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*
Cardiovascular Division and
Department of Biomedical Engineering, University of Virginia School of Medicine, Charlottesville, VA 22908;
Cardiovascular Research Institute, University of California, San Francisco, CA 94143; and
Pediatric Cardiology, Stanford University Medical Center, Stanford, CA 94305
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Induction of leukocyte rolling occurs rapidly in response to tissue injury or inflammation. Intravital microscopy has demonstrated leukocyte rolling in small venules several minutes following trauma induced by surgical exteriorization of the mouse cremaster muscle (6) or rat mesentery (7). This early rolling is almost entirely dependent upon recruitment of P-selectin to the luminal surface of vascular endothelial cells from preformed stores within the Weibel-Palade bodies (8). Mice deficient in P-selectin are characterized by deficient leukocyte rolling for up to 1 h after exteriorization of the cremaster muscle, after which E- and L-selectin-dependent rolling is observed (6, 8, 9).
Cell surface expression of P-selectin occurs in response to many stimuli, including cytokines, LPS, thrombin, and histamine (8, 10, 11, 12). Thrombin induces P-selectin expression by a cascade of events initiated by binding to the thrombin receptor, or protease-activated receptor-1 (PAR1),4 on the endothelial cell surface (13). This receptor belongs to a family of unique G protein-coupled receptors that undergo proteolytic cleavage of the amino-terminal exodomain by serine proteases to produce a new amino terminus that functions as a tethered ligand to activate receptor function (14, 15). Proteolytic activation of PAR1 by thrombin results in activation of phospholipases A2, C, and D (15, 16). Subsequent inositol triphosphate production and elevation of cytosolic Ca2+ (16) are most likely responsible for mobilization of granular stores of endothelial P-selectin (15). PAR1 agonists, such as the unmasked amino-terminal tethered ligand (SFLLRN), have been shown to promote P-selectin expression in cultured endothelial cells (17) and to induce P-selectin-dependent rolling in the mesentery of the rat (13).
Histamine is another vasoactive substance that triggers cell surface
expression of P-selectin on cultured endothelial cells
(12) and induces leukocyte rolling in rat mesentery
(7). Release of histamine upon degranulation of
perivascular mast cells can induce P-selectin-mediated leukocyte
rolling seen early after exteriorization of the mesenteric membrane in
rats (7). Treatment with histamine receptor antagonists
does not, however, abolish this rolling, suggesting that, in addition
to histamine, other substances may mediate venular P-selectin
expression in the setting of trauma or inflammation. One potential mast
cell-derived product is tryptase, which activates another member of the
protease-activated receptor family, PAR2. This receptor is structurally
homologous to PAR1 with a proteolytic cleavage site located between
arginine (Arg36) and serine
(Ser37) residues near the amino terminus
(18). Activation of PAR2 by tryptase, trypsin, or the
hexapeptide agonist SLIGRL that corresponds to the murine tethered
ligand sequence results in increases in intracellular
Ca+, similar to the response to PAR1 activation
(16, 19, 20). Accordingly, it has been demonstrated that
treatment of HUVECs with trypsin or the PAR2 peptide agonist SLIGRL
results in cell surface expression of P-selectin and von Willebrand
factor from Weibel-Palade bodies (21). The role of PAR2 in
promoting inflammatory responses is further suggested by the
demonstration that mRNA for PAR2 is up-regulated in HUVECs in response
to IL-1, TNF-
, and LPS (22). More recently, superfusion
of rat mesentery with PAR2 agonist peptides has been shown to increase
leukocyte rolling and adhesion in venules (23).
