Characterization of protease-activated receptor-2 immunoreactivity in normal human tissues.

PAR-2 is a second member of a novel family of G-protein-coupled receptors characterized by a proteolytic cleavage of the amino terminus, thus exposing a tethered peptide ligand that autoactivates the receptor. The physiological and/or pathological role(s) of PAR-2 are still unknown. This study provides tissue-specific cellular localization of PAR-2 in normal human tissues by immunohistochemical techniques. A polyclonal antibody, PAR-2C, was raised against a peptide corresponding to the amino terminal sequence SLIGKVDGTSHVTGKGV of human PAR-2. Significant PAR-2 immunoreactivity was detected in smooth muscle of vascular and nonvascular origin and stromal cells from a variety of tissues. PAR-2 was also present in endothelial and epithelial cells independent of tissue type. Strong immunolabeling was observed throughout the gastrointestinal tract, indicating a possible function for PAR-2 in this system. In the CNS, PAR-2 was localized to many astrocytes and neurons, suggesting involvement of PAR-2 in neuronal function. A role for PAR-2 in the skin was further supported by its immunolocalization in the epidermis. PAR-2C antibody exemplifies an important tool to address the physiological role(s) of PAR-2.

Biological consequences of thrombin receptor deficiency in mice.

The thrombin receptor (ThrR) is a membrane-bound, G-protein-coupled receptor for the serine protease thrombin. This receptor is expressed in a wide variety of cells and tissues, and elicits a range of physiological responses associated with tissue injury, inflammation, and wound repair. To achieve a better understanding of the physiological role of the ThrR, we have employed homologous recombination to create mice with a disrupted ThrR gene. Following heterozygous (+/-) intercrosses, a total of 351 surviving offspring were genotyped. Only 7% of these offspring were identified as homozygous (-/-) for the disrupted allele, indicating a profound effect on embryonic development. Paradoxically, adult ThrR-/- mice appeared to be normal by anatomical and histological analysis, including their platelet number and function. Similarly, ThrR deficiency had no detectable effect in adult ThrR-/- mice on basal heart rate, arterial blood pressure, vasomotor responses to angiotensin II and acetycholine, and coagulation parameters, even though the ThrR is expressed in many cardiovascular tissue types. In addition, the loss of ThrR function in the peripheral vasculature of adult ThrR-/- mice was confirmed by the absence of various standard hemodynamic effects of the ThrR-activating peptides SFLLRN-NH2 and TFLLRNPNDK-NH2. Our results indicate that ThrR deficiency has a strong impact on fetal development; however. ThrR-/- mice that proceed to full development display surprisingly little change in phenotype compared to the wild-type.

Activation of 15-lipoxygenase by low density lipoprotein in vascular endothelial cells. Relationship to the oxidative modification of low density lipoprotein.

Oxidatively-modified low density lipoprotein (LDL) is thought to play a significant role in the formation of lipid-laden macrophages, the primary cellular component of atherosclerotic fatty lesions. Recently, lipoxygenases have been implicated as a major enzymatic pathway involved in rabbit endothelial cell-mediated LDL modification. We investigated the effect of LDL on porcine aortic endothelial cell (PAEC) and human umbilical vein (HUVEC) and aortic endothelial cell (HAEC) lipoxygenase activity. By thin layer chromatography, we observed that human LDL stimulated the metabolism of radiolabeled arachidonic acid to 12 + 15-hydroxyeicosatetraenoic acid (HETE) in indomethacin-treated PAEC. Furthermore, radiolabeled linoleic acid, a specific substrate for the 15-lipoxygenase, was metabolized to its respective product 13-hydroxyoctadecadienoic acid (13-HODE) in the presence of LDL. Increased product formation in both studies was inhibited by the lipoxygenase blockers nordihydroguaiaretic acid (NDGA) and RG 6866. 15-HETE was confirmed as the predominant HETE product in LDL-treated cells by high performance liquid chromatography. Both porcine- and human-derived LDL stimulated the CL release of 15-HETE from cells as determined by radioimmunoassay. Release of immunoreactive 15-HETE was inhibited by NDGA, RG 6866, and 5,8,11,14-eicosatetraynoic acid (ETYA) but not by the selective 5-lipoxygenase inhibitor RG 5901. These lipoxygenase inhibitors had similar effects on the modification of LDL. Our results suggest that the oxidative modification of LDL by endothelial cells may be mediated in part through activation of 15-lipoxygenase.

