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.

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.

The Contributions of the P4-P2 Positions in PAR1 and 4 for Thrombin Recognition and Cleavage.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3574-3574
Author(s):  
Marvin T. Nieman ◽  
Alvin H. Schmaier
Keyword(s):  
Amino Acids ◽  
Cleavage Site ◽  
Receptor Activation ◽  
Breakdown Product ◽  
Wild Type ◽  
Binding Region ◽  
Alanine Scanning Mutagenesis ◽  
Important Amino Acid ◽  
Thrombin Cleavage

Abstract The angiotensin converting enzyme breakdown product of bradykinin, bradykinin 1–5 (RPPGF), inhibits thrombin induced human or mouse platelet aggregation by preventing proteolysis of PAR1 and PAR4 by binding adjacent to the thrombin cleavage site and thus preventing activation of these receptors (Am. J. Physiol.285:H183–H193, 2003; J. Pharmacol. Exp. Ther.311:492–501, 2004; FEBS Lett. 579:25–29, 2005). Alanine scanning mutagenesis determined that RPPGF binds to Pro46 at the P2 position of PAR4 to block thrombin cleavage. New studies determined the amino acids required for thrombin to bind and cleave PAR4. Wild type PAR4 exodomain is cleaved by thrombin with a Km of 17 μM, kcat of 3.5 s−1 and kcat/Km of 2x105 M−1 s−1. In contrast, PAR4 exodomain in which the P2 (PAR4-P46A) is changed to alanine, is not efficiently cleaved with 10 nM alpha thrombin. Alteration of PAR4’s P4 position (e.g., PAR4-P44A) does not influence the PAR4’s rate of cleavage. The ability of the PAR4 exodomain to inhibit thrombin proteolysis of H-D-Phe-Pip-pNA was also determined as an independent measure of the thrombin/PAR4 interaction. Wild type PAR4 exodomain and PAR4-P44A are competitive inhibitors of thrombin hydrolysis of the chromogenic substrate with a Ki of 23 ± 6 micromolar and 19.6 ± 4 μM respectively. In contrast, PAR4-P46A and PAR4-P44A/P46A have a Ki > 300 μM nd the nature of the inhibition changes from competitive to noncompetitive. Taken together, these data demonstrate that Pro46 of PAR4 is important for alpha thrombin to bind and orient PAR4 in its active site for efficient cleavage. Further studies examined the role of the combined amino acids in the P4 to P3 positions of PAR4 and PAR1 to contribute to the rate of thrombin cleavage. Chimeric molecules were prepared in which the P4 and P3 positions of PAR4 (ProAla) are replaced with those from PAR1 (LeuAsp) to generate PAR4-LD. The reciprocal chimera was also made (PAR1-PA). PAR4-LD is proteolyzed by 10 nM alpha thrombin more efficiently than PAR4-wt. Alternatively, the rate of PAR1-PA proteolysis by alpha thrombin is less efficient than PAR1-wt but better than PAR4-wt due to the presence of the exosite I binding region in the PAR1 exodomain. However, if the exosite I binding domain is removed from PAR1-PA the rate of cleavage is like that seen with PAR4-wt exodomain. These data indicate PAR1 is cleaved by alpha thrombin at a faster rate than PAR4 due to the amino acids in the P4 and P3 positions as well as the exosite I binding region. In sum, these data demonstrate that the P2 position of PAR4 is the most important amino acid in thrombin binding to the region adjacent to the thrombin cleavage site. However the combined P4 and P3 positions are also important determinants of the rate of receptor cleavage. These data indicate that the amino acids around the thrombin cleavage site of both PAR4 and 1 influence its rate of cleavage. Designing thrombin receptor activation antagonists directed to the thrombin cleavage site on PAR1 and 4 should be effective anti-platelet agents.