In this study, we sought to further characterize the role of PAR2 receptor activation in initiating the inflammatory cascade. Early P-selectin-dependent leukocyte rolling was evaluated by intravital microscopy of the cremaster muscle of wild-type and PAR2-deficient mice generated by gene targeting and homologous recombination. Leukocyte rolling in venules was also assessed in wild-type and PAR2-deficient mice following perivascular microinjections of the PAR2 hexapeptide agonist SLIGRL, which possesses little cross-activity with PAR1 (19).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A bacterial artificial chromosome containing the PAR2 gene
(Par2g) was obtained by PCR screen of a 129/SvJ mouse
genomic library (Genome Systems, St. Louis, MO). A 7-kb
XhoI/HindII fragment of the Par2g intron and a
1.2-kb fragment of Par2g 3' untranslated sequence generated by PCR
(forward primer, 5'-TGGTAGGGATGCACCAACATG-3' corresponding to nts
1379–1399 of GB Z48043; reverse primer,
5'-GGTAGGTTAAAGCAGACAGGGGAC-3' corresponding to nts 2440–2463 of
GB Z48043) were cloned into the pNTK vector to create the targeting
vector (Fig. 1
A). A 0.8-kb
XbaI fragment of genomic sequence 3' of the short arm of
homology was used as a probe to identify both the wild-type and
targeted alleles. RF8 embryonic stem cells (24)
(129/SvJae) were electroporated with the targeting construct, and
clones resistant to G418 and FIAU were selected and screened by
Southern blot (Fig. 1B). A male mouse highly chimeric for
PAR2+/- embryonic stem cells was bred to C57BL/6
females to generate PAR2+/- mice that were bred
to generate PAR2-/- mice. Hemodynamic responses
to the PAR2-activating peptide SLIGRL and control peptide LSIGRL were
measured (25) and compared with control mice to confirm
the PAR2-/- phenotype (Fig. 1C).
Mice homozygous for a null mutation in the PAR2 gene were viable,
fertile, of normal size and vitality, and did not show any signs of
disease under specific pathogen-free vivarium conditions.
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The study protocol was approved by the animal research committee at the University of Virginia. Twelve male PAR2-deficient (PAR2-/-) mice and 14 littermate wild-type mice weighing between 24 and 32 g were anesthetized with an i.p. injection (12.5 µl/g) of a solution containing ketamine hydrocholoride (10 mg/ml), xylazine (1 mg/ml), and atropine (0.02 mg/ml). Body temperature was maintained at 37°C with a heating pad. A jugular vein was cannulated for administration of fluids and 0.1 mg pentobarbital approximately every 45 min as needed to maintain anesthesia. One carotid artery was cannulated for blood pressure monitoring. Blood samples were withdrawn from the carotid arterial catheter at the onset and conclusion of the experiment to analyze systemic leukocyte concentrations using hemocytometer (Fisher Scientific, Pittsburgh, PA) measurements of Kimura-stained samples.
Either the right or the left cremaster muscle was prepared for intravital microscopy, as previously described (6). The muscle was exteriorized through a scrotal incision and secured to a translucent pedestal. A longitudinal incision was made in the muscle, the edges were secured to the pedestal, and the epididymis and testicle were gently pinned to the side. The preparation was superfused continuously with isothermic bicarbonate-buffered saline.
Microscopic observations were made using an intravital microscope
(Axioskop FS; Zeis, Oberkochen, Germany) with a saline immersion
objective (SW 40/0.75 numerical aperture). Video recordings were made
using a high-resolution camera (VE-1000CD, Dage-MTI) connected to an
S-VHS recorder (Panasonic; Matsushita Electric Co.). Centerline venular
RBC velocities were measured using a dual photodiode (26)
and converted to mean blood flow velocities by multiplying by an
empirical factor of 0.625 (27). Shear rates
(
w) were determined as:
w =
2.12(8Vb)/d, where
Vb is the mean blood velocity,
d is the vessel diameter, and 2.12 is a correction factor
for the shape of the velocity profile (28).
Venular diameters were measured off-line using video calipers.
Freeze-frame advancing allowed tracking of individual rolling
leukocytes over a distance of 30–100 µm. The total distance traveled
was divided by the elapsed time to derive the mean rolling velocity.
The number of rolling leukocytes (rn) was
determined by counting leukocytes crossing a line perpendicular to the
vessel during 1 min. Leukocyte rolling flux fraction (F),
which reflects the percentage of leukocytes passing through a venule
that are rolling, was calculated by F =
rn/(0.25
d2 · Vb · 60 · CL),
where d is vessel diameter, Vb
is centerline blood velocity, and CL is
the systemic blood leukocyte concentration (6).
Eight wild-type and nine PAR2-/- mice were used
to assess leukocyte rolling in response to surgical trauma
(6). Intravital microscopic observations were initiated
within 8 min of exterioration of the cremaster muscle in all animals.
