Inhibition of thrombin and SFLLR-peptide stimulation of platelet aggregation, phospholipase A2 and Na+/H+ exchange by a thrombin receptor antagonist

Seiler, Steven M.; Peluso, Marianne; Michel, Inge M.; Goldenberg, Harold; Fenton, John W.; Riexinger, Douglas; Natarajan, Sesha

doi:10.1016/0006-2952(94)00473-y

Cited by 44 publications

(32 citation statements)

References 40 publications

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“…on the inflammatory signaling pathways: IFN-␥ induces expression of SOCS family proteins as well as expression of inflammatory mediators (Sakamoto et al, 1998;Yasukawa et al, 2000). Thrombin has been reported to induce expression of inflammatory mediators in several types of cells, including astrocytes, microglia, and platelets (Seiler et al, 1995;Ryu et al, 2000;Meli et al, 2001). The results of the present study indicate that thrombin induces the expression of CIS protein, a member of SOCS family, in rat brain astrocytes ( Figs.…”

Section: Discussionsupporting

confidence: 60%

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Thrombin induces expression of cytokine‐induced SH2 protein (CIS) in rat brain astrocytes: Involvement of phospholipase A₂, cyclooxygenase, and lipoxygenase

Yang

Jou

et al. 2004

Glia

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Previously we have reported that thrombin induces inflammatory mediators in brain glial cells (Ryu et al. 2000. J Biol Chem 275:29955). In the present study, we found that thrombin induced a negative regulator of a cytokine signaling molecule, cytokineinduced SH2 protein (CIS), in rat brain astrocytes. In response to thrombin, CIS expression was increased at both the mRNA and protein levels. Although STAT5 is known to regulate CIS expression, thrombin did not activate STAT5, and inhibitors of JAK2 (AG490) and JAK3 (WHI-P97 and WHI-P154) had little effect on thrombin-induced CIS expression. In contrast, cytosolic phospholipase A 2 (cPLA 2 ), cyclooxygenase (COX), and lipoxygenase (LO) play a role in CIS expression, since inhibitors of cPLA 2 , cyclooxygenase (COX), and LO significantly reduced CIS expression. Reactive oxygen species (ROS) scavengers (N-acetylcysteine [NAC] and trolox) reduced thrombin-induced CIS expression, and inhibitors of COX and LO reduced ROS produced by thrombin. Furthermore, prostaglandin E 2 (PGE 2 ) and leukotriene B 4 (LTB 4 ), products of COX and LO, respectively, potentiated thrombin-induced CIS expression, indicating that ROS, and PGE 2 and LTB 4 generated by COX and LO, mediate CIS expression. Since interferon-␥ (IFN-␥)-induced GAS-luciferase activity and tyrosine phosphorylation of STAT1 and STAT3 were lower in CIS-transfected cells compared to control vector-transfected cells, CIS could have anti-inflammatory activity. These data suggest that thrombin-stimulation of ROS and prostaglandin and leukotriene production via the cPLA 2 , COX and LO pathways results in CIS expression. More importantly, CIS expression may be a negative feedback mechanism that prevents prolonged inflammatory responses.

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Section: Discussionsupporting

confidence: 60%

“…The intracellular signaling mechanisms activated by thrombin are diverse and include cPLA 2 (Seiler et al, 1995). Thus, we next investigated the involvement of PLA 2 in thrombin-induced CIS expression.…”

Section: Thrombin Induces Cis Expression Via a Statand Mapk-independementioning

confidence: 99%

Thrombin induces expression of cytokine‐induced SH2 protein (CIS) in rat brain astrocytes: Involvement of phospholipase A₂, cyclooxygenase, and lipoxygenase

Yang

Jou

et al. 2004

Glia

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“…These data suggest that small aliphatic side chains are preferred at position 1. Substitution of a mercaptoproprionic acid for serine in SFLLRN nearly abolished agonist activity for PAR1 (15,16). The cognate substitution of mercaptoproprionic acid for glycine in GYPGKF also resulted in loss of agonist function.…”

