Cockroach allergen serine proteinases: Isolation, sequencing and signalling via proteinase-activated receptor-2.

BACKGROUND: Allergy to the German cockroach (Blattella germanica) is a significant asthma risk factor for inner-city communities. Cockroach, like other allergens, contains trypsin-like enzyme activity that contributes to allergenicity and airway inflammation by activating proteinase-activated receptors (PARs). To date, the enzymes responsible for the proteolytic activity of German cockroach allergen have not been characterized. OBJECTIVES: We aimed to identify, isolate and characterize the trypsin-like proteinases in German cockroach allergen extracts used for clinical skin tests. For each enzyme, we sought to determine (1) its substrate and inhibitor enzyme kinetics (Km and IC50), (2) its amino acid sequence and (3) its ability to activate calcium signalling and/or ERK1/2 phosphorylation via PAR2. METHODS: Using a trypsin-specific activity-based probe, we detected three distinct enzymes that were isolated using ion-exchange chromatography. Each enzyme was sequenced by mass spectometery (deconvoluted with an expressed sequence tag library), evaluated kinetically for its substrate/inhibitor profile and assessed for its ability to activate PAR2 signalling. FINDINGS: Each of the three serine proteinase activity-based probe-labelled enzymes isolated was biochemically distinct, with different enzyme kinetic profiles and primary amino acid sequences. The three enzymes showed a 57%-71% sequence identity with a proteinase previously cloned from the American cockroach (Per a 10). Each enzyme was found to activate both Ca++ and MAPK signalling via PAR2. CONCLUSIONS AND RELEVANCE: We have identified three different serine proteinases from the German cockroach that may, via PAR2 activation, play different roles for allergen sensitization in vivo and may represent attractive therapeutic targets for asthma.

Functional inhibition of PAR2 alleviates allergen-induced airway hyperresponsiveness and inflammation.

BACKGROUND: Proteinase-activated receptor 2 (PAR2 ) is a G protein-coupled receptor activated by trypsin-like serine proteinases. PAR2 activation has been associated with inflammation including allergic airway inflammation. We have also shown that PAR2 activation in the airways leads to allergic sensitization. The exact contribution of PAR2 in the development of eosinophilic inflammation and airway hyperresponsiveness (AHR) in sensitized individuals is not clear. OBJECTIVE: To investigate whether functional inhibition of PAR2 during allergen challenge of allergic mice would inhibit allergen-induced AHR and inflammation in mouse models of asthma. METHODS: Mice were sensitized and challenged with ovalbumin (OVA) or cockroach extract (CE). To investigate the role of PAR2 in the development of AHR and airway inflammation, we administered blocking anti-PAR2 antibodies, or a cell permeable peptide inhibitor of PAR2 signalling, pepducin, i.n. before allergen challenges and then assessed AHR and airway inflammation. RESULTS: Administration of anti-PAR2 antibodies significantly inhibited OVA- and CE-induced AHR and airway inflammation. In particular, two anti-PAR2 antibodies, the monoclonal SAM-11 and polyclonal B5, inhibited AHR, airway eosinophilia, the increase of cytokines in the lung tissue and antigen-specific T cell proliferation, but had no effect on antigen-specific IgG and IgE levels. Pepducin was also effective in inhibiting AHR and airway inflammation in an OVA model of allergic airway inflammation. CONCLUSIONS AND CLINICAL RELEVANCE: Functional blockade of PAR2 in the airways during allergen challenge improves allergen-induced AHR and inflammation in mice. Therefore, topical PAR2 blockade in the airways, through anti-PAR2 antibodies or molecules that interrupt PAR2 signalling, has the potential to be used as a therapeutic option in allergic asthma.

Proteinase-activated receptor-2 activation participates in allergic sensitization to house dust mite allergens in a murine model.