Venules with diameters between 25 and 40 µm were recorded under
transillumination for 
1 min each, followed by measurement of
centerline blood velocity. Video recordings and velocity measurements
were made in three to five different venules in each animal within
1 h after exterioration of the cremaster.
Peptide microinjection
Six wild-type and three PAR2-/- mice
were studied to assess leukocyte rolling and adhesion in response to
microinjections of a PAR2 agonist. Solutions (100 nM) containing either
the murine PAR2-specific hexapeptide agonist SLIGRL (18, 19) or an inactive scrambled control peptide LSIGRL in normal
saline were loaded into glass micropipettes, which were pulled from
standard borosilicate glass with an outer diameter of 1 mm (Stoelting,
Wood Dale, IL) on a vertical pipette puller (Stoelting). The blunt end
of each pipette was fire polished by briefly passing the end through a
flame. The tip of the pipette was then beveled using a micropipette
grinder (model EG-40; Narishige, Greenville, NY) with a 0.3-µm
abrasive foil (6775; AH Thomas). The pipettes were filled with 
10
µl of either SLIGRL or LSIGRL peptide solution. Tubing was threaded
through the pipette holder and then secured on the end of the filled
pipette. At its opposite end, the tubing was connected to a 10-ml
syringe and three-way stopcock system. The pipette holder was inserted
within a piezo-driven micromanipulator (model DC-3k
Märzhäuser-Wetzlar). The pipettes were positioned adjacent
(within 20 µm) to venules between 25 and 40 µm in diameter.
Solutions were microinjected into perivenular tissue for 30 s.
Video recordings and velocity measurements were made immediately before
and 2 min following injection of either SLIGRL or LSIGRL, which were
performed in random order. Up to five microinjections were performed
per animal.
Peritoneal inflammation
Peritonitis was created in five wild-type and five PAR2-/- mice by i.p. injection of 1 ml of autoclaved 4% thioglycolate broth. At 4 h, mice were sacrificed and the peritoneum was lavaged with 5 ml of PBS containing 5 mM EDTA. Cell counts were made using hemocytometer measurements of Kimura-stained samples, and cellular differentials were assessed on Wright-stained smears.
Flow cytometry
Human microvascular endothelial cells (HMEC-1) (29) were grown to confluence in cell culture flasks. The growth medium consisted of MCDB 131 (Life Technologies, Rockville, MD), 10 ng/ml epidermal growth factor (Collaborative Biomedical Products, Bedford, MA), 1 µg hydrocortisone, 10% FBS, 1% penicillin, 1% streptomycin, and 10 mM L-glutamine. Human peripheral blood (2 ml) containing 10 U/ml heparin was added to flasks containing HMEC-1 cells or without cells (control). Either SLIGRL or an inactive control peptide (final concentration, 100 nM) was added to flasks with and without HMEC-1 cells, and the suspensions were incubated for 45 min at 37°C. Cells in the suspension were separately stained using PE-conjugated mAbs against L-selectin (DREG56; PharMingen, San Diego, CA) and Mac-1 (ICRF44; PharMingen). RBC were lysed with a solution of 0.15 M NH4Cl, 0.01 M NaHCO3, and 0.001 M disodium EDTA. Cells were washed with BSA/PBS and fixed in 1% paraformaldehyde/PBS. Samples (104 cells) were analyzed by flow cytometry (FACScalibur; Becton Dickinson, Mountain View, CA) gated to the characteristic forward and side scatter of granulocytes. Results are displayed as histograms of PE fluorescence.
Statistical methods
Data are expressed as means ± SD. Differences in hemodynamic parameters, leukocyte counts, and leukocyte rolling or adhesion parameters were compared by Student’s t test. Interval comparisons of leukocyte rolling flux fraction at the different time points were made by repeated-measures ANOVA. Differences were considered significant at p < 0.05 (two sided).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A total of 64 venules was studied in wild-type mice and 54 venules
in PAR2-/- mice for experiments investigating
leukocyte rolling in response to surgical trauma. The mean venular
diameters, venular blood velocities, and shear rates were similar
between wild-type and PAR2-/- mice (Table I).