Section: Resultsmentioning

confidence: 99%

Structure-Function Analysis of Protease-activated Receptor 4 Tethered Ligand Peptides

Faruqi¹,

Weiss²,

Shapiro³

et al. 2000

Journal of Biological Chemistry

186

130

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Thrombin activates protease-activated receptors (PARs) by specific cleavage of their amino-terminal exodomains to unmask a tethered ligand that binds intramolecularly to the body of the receptor to effect transmembrane signaling. Peptides that mimic such ligands are valuable as agonists for probing PAR function, but the tethered ligand peptide for PAR4, GYPGKF, lacks potency and is of limited utility. In a structureactivity analysis of PAR4 peptides, AYPGKF was ϳ10-fold more potent than GYPGKF and, unlike GYPGKF, elicited PAR4-mediated responses comparable in magnitude to those elicited by thrombin. AYPGKF was relatively specific for PAR4 in part due to the tyrosine at position 2; substitution of phenylalanine or p-fluorophenylalanine at this position produced peptides that activated both PAR1 and PAR4. Because human platelets express both PAR1 and PAR4, it might be desirable to inhibit both receptors. Identifying a single agonist for both receptors raises the possibility that a single antagonist for both receptors might be developed. The AY-PGKF peptide is a useful new tool for probing PAR4 function. For example, AYPGKF activated and desensitized PAR4 in platelets and, like thrombin, triggered phosphoinositide hydrolysis but not inhibition of adenylyl cyclase in PAR4-expressing cells. The latter shows that, unlike PAR1, PAR4 couples to G q and not G i .The coagulation protease thrombin elicits biological responses at least in part via G protein-coupled protease-activated receptors (PARs) 1 (1-5). PAR1, the prototype for this family, is activated when thrombin cleaves its amino-terminal exodomain at the R41/S42 peptide bond (1). This serves to unmask a new amino terminus beginning with the sequence SFLLRN, which serves as a tethered peptide agonist, binding intramolecularly to the body of the receptor to effect transmembrane signaling. PARs are thus in essence peptide receptors that carry their own ligands, which remain silent until unmasked by site-specific receptor cleavage. Synthetic peptides that mimic these tethered ligands function as PAR agonists and activate their receptors independent of protease and receptor cleavage (1, 4 -6). Such peptides have been extremely useful for probing the roles of PARs in cells and tissues.PAR4 is a recently characterized thrombin receptor that is expressed in human platelets along with PAR1 (4, 5, 7). Human PAR4 is activated when thrombin cleaves its amino-terminal exodomain at the Arg-47/Gly-48 peptide bond to unmask the tethered ligand GYPGQV (4, 5). The synthetic peptide GYPGQV does function as an agonist for PAR4, but a concentration of 200 -500 M is required for activity. This lack of potency severely limits the utility of this peptide for probing PAR4 function in culture systems and virtually precludes its use in vivo. Structure-function relationships for the PAR4 tethered ligand have not been explored. We here report such a study. In addition to identifying requirements for agonist activity and specificity, this study provides a new PAR4 specific agonist peptide, AY...

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“…Therefore, the development of potent, smallmolecule PAR-1 antagonists would be worthwhile to establish the potential therapeutic utility of blocking thrombin's action on cells, as opposed to blocking thrombin's actions on hemostatic proteins. Among the few examples of PAR-1 antagonists reported (20,21,(34)(35)(36)(37)(38), the molecules of interest are deficient as drug candidates because of two or more of the following attributes: weak potency; peptide constitution; failure to block the action of thrombin on platelets in a consistent manner, although reasonably blocking the action of agonist peptides; mixed agonist͞antagonist activity; and lack of good receptor͞ functional selectivity. The report of Bernatowicz et al (35) stands out by providing peptide compounds with potent antagonist activity in platelet aggregation induced by a PAR-1 agonist peptide (SFLLRNP-NH 2 ) and with high PAR-1 affinity.…”