BACKGROUND: Many aeroallergens contain proteinase activity and are able to induce allergic sensitization when presented to mucosal surfaces. Some of these allergens activate proteinase-activated receptor-2 (PAR2 ). OBJECTIVE: To determine the role of PAR2 activation in a murine house dust mite (HDM) allergy model. METHODS: We sensitized and challenged PAR2 -deficient mice with HDM, and examined allergic outcomes compared to wild-type animals. To focus on the role of PAR2 in allergic sensitization, we administered a PAR2 blocking antibody to wild-type animals during the sensitization phase and examined the outcomes immediately after sensitization or following subsequent allergen challenge. RESULTS: We found PAR2 -deficient mice sensitized and challenged with HDM failed to develop airway inflammation, did not produce HDM-specific IgG1 and had less IL-4 mRNA in the lungs than wild-type animals. Prevention of PAR2 activation during sensitization in wild-type mice diminished the levels of Th2 mediators, including IL-4, IL-5 and IL-13, in the lungs. Blocking PAR2 during the sensitization phase also led to decreased manifestations of allergic disease, including airway hyperresponsiveness (AHR) and airway inflammation following subsequent allergen challenge. HDM-induced proliferation of splenocytes obtained from animals sensitized in the presence of PAR2 antibody was reduced relative to those that did not receive antibody. The effect of PAR2 blockade could be transferred to naïve mice through splenic CD4(+) T cells from sensitized mice. CONCLUSIONS AND CLINICAL RELEVANCE: PAR2 activation plays a key role during the sensitization phase of our HDM allergy model, leading to increased lung cytokine production and augmented lung reactivity. PAR2 activation is a common mechanism for sensitization to a wide variety of allergens and is therefore a potential pharmacological target to prevent allergy.

Proteinase-activated receptor-2 and hyperalgesia: A novel pain pathway.

Using a combined pharmacological and gene-deletion approach, we have delineated a novel mechanism of neurokinin-1 (NK-1) receptor-dependent hyperalgesia induced by proteinase-activated receptor-2 (PAR2), a G-protein-coupled receptor expressed on nociceptive primary afferent neurons. Injections into the paw of sub-inflammatory doses of PAR2 agonists in rats and mice induced a prolonged thermal and mechanical hyperalgesia and elevated spinal Fos protein expression. This hyperalgesia was markedly diminished or absent in mice lacking the NK-1 receptor, preprotachykinin-A or PAR2 genes, or in rats treated with a centrally acting cyclooxygenase inhibitor or treated by spinal cord injection of NK-1 antagonists. Here we identify a previously unrecognized nociceptive pathway with important therapeutic implications, and our results point to a direct role for proteinases and their receptors in pain transmission.

Agonists of proteinase-activated receptor 2 induce inflammation by a neurogenic mechanism.

Trypsin and mast cell tryptase cleave proteinase-activated receptor 2 and, by unknown mechanisms, induce widespread inflammation. We found that a large proportion of primary spinal afferent neurons, which express proteinase-activated receptor 2, also contain the proinflammatory neuropeptides calcitonin gene-related peptide and substance P. Trypsin and tryptase directly signal to neurons to stimulate release of these neuropeptides, which mediate inflammatory edema induced by agonists of proteinase-activated receptor 2. This new mechanism of protease-induced neurogenic inflammation may contribute to the proinflammatory effects of mast cells in human disease. Thus, tryptase inhibitors and antagonists of proteinase-activated receptor 2 may be useful anti-inflammatory agents.

Does conventional early life academic excellence predict later life scientific discovery? An assessment of the lives of great medical innovators.

BACKGROUND: Perhaps, as never before, we need innovators. With our growing population numbers, and with increasing pressures on our education systems, are we in danger of becoming more rigid and formulaic and increasingly inhibiting innovation? When young can we predict who will become the great innovators? For example, in medicine, who will change clinical practice? AIMS: We therefore determined to assess whether the current academic excellence approach to medical school entrance would have captured previous great innovators in medicine, assuming that they should all have well fulfilled current entrance requirements. METHODS: The authors assembled a list of 100 great medical innovators which was then approved, rejected or added to by a jury of 12 MD fellows of the Royal Society of Canada. Two reviewers, who had taken both the past and present Medical College Admission Test as part of North American medical school entrance requirements, independently assessed each innovator's early life educational history in order to predict the innovator's likely success at medical school entry, assuming excellence in all entrance requirements. RESULTS: Thirty-one percent of the great medical innovators possessed no medical degree and 24% would likely be denied entry to medical school by today's standards (e.g. had a history of poor performance, failure, dropout or expulsion) with only 24% being guaranteed entry. Even if excellence in only one topic was required, the figure would only rise to 41% certain of medical school entry. CONCLUSION: These data show that today's medical school entry standards would have barred many great innovators and raise questions about whether we are losing medical innovators as a consequence. Our findings have important implications for promoting flexibility and innovation for medical education, and for promoting an environment for innovation in general.