Effect of ticlopidine ex vivo on platelet intracellular calcium mobilization.

The antiplatelet compound ticlopidine exerts its potent inhibitory activity through an as yet undetermined mechanism(s). The goal of this study was to determine the effect, if any, of ticlopidine ex vivo on platelet calcium mobilization. Ticlopidine inhibited ADP-induced platelet aggregation by 50-80%. In the presence of 1 mM EGTA, ticlopidine inhibited ADP- and thrombin-stimulated increases in [Ca2+]i in fura-2 loaded platelets. We evaluated further the effect of ticlopidine on calcium mobilization by examining both agonist-stimulated formation of inositol trisphosphate in intact platelets and the ability of inositol trisphosphate to release 45Ca from intracellular sites in permeabilized cells. We show here that while ticlopidine significantly affected agonist-induced intracellular calcium mobilization in intact platelets, the drug was without effect on agonist-stimulated formation of inositol trisphosphate in intact platelets and on inositol trisphosphate-induced 45Ca release in saponin-permeabilized platelets. Our study demonstrates that ticlopidine exerts at least part of its effect via inhibition of intracellular calcium mobilization but that its site of action remains to be determined.

Proaggregatory effect of epinephrine on rabbit platelets inhibited by ticlopidine.

Ticlopidine is a potent inhibitor of ADP-induced aggregation of rabbit platelets ex vivo. In vivo, however, multiple agonists play a role in platelet activation. In this study, we examined the effect of epinephrine on the antiplatelet action of ticlopidine in rabbit platelets. Epinephrine reversed the inhibitory effect of drug on ADP-induced platelet aggregation. The potentiating effect of epinephrine was mediated through alpha 2-adrenergic receptors, was reversed by pretreatment with the Na+/H+ exchange inhibitor dimethylamiloride, and was mimicked by agents that increased intracellular sodium or pH. Ticlopidine had no effect on resting intracellular pH, an indication that the effect of epinephrine was not compensating for a drug-induced intracellular acidification. While this potentiation was also found to be inhibited by aspirin, it did not involve enhanced release of thromboxane A2. Our results demonstrate that epinephrine can overcome the inhibitory effect of ticlopidine on ADP-induced aggregation through a mechanism involving activation of Na+/H+ exchange and through an as yet unidentified mechanism sensitive to aspirin.

Species differences in platelet responses to thrombin and SFLLRN. receptor-mediated calcium mobilization and aggregation, and regulation by protein kinases.

The thrombin receptor on human platelets is activated by thrombin to stimulate platelet aggregation through the tethered ligand SFLLRN. This study examined the effects of thrombin and SFLLRN on aggregation and calcium mobilization ([Ca2+]i) in rat, guinea pig, rabbit, dog, monkey, and human platelets, and the role of protein kinases in regulating these functions. Thrombin induced platelet aggregation and [Ca2+]i in all species studied; however, only guinea pig, monkey and human platelets were responsive to SFLLRN. Similar species specific effects were obtained with [Ca2+]i studies. The kinetic profile for [Ca2+]i differed among species, suggesting that regulatory mechanisms for calcium differed between agonists and among species. Staurosporine, a non-selective inhibitor of protein kinases, inhibited platelet aggregation induced by thrombin or SFLLRN in all species. Staurosporine inhibited thrombin-induced [Ca2+]i in guinea pigs, had no effect in rat, and increased [Ca2+]i in all other species. Staurosporine inhibited SFLLRN-induced [Ca2+]i in guinea pig, yet had no effect in monkey or human. Tyrphostin 23, a specific inhibitor of tyrosine protein kinases, inhibited thrombin-induced aggregation of rabbit, monkey, dog and human platelets. SFLLRN-induced aggregation was also inhibited by tyrphostin 23. Tyrphostin 23 inhibited [Ca2+]i induced by either thrombin or SFLLRN in all species. Based on the differential response to agonist stimulation, we propose that thrombin can activate platelets via SFLLRN-dependent and independent mechanisms, which could involve yet unrecognized subtypes of the thrombin receptor or distinct cellular activating mechanisms. Furthermore, differential regulation of calcium mobilization and aggregation was observed in those platelets responding to either thrombin or SFLLRN.