Section: Discussionmentioning

confidence: 99%

Design, synthesis, and biological characterization of a peptide-mimetic antagonist for a tethered-ligand receptor

Andrade‐Gordon¹,

Maryanoff²,

Derian³

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

179

127

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Protease-activated receptors (PARs) represent a unique family of seven-transmembrane G protein-coupled receptors, which are enzymatically cleaved to expose a truncated extracellular N terminus that acts as a tethered activating ligand. PAR-1 is cleaved and activated by the serine protease ␣-thrombin, is expressed in various tissues (e.g., platelets and vascular cells), and is involved in cellular responses associated with hemostasis, proliferation, and tissue injury. We have discovered a series of potent peptide-mimetic antagonists of PAR-1, exemplified by RWJ-56110. Spatial relationships between important functional groups of the PAR-1 agonist peptide epitope SFLLRN were employed to design and synthesize candidate ligands with appropriate groups attached to a rigid molecular scaffold. Prototype RWJ-53052 was identified and optimized via solid-phase parallel synthesis of chemical libraries. RWJ-56110 emerged as a potent, selective PAR-1 antagonist, devoid of PAR-1 agonist and thrombin inhibitory activity. It binds to PAR-1, interferes with PAR-1 calcium mobilization and cellular function (platelet aggregation; cell proliferation), and has no effect on PAR-2, PAR-3, or PAR-4. By flow cytometry, RWJ-56110 was confirmed as a direct inhibitor of PAR-1 activation and internalization, without affecting N-terminal cleavage. At high concentrations of ␣-thrombin, RWJ-56110 fully blocked activation responses in human vascular cells, albeit not in human platelets; whereas, at high concentrations of SFLLRN-NH 2, RWJ-56110 blocked activation responses in both cell types. Thus, thrombin activates human platelets independently of PAR-1, i.e., through PAR-4, which we confirmed by PCR analysis. Selective PAR-1 antagonists, such as RWJ-56110, should serve as useful tools to study PARs and may have therapeutic potential for treating thrombosis and restenosis.

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Inhibition of thrombin and SFLLR-peptide stimulation of platelet aggregation, phospholipase A2 and Na+/H+ exchange by a thrombin receptor antagonist

Cited by 44 publications

References 40 publications

Thrombin induces expression of cytokine‐induced SH2 protein (CIS) in rat brain astrocytes: Involvement of phospholipase A₂, cyclooxygenase, and lipoxygenase

Thrombin induces expression of cytokine‐induced SH2 protein (CIS) in rat brain astrocytes: Involvement of phospholipase A₂, cyclooxygenase, and lipoxygenase

Structure-Function Analysis of Protease-activated Receptor 4 Tethered Ligand Peptides

Design, synthesis, and biological characterization of a peptide-mimetic antagonist for a tethered-ligand receptor

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Inhibition of thrombin and SFLLR-peptide stimulation of platelet aggregation, phospholipase A2 and Na+/H+ exchange by a thrombin receptor antagonist

Cited by 44 publications

References 40 publications

Thrombin induces expression of cytokine‐induced SH2 protein (CIS) in rat brain astrocytes: Involvement of phospholipase A2, cyclooxygenase, and lipoxygenase

Thrombin induces expression of cytokine‐induced SH2 protein (CIS) in rat brain astrocytes: Involvement of phospholipase A2, cyclooxygenase, and lipoxygenase

Structure-Function Analysis of Protease-activated Receptor 4 Tethered Ligand Peptides

Design, synthesis, and biological characterization of a peptide-mimetic antagonist for a tethered-ligand receptor

Contact Info

Product

Resources

About

Thrombin induces expression of cytokine‐induced SH2 protein (CIS) in rat brain astrocytes: Involvement of phospholipase A₂, cyclooxygenase, and lipoxygenase

Thrombin induces expression of cytokine‐induced SH2 protein (CIS) in rat brain astrocytes: Involvement of phospholipase A₂, cyclooxygenase, and lipoxygenase