for development of small molecule modulators of PAR4.

scholarly journals Molecular basis for activation and biased signaling at the thrombin-activated GPCR proteinase activated receptor-4 (PAR4)

2019 ◽  
Vol 295 (8) ◽  
pp. 2520-2540 ◽  
Author(s):  
Pierre E. Thibeault ◽  
Jordan C. LeSarge ◽  
D'Arcy Arends ◽  
Michaela Fernandes ◽  
Peter Chidiac ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Calcium Signaling ◽  
Molecular Basis ◽  
Mapk Pathway ◽  
Cathepsin G ◽  
Site Directed Mutagenesis ◽  
Mitogen Activated Protein Kinase ◽  
Agonist Peptide ◽  
Biased Signaling ◽  
Tethered Ligand

Proteinase-activated receptor (PAR)-4 is a member of the proteolytically-activated PAR family of G-protein–coupled receptors (GPCR) that represents an important target in the development of anti-platelet therapeutics. PARs are activated by proteolytic cleavage of their receptor N terminus by enzymes such as thrombin, trypsin, and cathepsin-G. This reveals the receptor-activating motif, termed the tethered ligand that binds intramolecularly to the receptor and triggers signaling. However, PARs are also activated by exogenous application of synthetic peptides derived from the tethered-ligand sequence. To better understand the molecular basis for PAR4-dependent signaling, we examined PAR4-signaling responses to a peptide library derived from the canonical PAR4-agonist peptide, AYPGKF-NH2, and we monitored activation of the Gαq/11-coupled calcium-signaling pathway, β-arrestin recruitment, and mitogen-activated protein kinase (MAPK) pathway activation. We identified peptides that are poor activators of PAR4-dependent calcium signaling but were fully competent in recruiting β-arrestin-1 and -2. Peptides that were unable to stimulate PAR4-dependent calcium signaling could not trigger MAPK activation. Using in silico docking and site-directed mutagenesis, we identified Asp230 in the extracellular loop-2 as being critical for PAR4 activation by both agonist peptide and the tethered ligand. Probing the consequence of biased signaling on platelet activation, we found that a peptide that cannot activate calcium signaling fails to cause platelet aggregation, whereas a peptide that is able to stimulate calcium signaling and is more potent for β-arrestin recruitment triggered greater levels of platelet aggregation compared with the canonical PAR4 agonist peptide. These findings uncover molecular determinants critical for agonist binding and biased signaling through PAR4.

scholarly journals An internally quenched peptide as a new model substrate for rhomboid intramembrane proteases

2018 ◽  
Vol 399 (12) ◽  
pp. 1389-1397 ◽  
Author(s):  
Elena Arutyunova ◽  
Zhenze Jiang ◽  
Jian Yang ◽  
Ayodeji N. Kulepa ◽  
Howard S. Young ◽  
...  
Keyword(s):  
Serine Proteases ◽  
Catalytic Mechanism ◽  
Mass Spectrometry Analysis ◽  
Aqueous Environment ◽  
Growth Factor Signaling ◽  
Rhomboid Protease ◽  
Spectrometry Analysis ◽  
Intramembrane Proteases ◽  
Rhomboid Proteases ◽  
Lipid Environment

AbstractRhomboids are ubiquitous intramembrane serine proteases that cleave transmembrane substrates. Their functions include growth factor signaling, mitochondrial homeostasis, and parasite invasion. A recent study revealed that theEscherichia colirhomboid protease EcGlpG is essential for its extraintestinal pathogenic colonization within the gut. Crystal structures of EcGlpG and theHaemophilus influenzaerhomboid protease HiGlpG have deciphered an active site that is buried within the lipid bilayer but exposed to the aqueous environment via a cavity at the periplasmic face. A lack of physiological transmembrane substrates has hampered progression for understanding their catalytic mechanism and screening inhibitor libraries. To identify a soluble substrate for use in the study of rhomboid proteases, an array of internally quenched peptides were assayed with HiGlpG, EcGlpG and PsAarA fromProvidencia stuartti. One substrate was identified that was cleaved by all three rhomboid proteases, with HiGlpG having the highest cleavage efficiency. Mass spectrometry analysis determined that all enzymes hydrolyze this substrate between norvaline and tryptophan. Kinetic analysis in both detergent and bicellular systems demonstrated that this substrate can be cleaved in solution and in the lipid environment. The substrate was subsequently used to screen a panel of benzoxazin-4-one inhibitors to validate its use in inhibitor discovery.

scholarly journals A novel mechanism regulating human platelet activation by MMP-2–mediated PAR1 biased signaling

Blood ◽  
2017 ◽  
Vol 129 (7) ◽  
pp. 883-895 ◽  
Author(s):  
Manuela Sebastiano ◽  
Stefania Momi ◽  
Emanuela Falcinelli ◽  
Loredana Bury ◽  
Marc F. Hoylaerts ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Cleavage Site ◽  
Human Platelet ◽  
The Novel ◽  
Thrombin Cleavage ◽  
Pathway Activation ◽  
Key Points ◽  
Biased Signaling ◽  
Tethered Ligand

Key Points Active MMP-2 enhances platelet activation by cleaving PAR1 at an extracellular site different from the thrombin cleavage site. The novel PAR1-tethered ligand exposed by MMP-2 selectively stimulates PAR1-dependent Gq and G12/13 pathway activation.

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