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Leukocyte rolling was present in the initial venule studied in all
animals, even at the earliest time point studied (4 min after
exteriorization of the cremaster muscle). Measurements of rolling
leukocyte flux fraction were grouped into the time intervals of 0–15,
16–30, and >30 min after exteriorization of the cremaster muscle,
providing an approximately even distribution of venules studied at each
interval (Fig. 2). In the
PAR2-/- mice, the mean leukocyte rolling flux
fraction at 0–15 min was significantly lower compared with that in
wild-type mice (12 ± 2 vs 30 ± 6%, p <
0.05). Although a significant reduction in leukocyte rolling flux
fraction in the mutant mice persisted at 16–30 min (14 ± 3 vs
34 ± 8%, p < 0.05), no difference was found
after 30 min. This was due to a relative increase in the flux fraction
in the mutant mice (Fig. 2).
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) illustrate that rolling velocity was
significantly higher in PAR2-/- compared with
wild-type mice only at the earliest time interval (0–15 min). At this
time interval, the mean leukocyte rolling velocities in
PAR2-/- and control mice were 67 ± 46 and
52 ± 36 µm/s (p < 0.01), respectively.
In PAR2-/- mice, mean rolling velocity
decreased with time such that there was no significant difference in
rolling velocities after 15 min (Fig. 2
).
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Perivenular microinjections of the active peptide agonist SLIGRL
were performed in five and four venules in wild-type and
PAR2-/- mice, respectively. Microinjections of
the inactive control peptide LSIGRL were performed in four venules in
wild-type mice. There were no differences in baseline vessel diameter,
blood flow velocity, or shear rate between the different groups (Table II). Blood flow velocity and shear rate
were unchanged 2 min following injection of either the active or
control peptide (Table II). Examples of images obtained by intravital
microscopy of a single venule in the cremaster muscle of a wild-type
mouse immediately before and 2 min following perivascular injection of
SLIGRL are shown in Fig. 4. These images
demonstrate extensive leukocyte recruitment to the venular endothelial
surface within 2 min of PAR2 activation. In wild-type mice, rolling
leukocyte flux fraction more than doubled (from 34 ± 11 to
71 ± 24%, p < 0.05) in venules following
treatment with SLIGRL, whereas flux fraction was unchanged with
injections of the inactive peptide LSIGRL (Fig. 5A). A minimal increase in
rolling leukocyte flux fraction (from 21 ± 8 to 30 ± 2%)
was found in PAR2-/- mice following injection
of SLIGRL (Fig. 5A).
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B),
and an increase in the number of adhered leukocytes (Fig. 5C). The mean leukocyte rolling velocity and number of
adhered leukocytes did not significantly change with microinjections of
either the control peptide in wild-type mice or of SLIGRL in
PAR2-/- mice (Fig. 5, B and
C). Peritoneal leukocyte recruitment
All mice survived to 4 h after induction of peritonitis. Total leukocyte counts in the peritoneal lavage at 4 h were not significantly different in wild-type compared with PAR2-/- mice (2.53 ± 0.49 vs 2.63 ± 0.85 [x 106] per ml, p = 0.82). The proportions of neutrophils, lympocytes, and monocytes/macrophages were also similar (data not shown).
Leukocyte activation by PAR2 agonist peptide
To investigate whether activation of PAR2 activates neutrophils,
expression of L-selectin and Mac-1 was assessed using flow cytometry.
Upon neutrophil activation, L-selectin is shed from the cell surface,
resulting in reduced levels of expression (30), whereas
Mac-1 surface expression is up-regulated (31). SLIGRL did
not change expression of L-selectin or Mac-1 on neutrophils (Fig. 6, top panels). When SLIGRL
was added to neutrophils incubated with HMEC-1 cells, L-selectin
expression was reduced and Mac-1 was up-regulated (bottom
panels). This suggests indirect neutrophil activation via
endothelial-derived mediators. Mac-1 and L-selectin expression were not
affected by the inactive peptide LSIGRL (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A direct test of the in vivo effect of PAR2 activation was achieved by injecting a specific PAR2-activating peptide, SLIGRL, corresponding to the N terminus of the cleaved receptor (18). SLIGRL, but not control peptide, elicited three distinct responses in wild-type mice. First, the number of rolling leukocytes in small venules per unit of time, or rolling leukocyte flux, increased. Second, leukocyte rolling velocity decreased by about 50%. Third, a significant number of rolling leukocytes became firmly adherent. The first two observations are entirely consistent with increased surface expression of P-selectin in response to SLIGRL (21).