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.

scholarly journals Dual coupling of the α-thrombin receptor to signal-transduction pathways involving phosphatidylinositol and phosphatidylcholine metabolism

1998 ◽  
Vol 337 (1) ◽  
pp. 97-104 ◽  
Author(s):  
Jie CHENG ◽  
Joseph J. BALDASSARE ◽  
Daniel M. RABEN
Keyword(s):  
Signal Transduction ◽  
Cleavage Site ◽  
Thrombin Receptor ◽  
Signal Transduction Pathways ◽  
Metabolizing Enzymes ◽  
Thrombin Cleavage ◽  
Messenger Molecules ◽  
Mitogenic Signalling ◽  
Signalling Cascade ◽  
Phosphatidylcholine Metabolism

Addition of α-thrombin to quiescent IIC9 cells results in the activation of lipid-metabolizing enzymes associated with signal-transduction cascades. These enzymes include phosphatidylinositol (PI)-specific phospholipase C (PI-PLC), phosphatidylcholine (PC)-specific phospholipases C and D and phospholipase A2 (PLA2). Whereas the α-thrombin receptor has been shown to couple with PI-PLCs, it is not clear whether this receptor, or a putative second receptor, couples to one or more of the other phospholipases. In this report we determine whether the cloned receptor couples to all or a subset of these enzymes. We show that (i) an α-thrombin-receptor-activating peptide also elicits the above responses and (ii) addition of enterokinase to IIC9 cells, stably transfected with an α-thrombin receptor (enterokinase- responsive α-thrombin receptor, EKTR) containing an enterokinase cleavage site in place of an α-thrombin cleavage site, stimulates both PI and PC hydrolysis, including PLA2. Enterokinase also induces mitogenesis in the IIC9s transfected with EKTR. These results indicate that, in addition to initiating a mitogenic signalling cascade, the cloned α-thrombin receptor couples to enzymes involved in generating PC-derived, as well as PI-derived, second-messenger molecules in IIC9s. Additionally, using the cells transfected with EKTR, we further demonstrate that only activated, i.e. cleaved, receptors are desensitized.

Aprotinin: is it prothrombotic?

Perfusion ◽  
2001 ◽  
Vol 16 (5) ◽  
pp. 401-409 ◽  
Author(s):  
M Poullis ◽  
R C Landis ◽  
K M Taylor
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Mechanism Of Action ◽  
Adenosine Diphosphate ◽  
Platelet Membrane ◽  
Calcium Flux ◽  
Thrombin Receptor ◽  
Proteolytic Activation ◽  
Agonist Peptide ◽  
Receptor Family

Controversy continues as to whether aprotinin (Trasylol) is prothrombotic. The recent discovery of the thrombin receptor family, known as the protease-activated receptor family (PAR) has been essential in aiding our understanding of the mechanism of action of aprotinin. Our results show that aprotinin has no effect on platelet aggregation induced by adrenaline, adenosine diphosphate, phorbol-12-myristate-13-acetate, collagen or PAR 1 agonist peptide. However, aprotinin inhibits thrombin-induced platelet activation as assessed by macroaggregation, microaggregation and platelet membrane calcium flux. Aprotinin inhibits proteolytic activation of platelets, but platelets can still be activated by non-proteolytic mechanisms.

scholarly journals Role of GRK6 in the Regulation of Platelet Activation through Selective G Protein-Coupled Receptor (GPCR) Desensitization

2020 ◽  
Vol 21 (11) ◽  
pp. 3932 ◽  
Author(s):  
Preeti Kumari Chaudhary ◽  
Sanggu Kim ◽  
Youngheun Jee ◽  
Seung-Hun Lee ◽  
Kyung-Mee Park ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
G Protein ◽  
Platelet Activation ◽  
Functional Role ◽  
Thrombus Formation ◽  
Receptor Activation ◽  
G Protein Coupled ◽  
Gpcr Desensitization ◽  
Stimulation Of