Inability of oxytocin to activate pyruvate dehydrogenase in the Brattleboro rat.

Oxytocin has insulin-like activity in that it stimulates lipogenesis and increases pyruvate dehydrogenase activity. However, in adipocytes from homozygous Brattleboro rats oxytocin is incapable of stimulating lipogenesis or pyruvate dehydrogenase activity, although insulin stimulation of both processes is normal and the antilipolytic activity of oxytocin is normal. Thus, the Brattleboro rat provides a new genetic model for the study of oxytocin action, wherein recognition of the chemical mediator is partially defective.

Adult hemoglobin synthesis by reticulocytes from the human fetus at midtrimester.

The synthesis of adult-type hemoglobin was measured in small samples of peripheral blood cells from 9- to 18-week human fetuses. Hemoglobin indistinguishable from hemoglobin A was identified by ion-exchange chromatography, electrophoresis at pH 8.6, tryptic peptide analysis, and the insensitivity of its synthesis to the action of O-methylthreonine. Synthesis of hemoglobin A accounted for 8 to 14 percent of total hemoglobin synthesis and was demonstrated in as little as 10 microliters of fetal blood. These studies provide sensitive methods for the detection of beta chain types in hemoglobin synthesized by the human fetus at midtrimester. If methods to obtain small quantities of fetal blood at midtrimester become available, these techniques should be applicable to the antenatal detection of sickle cell anemia and related hemoglobinopathies.

Role of protease-activated receptors in inflammatory responses, innate and adaptive immunity.

Serine proteases are well known as enzymes involved in digestion of dietary proteins, blood coagulation, and homeostasis. Only recent groundbreaking studies revealed a novel role of serine proteases as signaling molecules acting via protease-activated receptors (PARs). Important effects of PAR activation on leukocyte motility, cytokine production, adhesion molecule expression, and a variety of other physiological or pathophysiological functions have been described in vitro and in vivo. The crucial role of PAR activation during disease progression was revealed in animal models of different gastrointestinal pathologies, neuroinflammatory and neurodegenerative processes, skin, joint and airway inflammation, or allergic responses. This review focuses on the findings related to the impact of PAR deficiency in animal models of inflammatory and allergic diseases. Additionally, we observe the role of PAR activation in the regulation of functional responses of innate and adaptive immune cells in vitro. Understanding the mechanisms by which PARs exert the effects of serine proteases on immune cells may lead to new therapeutic strategies in inflammation, immune defense, and allergy.

Proteinases and signalling: pathophysiological and therapeutic implications via PARs and more.

Proteinases like thrombin, trypsin and tissue kallikreins are now known to regulate cell signaling by cleaving and activating a novel family of G-protein-coupled proteinase-activated receptors (PARs 1-4) via exposure of a tethered receptor-triggering ligand. On their own, short synthetic PAR-selective PAR-activating peptides (PAR-APs) mimicking the tethered ligand sequences can activate PARs 1, 2 and 4 and cause physiological responses both in vitro and in vivo. Using the PAR-APs as sentinel probes in vivo, it has been found that PAR activation can affect the vascular, renal, respiratory, gastrointestinal, musculoskeletal and nervous systems (both central and peripheral nervous system) and can promote cancer metastasis and invasion. In general, responses triggered by PARs 1, 2 and 4 are in keeping with an innate immune inflammatory response, ranging from vasodilatation to intestinal inflammation, increased cytokine production and increased or decreased nociception. Further, PARs have been implicated in a number of disease states, including cancer and inflammation of the cardiovascular, respiratory, musculoskeletal, gastrointestinal and nervous systems. In addition to activating PARs, proteinases can cause hormone-like effects by other signalling mechanisms, like growth factor receptor activation, that may be as important as the activation of PARs. We, therefore, propose that the PARs themselves, their activating serine proteinases and their associated signalling pathways can be considered as attractive targets for therapeutic drug development. Thus, proteinases in general must now be considered as 'hormone-like' messengers that can signal either via PARs or other mechanisms.

