Thrombin-induced events in non-platelet cells are mediated by the unique proteolytic mechanism established for the cloned platelet thrombin receptor.

Hung, David T.; Vu, T.-K. H.; Nelken, N. A.; Coughlin, Shaun R.

doi:10.1083/jcb.116.3.827

Cited by 152 publications

(84 citation statements)

References 22 publications

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“…PAR-1 and PAR-2 activation results in many of these biochemical events, indicating that they are likely participants in the balance of tumor containment and/or metastasis. [53][54][55][56][57][58] Moreover, the expression of tissue factor, an essential co-factor for plasma coagulation factor VII/VIIa, was reported to be consistently observed in stromal cells of invasive breast carcinomas but not in the benign breast tumors. 58 The increased presence of tissue factor/factor VIIa within the TME, which in turn can generate thrombin via the extrinsic coagulation pathway on fibroblasts, parallels our observation of increased PAR-1 expression.…”

Section: Discussionmentioning

confidence: 99%

Differential Expression of Protease-Activated Receptors-1 and -2 in Stromal Fibroblasts of Normal, Benign, and Malignant Human Tissues

D’Andrea¹,

Derian²,

Santulli³

et al. 2001

The American Journal of Pathology

131

102

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The serine proteases thrombin and trypsin are among many factors that malignant cells secrete into the extracellular space to mediate metastatic processes such as cellular invasion, extracellular matrix degradation, angiogenesis, and tissue remodeling. Malignant cells solicit the help of other cell types, such as stromal fibroblasts, mast cells, monocytes, and vascular cells, to facilitate their invasion into the surrounding tissue 1 because unrestrained growth of the tumor, by itself, does not result in invasion and metastasis. 2 The interface between the invading malignant cells and the hosting stromal cells, referred to as the tumor microenvironment (TME), 3 possesses a vast array of well-orchestrated cell signaling molecules which function to facilitate the ability of the proliferating tumor front to invade the stroma, as well as to degrade and remodel the extracellular matrix. 1 Of the many factors secreted by tumor cells, the two proteolytic enzymes, thrombin and trypsin, have been correlated to the stage and type of carcinoma and are associated with cell invasion and extracellular matrix degradation. 4,5 Furthermore, the ratio of proteases to their inhibitors in the TME can favor capillary sprout elongation and lumen formation during angiogenesis. 2 Thrombin is known to influence the behavior of all of the cells identified within the TME. For instance, thrombin activates platelets to adhere to other cells or extracellular matrix, increases vascular permeability and expression of adhesion molecules, attracts monocytes, stimulates mitogenic activity of endothelial cells and fibroblasts, and degranulates mast cells. 6 -9 Thrombin also influences the rate of deposition of connective tissue proteins and the development of tissue fibrosis during normal wound healing; a process similar to cellular metastasis. 10,11 Many of thrombin's effects are mediated through the seven transmembrane G-protein coupled receptors, protease-activated receptor (PAR)-1, via proteolytic cleavage of the amino-terminal extension unveiling a new amino terminus that activates the receptor through a tethered peptide ligand mechanism. 12 In vitro studies have demonstrated increased tumor cell adhesion to endothelium, extracellular matrix and platelets, enhanced metastatic capacity of tumor cells, and activated cell growth and stimulation of angiogenesis in response to thrombin and PAR-1 agonist peptides. 13-18 PAR-1 has been localized in smooth muscle cells, 19 pancreas tumor cells, 20 -21 carcinoma and melanoma cell lines 16 and recently in human mast cells. 22 In breast carcinoma cells, the level of PAR-1 expression has been correlated to the degree of invasiveness. 23 Furthermore, B16F10 melanoma cells, transfected with PAR-1, enhanced thrombin-treated tumor cell adhesion to fibronectin 2.5-fold in vitro and pulmonary metastasis as high as 39-fold in vivo compared to the control thrombin-treated tumor cells. 24 Trypsin can stimulate fibroblasts to secrete procollagen, stimulate mast cells to degranulate, and is secreted

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Section: Discussionmentioning

confidence: 99%

Differential Expression of Protease-Activated Receptors-1 and -2 in Stromal Fibroblasts of Normal, Benign, and Malignant Human Tissues

D’Andrea¹,

Derian²,

Santulli³

et al. 2001

The American Journal of Pathology

131

102

View full text Add to dashboard Cite

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“…The G protein-coupled thrombin receptor, protease-activated receptor-1 (PAR1), can mediate many of the actions of thrombin on cells including chemotaxis (12)(13)(14). Unlike most GPCRs, PAR1 is activated by an unusual proteolytic mechanism in which thrombin cleaves the amino-terminal exodomain of PAR1 to expose a new amino terminus that then serves as a tethered ligand (15,16).…”

Section: From the Cardiovascular Research Institute University Of Camentioning

confidence: 99%

The Carboxyl Tail of Protease-activated Receptor-1 Is Required for Chemotaxis

Sambrano

Coughlin

1999

Journal of Biological Chemistry

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The G protein-coupled thrombin receptor, proteaseactivated receptor 1 (PAR1), mediates many of the actions of thrombin on cells including chemotaxis. In contrast to the reversible agonist binding that regulates signaling by most G protein-coupled receptors (GPCRs), PAR1 is activated by an irreversible proteolytic mechanism. Although activated PAR1 is phosphorylated, uncoupled, and internalized like typical GPCRs, signal termination is additionally dependent on lysosomal degradation of cleaved and activated receptors. In the present study we exploit two PAR1 mutants to examine the link between chemotaxis and receptor shutoff. One, a carboxyl tail deletion mutant (Y397Z), is defective in phosphorylation and internalization. The other, a carboxyl tail chimeric receptor (P/S), is phosphorylated and internalized upon activation but recycles to the plasma membrane like reversibly activated GPCRs. Expression of these receptors in a hematopoietic cell line disrupted cell migration along thrombin gradients. Thrombin activation of cells expressing P/S or Y397Z resulted in persistent signaling independent of the continued presence of thrombin. Signaling in response to the soluble agonist peptide SFLLRN was reversible for P/S but persisted for Y397Z. Strikingly, cells expressing P/S responded chemokinetically to thrombin but chemotactically to SFLLRN. In contrast, Y397Z-mediated migration was largely chemokinetic to both agonists. These studies suggest that termination of PAR1 signaling at the level of the receptor is necessary for gradient detection and directional migration.

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“…Agonists were then added together with 20 mM LiCl in serum-free media and incubated at 37°C for 20 -120 min. Cells were extracted, and total [ 3 H]inositol phosphates were quantitated as described (11).…”

mentioning

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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Thrombin-induced events in non-platelet cells are mediated by the unique proteolytic mechanism established for the cloned platelet thrombin receptor.

Cited by 152 publications

References 22 publications

Differential Expression of Protease-Activated Receptors-1 and -2 in Stromal Fibroblasts of Normal, Benign, and Malignant Human Tissues

Differential Expression of Protease-Activated Receptors-1 and -2 in Stromal Fibroblasts of Normal, Benign, and Malignant Human Tissues

The Carboxyl Tail of Protease-activated Receptor-1 Is Required for Chemotaxis

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

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