Polyphosphoinositide hydrolysis in endothelial cells and carotid artery segments. Bradykinin-2 receptor stimulation is calcium-independent.

Bovine aortic and cerebral microvascular endothelial cells and cultured segments of canine common carotid artery possess functional receptors for the nonapeptide bradykinin which mediate a rapid increase in the formation of [3H]inositol 1-phosphate, [3H]inositol 1,4-bisphosphate, and [3H]inositol 1,4,5-trisphosphate from cell membranes containing isotopically labeled myo-inositol. Bradykinin stimulated the formation of [3H]inositol phosphates from cells in culture or tissues at threshold concentrations of 0.1 nM and 1 nM, and with a half-maximal effective concentration of 0.6-1.0 nM and 30 nM, respectively. In cultured cells, the formation of [3H]inositol trisphosphate and [3H]inositol bisphosphate preceded the formation of [3H]inositol monophosphate. Similarly, [3H]inositol phosphate formation was not inhibited by addition of calcium channel blockers, a calcium chelator, or an intracellular calcium antagonist. Calcium ionophore A23187 did not promote [3H]inositol phosphate accumulation. The receptor selectivity of the bradykinin response in cultured cells was most compatible with a type-2 mediated response. Kallidin stimulated with the same potency as bradykinin but was more potent than methionyl-lysyl-bradykinin or des-Arg9-bradykinin. The B1 receptor antagonists des-Arg9-[Leu8]-bradykinin and des-Arg10-[Leu9]-kallidin were without effect. The rapidity of the inositol phosphate response as well as the close correspondence between the bradykinin type-2 receptor mediated hydrolysis of polyphosphoinositides and changes in prostacyclin synthesis, vessel dilation, and permeability suggests that breakdown products of inositol lipids serve as second messengers mediating the effects of bradykinin on the vascular endothelium.

Thrombin receptor-dependent prostaglandin E2 synthesis in hamster fibroblasts: synergistic interactions with interleukin-1beta.

Cellular responses to alpha-thrombin are mediated through a G-protein-coupled receptor that undergoes proteolytic cleavage, unveiling a tethered peptide ligand with the amino-terminal sequence SFLLRN. The synthetic peptide SFLLRN can mimic many of thrombin's actions via directly stimulating the thrombin receptor. Thrombin has been implicated in several cellular events associated with tissue injury, including fibroblast growth, matrix deposition, and inflammatory responses. The role of the thrombin receptor in fibroblast-dependent release of the inflammatory mediator prostaglandin E2 was evaluated and compared to its well-characterized effect on cell proliferation. Both thrombin and SFLLRN stimulated [3H]thymidine incorporation into DNA but failed to induce prostaglandin E2 release from CCL39 cells. The inflammatory cytokine interleukin-1beta synergized with thrombin and SFLLRN to induce the release of prostaglandin E2, whereas it had no effect on thrombin receptor-mediated DNA synthesis. Interleukin-1beta had no direct effects on thrombin receptor-mediated phosphoinositide hydrolysis, suggesting that its effects were downstream from early signal transduction events. Thrombin and interleukin-1beta together significantly increased the expression of prostaglandin H synthase-2 in accordance with the prostaglandin E2 results. These studies indicate that the fibroblast thrombin receptor differentially couples to intracellular signaling pathways leading to distinct functional responses and that thrombin receptor-effector interactions could be modulated by interleukin-1beta.

Differential regulation of human keratinocyte growth and differentiation by a novel family of protease-activated receptors.