Epidermal growth factor-urogastrone causes vasodilatation in the anesthetized dog.

Epidermal growth factor-urogastrone (EGF-URO) administered intraarterially was a potent dilator in dog femoral (FEM), superior (cephalic) mesenteric (SMA), celiac (CAC), coronary (COR), carotid (CAR), and renal (REN) vascular beds. The effects of EGF-URO, which exhibited tachyphylaxis, could not be attributed either to recirculating EGF-URO or to the secondary release of other agonists or products of the cyclooxygenase pathway. Two vascular beds (FEM, SMA) showed a high maximum responsiveness to EGF-URO (maximum effect [Emax] approximately equal to 70% increase in flow) whereas another group (CAC, COR, CAR, and REN) exhibited lower responsiveness (Emax approximately equal to 20%). The ED50 for this effect of EGF-URO was in the range of 0.4 micrograms (FEM, SMA, CAR, and COR) to 0.9 micrograms (CAC and REN). In isolated dog COR helical strips, EGF-URO did not exhibit either a direct relaxing or a contractile effect. However, preincubation of strips with EGF-URO caused up to a 66% inhibition of contraction in response to norepinephrine (1 microM), with an ED50 for EGF-URO of 1 nM. This action of EGF-URO also showed marked tachyphylaxis. Our data point to a potential role for EGF-URO (and possibly for the structurally related alpha-transforming growth factor) in the regulation of blood flow in vivo.

Evaluation of the antiinflammatory and phospholipase-inhibitory activity of calpactin II/lipocortin I.

We have examined the ability of a highly purified 38-kD phospholipase-inhibitory protein (p38) isolated from human placental membranes that is also a preferred substrate for the epidermal growth factor-urogastrone (EGF-URO) receptor/kinase, to block the release of arachidonate from zymosan-stimulated murine peritoneal macrophages in vitro and to exhibit antiinflammatory activity in a carrageenin rat paw edema test in vivo. The ability of glucocorticoids to increase the amounts of this protein in macrophage cultures was also examined. p38 represents the naturally occurring, intact, NH2-terminally blocked human placental form of the protein termed calpactin II (or lipocortin I), for which partial amino acid sequence data and a complete amino acid sequence deduced from cDNA analysis have been reported. Our data demonstrated that, whereas p38 was an effective inhibitor of pancreatic phospholipase A2 in vitro, it was unable to inhibit either the release of arachidonate from cultured zymosan-stimulated mouse peritoneal macrophages or inflammation in a rat paw edema test. At comparatively high protein concentrations, p38 enhanced either arachidonate release from intact macrophages in vitro (0.5-10 micrograms/ml) or carrageenin-induced paw swelling in vivo (2.5 or 25 micrograms per injection). Furthermore, we were unable to detect induced amounts of p38 in cultures of glucocorticoid-treated peritoneal macrophages obtained from either mice or rats. Our data indicate that the antiphospholipase activity of p38 in vitro and the ability of p38 to serve as a receptor/kinase substrate may in no way relate to the putative ability of the protein to modify eicosanoid release from macrophages in vivo, so as to modulate the inflammatory process. Our data also raise the possibility that p38 (calpactin II) may not be a true representative of the lipocortin family of glucocorticoid-inducible antiinflammatory proteins, despite its ability to inhibit phospholipase A2 in vitro.

Integrating the GPCR transactivation-dependent and biased signalling paradigms in the context of PAR1 signalling.

Classically, receptor-mediated signalling was conceived as a linear process involving one agonist, a variety of potential targets within a receptor family (e.g. α- and ß-adrenoceptors) and a second messenger (e.g. cAMP)-triggered response. If distinct responses were stimulated by the same receptor in different tissues (e.g. lipolysis in adipocytes vs. increased beating rate in the heart caused by adrenaline), the differences were attributed to different second messenger targets in the different tissues. It is now realized that an individual receptor can couple to multiple effectors (different G proteins and different ß-arrestins), even in the same cell, to drive very distinct responses. Furthermore, tailored agonists can mould the receptor conformation to activate one signal pathway versus another by a process termed 'biased signalling'. Complicating issues further, we now know that activating one receptor can rapidly trigger the local release of agonists for a second receptor via a process termed 'transactivation'. Thus, the end response can represent a cooperative signalling process involving two or more receptors linked by transactivation. This overview, with a focus on the GPCR, protease-activated receptor-1, integrates both of these processes to predict the complex array of responses that can arise when biased receptor signalling also involves the receptor transactivation process. The therapeutic implications of this signalling matrix are also briefly discussed. Linked Articles This article is part of a themed section on Molecular Pharmacology of G Protein-Coupled Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v173.20/issuetoc.