Platelet G protein-coupled receptors (GPCRs) regulate platelet function by mediating the response to various agonists, including adenosine diphosphate (ADP), thromboxane A2, and thrombin. Although GPCR kinases (GRKs) are considered to have the crucial roles in most GPCR functions, little is known regarding the regulation of GPCR signaling and mechanisms of GPCR desensitization by GRKs in platelets. In this study, we investigated the functional role of GRK6 and the molecular basis for regulation of specific GPCR desensitization by GRK6 in platelets. We used GRK6 knockout mice to evaluate the functional role of GRK6 in platelet activation. Platelet aggregation, dense- and α-granule secretion, and fibrinogen receptor activation induced by 2-MeSADP, U46619, thrombin, and AYPGKF were significantly potentiated in GRK6−/− platelets compared to the wild-type (WT) platelets. However, collagen-related peptide (CRP)-induced platelet aggregation and secretion were not affected in GRK6−/− platelets. Interestingly, platelet aggregation induced by co-stimulation of serotonin and epinephrine which activate Gq-coupled 5HT2A and Gz-coupled α2A adrenergic receptors, respectively, was not affected in GRK6−/− platelets, suggesting that GRK6 was involved in specific GPCR regulation. In addition, platelet aggregation in response to the second challenge of ADP and AYPGKF was restored in GRK6−/− platelets whereas re-stimulation of the agonist failed to induce aggregation in WT platelets, indicating that GRK6 contributed to P2Y1, P2Y12, and PAR4 receptor desensitization. Furthermore, 2-MeSADP-induced Akt phosphorylation and AYPGKF-induced Akt, extracellular signal-related kinase (ERK), and protein kinase Cδ (PKCδ) phosphorylation were significantly potentiated in GRK6−/− platelets. Finally, GRK6−/− mice exhibited an enhanced and stable thrombus formation after FeCl3 injury to the carotid artery and shorter tail bleeding times, indicating that GRK6−/− mice were more susceptible to thrombosis and hemostasis. We conclude that GRK6 plays an important role in regulating platelet functional responses and thrombus formation through selective GPCR desensitization.

scholarly journals Synthetic alpha-thrombin receptor peptides activate G protein-coupled signaling pathways but are unable to induce mitogenesis.

1992 ◽  
Vol 3 (1) ◽  
pp. 95-102 ◽  
Author(s):  
V Vouret-Craviari ◽  
E Van Obberghen-Schilling ◽  
U B Rasmussen ◽  
A Pavirani ◽  
J P Lecocq ◽  
...  
Keyword(s):  
Adenylate Cyclase ◽  
Phospholipase C ◽  
G Protein ◽  
Synthetic Peptides ◽  
Cleavage Site ◽  
Amino Acid Sequences ◽  
Thrombin Receptor ◽  
Thrombin Cleavage ◽  
Ccl39 Cells ◽  
G Protein Coupled

alpha-Thrombin (thrombin) stimulates phospholipase C and modulates the activity of adenylate cyclase in a number of cell types via G protein-coupled receptors. It is also a potent growth factor, notably for a line of hamster fibroblasts (CCL39 cells). Recently, predicted amino acid sequences for human and hamster thrombin receptors have been reported that display a putative thrombin cleavage site in the N-terminal extracellular domain. Synthetic peptides corresponding to 14 residues carboxyl to the presumed thrombin cleavage site of the human receptor have been shown to activate platelets as well as the thrombin receptor expressed in Xenopus oocytes. In the present study we have examined the effects of synthetic peptides corresponding to the same region of the hamster receptor (S-42-L-55) and shorter peptides (2-7 residues) on signal transducing systems in CCL39 cells. Our results indicate that hamster receptor peptides of greater than or equal to 5 residues effectively stimulate phospholipase C in CCL39 cells via the thrombin receptor and induce rapid desensitization of the response. The same peptides also inhibit adenylate cyclase in a pertussis toxin-sensitive manner. Although the peptides are potent agonists of serotonin release in platelets, unlike thrombin, by themselves they are not mitogenic. However, they potentiate DNA synthesis in cooperation with growth factors possessing tyrosine kinase receptors. Hence, we conclude that the potent mitogenic action of thrombin cannot be accounted for solely by the activation of the cloned receptor. We postulate the existence of an additional receptor activated by thrombin, which is required for its full mitogenic potential.

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