Thrombin receptor (ThrR) and protease-activated receptor-2 (PAR-2) are members of a unique G protein-coupled receptor family, which are characterized by the unveiling of a tethered peptide ligand upon proteolysis of their NH2 terminus. We have previously shown that cultured human basal keratinocytes express both receptors (R.J. Santulli et al., Proc. Natl. Acad. Sci. USA, 92: 9151-9155, 1995); however, their functional role in epidermal physiology has yet to be described. In the present study, we determined the effects of receptor activation on keratinocyte cell growth and differentiation using thrombin (selective for ThrR), SLIGRL (selective for PAR-2), and SFLLRN (stimulates ThrR and PAR-2), as agonists. ThrR stimulation enhanced cell growth in a dose-dependent manner in the absence of growth factors (epidermal growth factor and bovine pituitary extract). In contrast, under the same conditions, activation of PAR-2 led to the inhibition of cell growth. This inhibitory activity by PAR-2 activation was also observed in the presence of growth factors. Activation of both receptors diminished protein expression of the differentiation marker transglutaminase type 1 induced by either calcium or IFN-gamma. Calcium-induced involucrin expression was also decreased. These results indicate that PAR-2 and ThrR differentially modulate keratinocyte function and may provide an important regulatory function in the epidermis by altering the functional state of keratinocytes.

Desensitization to substance P-induced vasodilation in vitro is not shared by endogenous tachykinin neurokinin A.

Two mammalian tachykinins, substance P (SP) and neurokinin A (NKA), were measured by radioimmunoassay in canine cephalic blood vessels and tested for their vasoactivity in vitro. Levels of immunoreactive SP were approximately 2-3 times greater than those of immunoreactive NKA in common carotid, basilar, and middle cerebral arteries. Both endogenous tachykinins relaxed precontracted segments of common carotid and basilar arteries in a dose-dependent manner with an EC50 of 8.9 X 10(-11) M and 7 X 10(-10) M, respectively, when added cumulatively. Relaxation was endothelial dependent for both substances and not blocked or enhanced by pretreatment with indomethacin, propranolol, lithium chloride, or atropine. Neither SP nor NKA released 3H-inositol phosphates from phospholipid membranes of canine carotid segments after preincubation with 3H-inositol. SP but not NKA or the C-terminal fragments SP(4-11) caused desensitization to subsequent additions of itself but not to the relaxation induced by sodium nitroprusside, calcitonin gene-related peptide, or bradykinin. These studies demonstrate that at least 2 peptides derived from beta-preprotachykinin are contained within cephalic blood vessels and that these products share similar vasoactive properties but differ in their ability to desensitize vascular tachykinin receptors.

Mitogenic responses mediated through the proteinase-activated receptor-2 are induced by expressed forms of mast cell alpha- or beta-tryptases.

The proteinase-activated receptor-2 (PAR-2) is the second member of a putative larger class of proteolytically activated receptors that mediate cell activation events by receptor cleavage or synthetic peptidomimetics corresponding to the newly generated N-terminus. To further study the previously identified mitogenic effects of PAR-2, we used the interleukin-3 (IL-3)-dependent murine lymphoid cell line, BaF3, for generation of stable cell lines expressing PAR-2 (BaF3/PAR-2) or the noncleavable PAR-2 mutant PAR-2(Arg36 --> Ala36). Only BaF3 cells expressing either wild-type or mutated receptor exhibited mitogenic responses when grown in IL-3-deficient media supplemented with PAR-2 activating peptide (SLIGRL, PAR39-44). This effect was dose dependent with an EC50 of approximately 80 micromol/L, sustained at 24, 48, and 72 hours, and was also demonstrable using thrombin receptor peptide TR42-47. Because tryptase shares approximately 70% homology with trypsin (previously shown to activate PAR-2), we studied recombinantly expressed forms of alpha- and beta-tryptases as candidate protease agonists for PAR-2. Hydrolytic activity of the chromogenic substrate tosyl-glycyl-prolyl-argly-4-nitroanilide acetate was present as a sharp peak at Mr approximately 130, confirming the presence of secretable and functionally active homotetrameric alpha- and beta-tryptases in transfected COS-1 cells. Dose-dependent proliferative responses were evident using either secreted form of tryptase with maximal responses seen at approximately 3 pmol/L (0.1 U/L). Receptor proteolysis was necessary and sufficient for mitogenesis because active site-blocked tryptase failed to induce this response, and proliferative responses were abrogated in BaF3 cells expressing PAR-2(Arg36 --> Ala36). These results specifically identify both forms of mast cell tryptases as serine protease agonists for PAR-2 and have implications for elucidating molecular mechanisms regulating cellular activation events mediated by proteases generated during inflammatory, fibrinolytic, or hemostatic-regulated pathways.