P-selectin is known to be up-regulated by surgical trauma caused by exteriorization of the cremaster muscle for intravital microscopy (7, 8). Leukocyte rolling in venules of the mouse cremaster muscle is largely P-selectin dependent for at least 60 min (6, 32). In this model, leukocyte rolling flux reaches a peak at about 50 min, which most likely corresponds to the peak of P-selectin expression (6). In the present study, peptide microinjections were performed after allowing the tissue to rest for at least 60 min after surgical exteriorization, a time when leukocyte rolling begins to decrease and P-selectin expression is less than maximal (6). The 2-fold increase in leukocyte rolling flux fraction with SLIGRL microinjection in this study is greater than that recently observed in rat mesenteric venules during superfusion with a PAR2 agonist (23). The greater response to the agonist in the current study most likely represents differences in both the dose and route of peptide administration as well as differences in the tissue and species studied.
A reasonable explanation for increased leukocyte rolling flux and decreased rolling velocity in response to SLIGRL is that activation of PAR2 increases surface expression of P-selectin on endothelial cells (13, 21). Endothelial cells are known to express PAR2 and respond to receptor activation by a rise in intracellular free calcium and activation of phospholipase C (16). Moreover, PAR2 activation by the peptide, SLIGKV, causes mobilization of intracellular free calcium, degranulation of Weibel-Palade bodies, and surface expression of von Willebrand factor in HUVEC (33). Since both von Willebrand factor and P-selectin are stored in Weibel-Palade bodies (34), it is likely that P-selectin expression is induced through the same mechanism. The conclusion that PAR2 activation induced increased surface expression of endothelial P-selectin is further supported by the similarity of the in vivo responses to PAR2 activation and to histamine (35) or leukotriene C4 (LTC4) (36), interventions that are known to cause acute up-regulation of P-selectin surface expression in endothelial cells (33, 37). The observed reduction in leukocyte rolling velocity following injection of SLIGRL is also consistent with increased expression of P-selectin. Our data obtained after activation of PAR2 parallel those seen previously in response to LTC4, which caused a 50% reduction in leukocyte rolling velocity due to increased P-selectin expression (36).
The increase in leukocyte adhesion with SLIGRL is not readily explained
by increased P-selectin expression. Even at high site densities,
P-selectin does not support firm adhesion (38). However,
endothelial stimulation with SLIGRL may induce endothelial cells to
express other adhesion molecules and mediators relevant to leukocyte
trafficking. For example, endothelial cell activation with histamine,
thrombin, or LTC4 all cause expression of
platelet-activating factor (PAF) (37, 39). PAF is known to
activate rolling leukocytes in a juxtacrine fashion (39, 40), resulting in activation of 
2
integrins that promote firm leukocyte adhesion. Leukocyte migration
into the peritoneal cavity of rats following i.p. injection of PAR2
agonists has been shown to be inhibited by pretreatment with the PAF
receptor antagonist WEB 2086 (23). Although endothelial
cells can produce PAF (37, 39), and PAF receptor
antagonist prevents neutrophil recruitment in response to PAR2 agonist
(23), it is not clear whether endothelial cells produce a
relevant neutrophil-activating signal in response to PAR2 activation.
Accordingly, we investigated whether rapid leukocyte adhesion observed
following SLIGRL microinjection is mediated by endothelial-derived
mediators. Our flow cytometry data demonstrate leukocyte activation
when exposed to SLIGRL and cultured endothelial cells, but not to
SLIGRL or endothelial cells alone. This finding is consistent with
PAR2-induced expression of PAF and possibly other mediators that can
activate rolling leukocytes to promote firm adhesion. Although PAR2 has
been reported to be present on neutrophils (41), we did
not find evidence of direct neutrophil activation by SLIGRL as assessed
by 2 integrin expression or L-selectin
shedding measured by flow cytometry.
