Of the four known protease-activated receptors (PARs), PAR1 and PAR4 are expressed by human platelets and mediate thrombin signaling. Whether these receptors are redundant, interact, or play at least partially distinct roles is unknown. It is possible that PAR1 and/or PAR4 might confer responsiveness to proteases other than thrombin. The neutrophil granule protease, cathepsin G, is known to cause platelet secretion and aggregation. We now report that this action of cathepsin G is mediated by PAR4. Cathepsin G triggered calcium mobilization in PAR4-transfected fibroblasts, PAR4-expressing Xenopus oocytes, and washed human platelets. An antibody raised against the PAR4 thrombin cleavage site blocked platelet activation by cathepsin G but not other agonists. Desensitization with a PAR4 activating peptide had a similar effect. By contrast, inhibition of PAR1 function had no effect on platelet responses to cathepsin G. When neutrophils were present, the neutrophil agonist fMet-Leu-Phe triggered calcium signaling in Fura-2-loaded platelets. Strikingly, this neutrophildependent platelet activation was blocked by the PAR4 antibody. These data show that PAR4 mediates platelet responses to cathepsin G and support the hypothesis that cathepsin G might mediate neutrophil-platelet interactions at sites of vascular injury or inflammation.Proteases regulate cellular behaviors in part via a family of G protein-coupled protease-activated receptors (PARs) 1 for which the thrombin receptor, PAR1, is the prototype. PAR1 is activated by an unusual mechanism in which thrombin cleaves the receptor amino-terminal exodomain to expose a new amino terminus that then serves as a tethered ligand (1, 2). The synthetic peptide SFLLRN, which mimics the first six amino acids of this newly unmasked amino terminus, can activate PAR1 independent of receptor cleavage and thrombin. The cognate P1Ј-P6Ј peptides of other PARs have been useful as agonists for probing the role of these receptors in various cellular responses (3-5).Four distinct PARs are now known. PAR1, PAR3, and the recently characterized PAR4 can be activated by thrombin (1, 5-7). PAR2 is activated by trypsin and trypsin-like enzymes (4). The importance of platelet activation in myocardial infarction and other thrombotic diseases has prompted considerable focus on identifying the PARs that mediate platelet activation by thrombin. Available data suggest that thrombin signaling in human platelets is mediated largely by hPAR1 and hPAR4 (8). PAR1 is necessary for platelet activation at low concentrations of thrombin, and PAR4 can mediate signaling at higher concentrations. The presence of two receptors raises the question of whether PAR1 and PAR4 are redundant, interact, or serve at least partially distinct functions in human platelets. In particular, it is possible that PAR4 might confer responsiveness to proteases other than thrombin.Platelet PARs may be exposed to a variety of proteases at sites of inflammation or coagulation, and leukocytes are one potential source. For example, neutr...

Protease-activated receptors 1 and 4 (PAR1 and PAR4) mediate thrombin signaling in human platelets. Whether these receptors are redundant, interact, or serve only partially overlapping functions is unknown. We report that PAR1 and PAR4 signal with distinct tempos. In transfected fibroblasts, PAR4 triggered substantially more phosphoinositide hydrolysis per activated receptor than PAR1 and was shut off more slowly than PAR1. Shutoff and internalization of PAR1 depends upon phosphorylation of its carboxyl tail upon receptor activation. In contrast to PAR1, phosphorylation of PAR4 was undetectable, and activation-dependent internalization of PAR4 was much slower than that seen for PAR1. Mutation of potential phosphorylation sites in the carboxyl tail of PAR1 enhanced PAR1 signaling, whereas analogous mutations in PAR4 had no effect. Thus PAR4 signaling is shut off less rapidly than PAR1, probably due to differences in receptor phosphorylation. PAR1 and PAR4 also signaled with distinct tempos in platelets. PAR1 triggered a rapid and transient increase in intracellular calcium, whereas PAR4 triggered a more prolonged response. Together, the tempo of these responses accounted for that triggered by thrombin. Thus differences in the rates at which PAR1 and PAR4 are shut off allow thrombin to trigger intracellular signaling with distinct temporal characteristics.Thrombin, a serine protease generated at sites of vascular injury, is the most potent activator of platelets (1, 2). This action of thrombin is thought to be critical for hemostasis and thrombosis. Cellular responses to thrombin are mediated by a family of G protein-coupled protease-activated receptors (PARs) 1 (3). Cleavage of the amino-terminal exodomain of a PAR unmasks a tethered ligand that binds to the body of the receptor to trigger signaling. Synthetic peptides that mimic these tethered ligands are agonists for their respective PARs and have been used as pharmacological probes of PAR function in various cell types.Three thrombin-activated receptors, PAR1, PAR3, and PAR4, have been identified (4 -8). PAR1 and PAR4 appear to account for most if not all thrombin signaling in human platelets (9). Both receptors are expressed by platelets, and activation of either PAR1 or PAR4 with their cognate agonist peptides was sufficient to trigger platelet ATP secretion and aggregation. PAR1-blocking antibodies inhibited platelet secretion and aggregation at low but not high concentrations of thrombin. PAR4-blocking antibodies alone had little effect on platelet activation, but inhibition of both PAR1 and PAR4 with blocking antibodies markedly attenuated platelet activation even at high concentrations of thrombin. In heterologous expression systems, higher concentrations of thrombin were required for cleavage of PAR4 than PAR1. Taken together, these observations suggest that PAR1 is the primary mediator of platelet activation at low concentrations of thrombin and that, in the absence of PAR1 function, PAR4 can mediate platelet activation at high concentrations of thromb...

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...

The activity of the small GTPase, Rac1, plays a role in various cellular processes including cytoskeletal rearrangement, gene transcription, and malignant transformation. In this report constitutively active Rac1 (Rac V12) is shown to stimulate the activation of STAT3, a member of the family of signal transducers and activators of transcription (STATs). The activity of Rac1 leads to STAT3 translocation to the nucleus coincident with STAT3-dependent gene expression. The expression of Vav (⌬1-187), a constitutively active guanine nucleotide exchange factor for the Rho GTPases, or activated forms of Ras or Rho family members, leads to STAT3-specific activation. The activation of STAT3 requires tyrosine phosphorylation at residue 705, but is not dependent on phosphorylation of Ser-727. Our studies indicate that Rac1 induces STAT3 activation through an indirect mechanism that involves the autocrine production and action of IL-6, a known mediator of STAT3 response. Rac V12 expression results in the induction of the IL-6 and IL-6 receptor genes and neutralizing antibodies directed against the IL-6 receptor block Rac1-induced STAT3 activation. Furthermore, inhibition of the nuclear factor-B activation or disruption of IL-6-mediated signaling through the expression of I B␣ S32AS36A and suppressor of cytokine signaling 3 , respectively, blocks Rac1-induced STAT3 activation. These findings elucidate a mechanism dependent on the induction of an autocrine IL-6 activation loop through which Rac1 mediates STAT3 activation establishing a link between oncogenic GTPase activity and Janus kinase͞STAT signaling.