Receptor-activating peptides distinguish thrombin receptor (PAR-1) and protease activated receptor 2 (PAR-2) mediated hemodynamic responses in vivo.

Vascular expression and cellular functions of the thrombin receptor (PAR-1) and protease activated receptor 2 (PAR-2) suggest similar but distinct vascular regulatory roles. The vascular actions of PAR-1 and PAR-2 in vivo were differentiated by monitoring mean arterial pressure (MAP) and heart rate (HR) of anesthetized mice in response to intravenous SFLLRN (0.1, 0.3, and 1 mumol/kg) and SLIGRL (0.1, 0.3, and 1 mumol/kg), the respective receptor-activating sequences for PAR-1 and PAR-2, and TFLLRNPNDK (0.3, 1, and 3 mumol/kg), a synthetic peptide selective for PAR-1. All peptides dose dependently decreased MAP (order of potency: SLIGRL > SFLLRN > TFLLRNPNDK). SLIGRL induced a more prolonged hypotension with a slow return to baseline, whereas SFLLRN- and TFLLRNPNDK-induced hypotension was followed by a rapid return towards baseline and a sustained moderate hypotension. SFLLRN and TFLLRNPNDK, but not SLIGRL, decreased HR. N omega-Nitro-L-arginine methyl ester HCl (L-NAME), an inhibitor of nitric oxide synthesis, attenuated the cumulative hypotensive response to SLIGRL but had no effect on the SFLLRN and TFLLRNPNDK hypotension. However, L-NAME revealed a rebound hypertension in response to SFLLRN and TFLLRNPNDK but not SLIGRL. In conclusion, activation of either PAR-1 or PAR-2 in vivo results in hypotension. In addition, only PAR-1 activation induced hypertension following L-NAME, reflecting concurrent PAR-1-mediated vasoconstriction. Thus, these different hemodynamic responses in vivo suggest distinct physiological or pathophysiological roles for PAR-1 and PAR-2 in local vascular regulation.

Differential expression of protease-activated receptors-1 and -2 in stromal fibroblasts of normal, benign, and malignant human tissues.

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. The degree of protease secretion from malignant cells has been correlated to their metastatic potential. Protease activated receptors (PAR)-1 and -2, which are activated by thrombin and trypsin respectively, have not been extensively characterized in human tumors in situ. We investigated the presence of PAR-1 and PAR-2 in human normal, benign and malignant tissues using immunohistochemistry and in situ hybridization. Our results demonstrate PAR-1 and PAR-2 expression in the tumor cells, mast cells, macrophages, endothelial cells, and vascular smooth muscle cells of the metastatic tumor microenvironment. Most notably, an up-regulation of PAR-1 and PAR-2 observed in proliferating, smooth muscle actin (SMA)-positive stromal fibroblasts surrounding the carcinoma cells was not observed in normal or benign conditions. Furthermore, in vitro studies using proliferating, SMA-positive, human dermal fibroblasts, and scrape-wounded human dermal fibroblasts demonstrated the presence of PAR-1 and PAR-2 not detected in quiescent, SMA-negative cultures. PAR-1 and PAR-2 in the cells forming the tumor microenvironment suggest that these receptors mediate the signaling of secreted thrombin and trypsin in the processes of cellular metastasis.