Characterization of the particulate and soluble acceptor for transcobalamin II from human placenta and rabbit liver.

We describe in both human placenta and rabbit liver membranes specific acceptors which bind the human transcobalamin II-vitamin B-12 (cobalamin) complex with an affinity of 2.3 . 10(9) (placenta) and 6.7 . 10(9) (liver) M-1 and which bind the rabbit transcobalamin II-cobalamin complex with an affinity of 1.1 . 10(9) (placenta) and 1.9 . 10(9) (liver) M-1, respectively. The binding requires Ca2+ and is sensitive to both 1 M NaCl and acid pH. A new ligand binding assay, based on the ability of the acceptor, but not transcobalamin II, to bind to concanavalin A, is described and is used to characterize the solubilized acceptors. The solubilized acceptors bind human transcobalamin II-cobalamin with high affinity (about 2-9 . 10(9) M-1) but do not bind free cobalamin; unsaturated transcobalamin II is bound with an affinity approximately one-third of that for transcobalamin II saturated with cobalamin. On gel filtration, the human acceptor saturated with transcobalamin II-cobalamin exhibits a Stokes radius of 6.7 nm, whereas the free acceptor has a Stokes radius of 5.1 nm. The rabbit liver acceptor either unsaturated or saturated with transcobalamin II-cobalamin exhibits a Stokes radius of 5.7 nm. Both acceptors bind to lectins such as concanavalin A, wheat germ agglutinin and phytohemagglutinin, indicating their glycoprotein nature, and both acceptors can be purified approximately 30-fold by affinity chromatography on wheat germ agglutinin-Sepharose columns. The concanavalin A assay, combined with lectin-Sepharose and transcobalamin II-cobalamin-Sepharose affinity chromatography will provide for the isolation and study of pure acceptors from a variety of tissue sources.

Tyrosine kinase pathways and the regulation of smooth muscle contractility.

Epidermal growth factor (EGF) mediates three tyrosine kinase-dependent smooth muscle response paradigms, two of which comprise a rapid increase in muscle tension and one of which is characterized by an agonist-mediated reduction in sensitivity to other agents. The three types of response are mediated via distinct signal transduction pathways, and marked tissue and species variation have been observed, even for a single growth factor agonist. Vasoactive agents, such as angiotensin II and vasopressin, that act via G protein-coupled receptors can also work via tyrosine kinase pathways to cause contraction in some of the same intact smooth muscle preparations that contract in response to EGF. In this review, Morley Hollenberg discusses the tyrosine kinase-modulated signal transduction pathways for EGF and also agonists that act via G protein-coupled receptors, and hypothesizes that there may be an intermediary non-receptor tyrosine kinase that may serve as a point of convergence for the contractile actions of these agents in smooth muscle.

Protease-activated receptors in inflammation, neuronal signaling and pain.

The ability of proteases to regulate cell function via protease-activated receptors (PARs) has led to new insights about the potential physiological functions of these enzymes. Several studies suggest that PARs play roles in both inflammation and tissue repair, depending on the cellular environment in which they act. The recent detection of PARs on peripheral and central neurons suggests that neuronal PARs might be involved not only in neurogenic inflammation and neurodegenerative processes, but also in nociception. Thus, the list of potential roles for PARs has lengthened considerably and their physiological course of action might be much broader than initially anticipated.

Proteinases, their receptors and inflammatory signalling: the Oxford South Parks Road connection.

In keeping with the aim of the Paton Memorial Lecture to 'facilitate the historical study of pharmacology', this overview, which is my distinct honour to write, represents a 'Janus-like' personal perspective looking both backwards and forwards at the birth and growth of 'receptor molecular pharmacology' with special relevance to inflammatory diseases. The overview begins in the Oxford Department of Pharmacology in the mid-1960s and then goes on to provide a current perspective of signalling by proteinases. Looking backwards, the synopsis describes the fruitful Oxford Pharmacology Department infrastructure that Bill Paton generated in keeping with the blueprint begun by his predecessor, J H Burn. Looking forwards, the overview illustrates the legacy of that environment in generating some of the first receptor ligand-binding data and providing the inspiration and vision for those like me who were training in the department at the same time. With apologies, I mention only in passing a number of individuals who benefitted from the 'South Parks Road connection' using myself as one of the 'outcome study' examples. It is also by looking forward that I can meet the complementary aim of summarizing the lecture presented at a 'BPS 2014 Focused Meeting on Cell Signalling' to provide an overview of the role of proteinases and their signalling mechanisms in the setting of inflammation.

Identification of classical, novel, and atypical protein kinase C isoenzymes in the bovine parathyroid.

Because phospholipid metabolism leading to the activation of protein kinase C (PKC) may play a key regulatory role in the degradation and secretion of PTH, we examined parathyroid cell fractions for the presence of various PKC isoenzymes. Hydroxylapatite chromatography identified the classical PKCs, alpha and beta, but not gamma in parathyroid cell extracts. Western blot analysis confirmed the presence of PKC alpha and beta in these extracts. Of the so-called novel PKCs, Western blot analysis revealed the presence of only one isoenzyme, novel PKC epsilon in parathyroid cell soluble extracts. Western blot analysis using an antibody to the C-terminus of the atypical isoenzyme, PKC zeta, identified a protein of lower molecular weight in addition to PKC zeta. This lower molecular weight protein presumably represents PKC lambda, which shares a high degree of C-terminal sequence similarity with PKC zeta. These findings suggest the possibility that members of all three groups of the PKC family are present and may play a regulatory role in the bovine parathyroid cell.

Receptors for insulin and epidermal growth factor-urogastrone in adult human fibroblasts do not change with donor age.

The ligand binding and biological responsiveness of cultured fibroblast monolayers were measured for porcine insulin and for murine epidermal growth factor--urogastrone in skin fibroblast cultures derived from young (22 to 31 years) and old (65 to 80 years) normal male volunteer donors. The receptor characteristics of the cells studied from the two groups of donors were not found to differ. In contrast with previous data, suggesting that there may be a genetically programmed aging of the insulin binding system, it is concluded that for both insulin and epidermal growth factor, receptor characteristics do not change with adult donor age.

Synthesis and contractile activities of cyclic thrombin receptor-derived peptide analogues with a Phe-Leu-Leu-Arg motif: importance of the Phe/Arg relative conformation and the primary amino group for activity.

Based on the minimal peptide sequence (Phe-Leu-Leu-Arg) that has been found to exhibit biological activity in a gastric smooth muscle contractile assay for thrombin receptor-activating peptides, the cyclic peptide analogues cyclo(Phe-Leu-Leu-Arg-Acp) (1), cyclo(Phe-Leu-Leu-Arg-epsilon Lys) (2), and cyclo(Phe-Leu-Leu-Arg-Gly) (3) have been synthesized by the solid-phase method using benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluoroborate or 2-(1H-benzo-triazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate as cyclization reagents. The contractile activities of compounds 1-3 have been compared with that of the linear thrombin receptor-activating peptide (TRAP) Ser-Phe-Leu-Leu-Arg-NH2 (compound 4) using a gastric smooth muscle strip assay. Compound 2, wherein the epsilon-amino group of lysine was coupled to the alpha-carboxyl of arginine, exhibited a contractile activity comparable to that of the linear TRAP, compound 4. However compound 1, wherein the aminocaproic linker group yielded a ring size the same as for compound 2 but without a primary amino group, exhibited a contractile activity 600-1000-fold lower than compounds 2 and 4. Compound 3, which exhibited partial agonist activity, was about 100-fold less potent than either compound 2 or 4. NMR spectroscopy of compound 2 revealed a proximity of the Phe and Arg side chains, leading to a molecular model generated by distance geometry and molecular dynamics, wherein the Phe and Arg residues are shown in proximity on the same side of the peptide ring. We conclude that the Phe and Arg side chains along with the primary amino group form an active recognition motif that is augmented by the presence of a primary amino group in the cyclic peptide. We suggest that a comparable cyclic conformation may be responsible for the interaction of linear TRAPs with the thrombin receptor.