Thrombin regulation of cell function through protease-activated receptors: implications for therapeutic intervention.

The serine protease thrombin is well recognized as being pivotal to the maintenance of hemostasis under both normal and pathological conditions. Its cellular actions are mediated through a unique family of protease-activated receptors (PARs). These receptors represent a novel family of G protein-coupled receptors that undergo proteolytic cleavage of their amino terminus and subsequent autoactivation by a tethered peptide ligand. This paper reviews the consequences of PAR activation in thrombosis, vascular injury, inflammation, tissue injury, and within the tumor microenvironment.

Inhibition of chemotactic peptide-induced neutrophil adhesion to vascular endothelium by cAMP modulators.

An early event associated with neutrophil-dependent tissue damage involves the adhesion of neutrophils to the vascular endothelium and the subsequent release of oxygen-derived free radicals and granule constituents. Elevations in intracellular cAMP are known to inhibit free radical release but not lysosomal enzyme release. The role of cAMP in FMLP-induced neutrophil adhesion was examined in this study by using an in vitro model of neutrophil-endothelial cell adhesion. FMLP stimulated a time- and concentration-dependent increase in human neutrophil adhesion to HUVEC. FMLP-mediated adhesion was inhibited by a diverse group of cAMP modulators: forskolin, isoproterenol, phosphodiesterase IV inhibitors (rolipram and Ro 20-1724), but not phosphodiesterase III inhibitors (milrinone and bemoradan). Endogenous adenosine has previously been shown to mediate FMLP-induced increases in cAMP enhanced in the presence of Ro 20-1724. In this study, adenosine deaminase prevented the inhibitory effects observed with rolipram and Ro 20-1724, implicating endogenous adenosine as a co-modulator of inhibition. FMLP stimulated neutrophil shape change and the surface expression of the beta 2 integrins CD11b/CD18 and CD11a/CD18. Both these responses were inhibited by rolipram but not bemoradan. With the use of 4,4'-diisothiocyanostilbene-2,2'disulfonic acid, we showed that mobilization of the intracellular pool of CD11b/CD18 paralleled adhesion. We conclude that neutrophil-endothelial cell adhesion is attenuated by elevating neutrophil intracellular cAMP and that inhibition of neutrophil CD11b/CD18 surface expression by cAMP accounts for this observed inhibition of adhesion.

The role of the neutrophil and formed elements of the blood in an in vitro model of reperfusion injury.

Using The globally ischaemic isolated guinea-pig heart we conducted studies to assess the role of activated neutrophils (PMNs) and the role of the endothelium in reperfusion injury. Reperfusion injury was induced by a 20 min period of global ischaemia followed by a 30 min reperfusion with Krebs' buffer supplemented with f-Met-Leu-Phe (fMLP) and heparinized blood. Ischaemia alone or blood alone resulted in a complete recovery in contractile function measured by developed pressure, fMLP (500 muM) and blood, administered to normoxic hearts did not affect contractile function. The combination of 100 muM fMLP and blood beginning at reperfusion and continuing for 30 min decreased the recovery in contractile function (max. 33 +/- 6% reovery) while buffer and 100 pM fMLP resulted in a complete recovery in function. In hearts infused with buffer and neutropenic blood incubated with 100 muM fMLP a complete recovery in function was observed. Isolated peritoneal neutrophils, 7-70 x 10(5) PMN/ min, incubated with 100 muM fMLP and Krebs' solution decreased contractile function in a concentration-related manner (max. 44 +/- 11% recovery). Platelets, plasma or red blood cells alone incubated with fMLP did not decrease recovery in developed pressure. Platelets and PMN incubated with 100 muM fMLP did not, while red blood cells and PMN did, elicit a reduction in recovery in contractile function (34 +/- 4% recovery). A 20 min period of global ischaemia destroys the functional integrity of the endothelium (response to Ach). Pre-treatment of the heart with sufficient H(2)O(2) to functionally damage the endothelium, followed by infusion of Krebs' solution supplemented with blood and 100 muM fMLP also elicited a reduction in recovery of contractile function (42 +/- 15% recovery). In summary, partially activated neutrophils play a major role in reperfusion injury and there exists a cooperativity between the RBC and PMN in this model.

Evidence for the presence of a protease-activated receptor distinct from the thrombin receptor in human keratinocytes.

Thrombin receptor activation was explored in human epidermal keratinocytes and human dermal fibroblasts, cells that are actively involved in skin tissue repair. The effects of thrombin, trypsin, and the receptor agonist peptides SFLLRN and TFRIFD were assessed in inositolphospholipid hydrolysis and calcium mobilization studies. Thrombin and SFLLRN stimulated fibroblasts in both assays to a similar extent, whereas TFRIFD was less potent. Trypsin demonstrated weak efficacy in these assays in comparison with thrombin. Results in fibroblasts were consistent with human platelet thrombin receptor activation. Keratinocytes, however, exhibited a distinct profile, with trypsin being a far better activator of inositolphospholipid hydrolysis and calcium mobilization than thrombin. Furthermore, SFLLRN was more efficacious than thrombin, whereas no response was observed with TFRIFD. Since our data indicated that keratinocytes possess a trypsin-sensitive receptor, we addressed the possibility that these cells express the human homologue of the newly described murine protease-activated receptor, PAR-2 [Nystedt, S., Emilsson, K., Wahlestedt, C. & Sundelin, J. (1994) Proc. Natl. Acad. Sci. USA 91, 9208-9212]. PAR-2 is activated by nanomolar concentrations of trypsin and possesses the tethered ligand sequence SLIGRL. SLIGRL was found to be equipotent with SFLLRN in activating keratinocyte inositolphospholipid hydrolysis and calcium mobilization. Desensitization studies indicated that SFLLRN, SLIGRL, and trypsin activate a common receptor, PAR-2. Northern blot analyses detected a transcript of PAR-2 in total RNA from keratinocytes but not fibroblasts. Levels of thrombin receptor message were equivalent in the two cell types. Our results indicate that human keratinocytes possess PAR-2, suggesting a potential role for this receptor in tissue repair and/or skin-related disorders.

Inhibitors of 2-ketoglutarate-dependent dioxygenases block aspartyl beta-hydroxylation of recombinant human factor IX in several mammalian expression systems.

While a role has been ascribed to the gamma-carboxyglutamate (Gla) residues in vitamin K-dependent coagulation proteins and the enzyme catalyzing this posttranslational modification has been identified and partially characterized, both the functional significance of a second posttranslationally synthesized amino acid found in these proteins, beta-hydroxyaspartate (Hya), and the aspartyl beta-hydroxylating enzyme remain to be determined. We now report that inhibitors of 2-ketoglutarate-dependent dioxygenases, such as dipyridyl, o-phenanthroline, and pyridine 2,4-dicarboxylate, block hydroxylation of Asp64 in recombinant factor IX molecules produced in three different mammalian expression systems. This hydroxylation was not inhibited by the specific copper chelators 2,9-dimethylphenanthroline or D-penicillamine. The Gla levels in these proteins were unaffected by these compounds and demonstrate that carboxylation proceeds independently of hydroxylation. Using these Hya-deficient recombinant factor IX molecules we demonstrate that this residue does not play a significant role in factor IX binding to endothelial cells under equilibrium conditions. From additional binding studies we have concluded that the Gla domain of factor IX is a major cell binding domain of factor IX. Furthermore, in contrast to studies demonstrating a marked loss of one-stage clotting activity in recombinant factors IX following site-directed mutations of Asp64 to neutral or basic residues (Rees, D. J. G., Jones, I. M., Handford, P. A., Walter, S. J., Esnouf, M. P., Smith, K. J., and Brownlee, G. J. (1988) EMBO J. 7, 2053-2061), we have not found a decrease of one-stage clotting activity with Hya-deficient factor IX. Hya-deficient proteins produced in this manner may prove to be more appropriate to elucidate the function of Hya than those produced by site-directed mutagenesis.