The generation and characterization of a PAR2 null mouse enabled us to study the impact of this receptor on the inflammatory response. Previous descriptions of the effects of a different null mutation in the PAR2 gene did not address the impact of the absence of PAR2 on inflammatory responses (42, 43). In this study, we show that leukocyte rolling is markedly attenuated in PAR2-/- mice, indicating that PAR2 makes a substantial contribution to the initial inflammatory response seen in wild-type mice. The rolling defect was not sustained beyond 30 min after exteriorization, a time point at which P-selectin-independent mechanisms of rolling become important (6). The absence of a sustained defect in leukocyte recruitment explains similar peritoneal inflammatory responses in wild-type and PAR2-/- mice 4 h following thioglycolate injection.
P-selectin-dependent rolling in venules of the mouse cremaster muscle and similar intravital microscopic preparations is induced by mast cell degranulation, and prevented by inhibition of mast cell degranulation (8, 44). Mast cells contain and, upon stimulation, release tryptase, a known activator of PAR2 (20). Mast cells also contain and release histamine, which also can induce P-selectin expression on endothelial cells (32). However, blocking histamine H1 or H2 receptors or both does not reduce trauma-induced leukocyte rolling (7, 8, 44), although histamine application can induce leukocyte rolling (7). This suggests that histamine is not the only and maybe not even the most important mediator released by mast cells that can activate endothelial cells. The present data suggest that mast cell-derived tryptase may be an important and early activator of endothelial cells in response to tissue trauma. In addition to sensing tryptase, PAR2 can be activated by the tissue factor/factor VIIa complex and by factor Xa (45). The ability of PAR2 to sense activation of the coagulation cascade provides another potential link between tissue trauma and cellular responses. PAR2 activation has been reported to promote vasodilation and sensitivity to endotoxin (46) as well as neurogenic inflammation (47). Our data show that PAR2 is an important receptor in inflammation and, indeed, appears necessary for some of the earliest inflammatory responses in vivo.
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Footnotes |
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2 Current address: Cardiovascular Division, University of Pennsylvania, Philadelphia, PA 19104-6069.
3 Address correspondence and reprint requests to Dr. Klaus Ley, Box 800759, Department of Biomedical Engineering, University of Virginia Medical Center, Charlottesville, VA 22908.
4 Abbreviations used in this paper: PAR, protease-activated receptor; HMEC, human microvascular endothelial cell; LTC4, leukotriene C4; PAF, platelet-activating factor.
Received for publication April 17, 2000. Accepted for publication August 29, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in vivo. Cell Adhes. Commun. 2:7.[Medline]
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Y. Kida, H. Inoue, T. Shimizu, and K. Kuwano Serratia marcescens Serralysin Induces Inflammatory Responses through Protease-Activated Receptor 2 Infect. Immun., January 1, 2007; 75(1): 164 - 174. [Abstract] [Full Text] [PDF]
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K. Hirano The Roles of Proteinase-Activated Receptors in the Vascular Physiology and Pathophysiology Arterioscler Thromb Vasc Biol, January 1, 2007; 27(1): 27 - 36. [Abstract] [Full Text] [PDF]
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 C A Dinarello and S-H Kim IL-32, a novel cytokine with a possible role in disease Ann Rheum Dis, November 1, 2006; 65(suppl_3): iii61 - iii64. [Abstract] [Full Text] [PDF]
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F. Cattaruzza, N. Cenac, E. Barocelli, M. Impicciatore, E. Hyun, N. Vergnolle, and C. Sternini Protective Effect of Proteinase-Activated Receptor 2 Activation on Motility Impairment and Tissue Damage Induced by Intestinal Ischemia/Reperfusion in Rodents Am. J. Pathol., July 1, 2006; 169(1): 177 - 188. [Abstract] [Full Text] [PDF]
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E. Camerer, I. Cornelissen, H. Kataoka, D. N. Duong, Y.-W. Zheng, and S. R. Coughlin Roles of protease-activated receptors in a mouse model of endotoxemia Blood, May 15, 2006; 107(10): 3912 - 3921. [Abstract] [Full Text] [PDF]
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W. Ruf Flow perturbation is linked to endothelial par signaling. Arterioscler Thromb Vasc Biol, May 1, 2006; 26(5): 962 - 964. [Full Text] [PDF]
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 H. T. Abey, D. P. Fairlie, J. D. Moffatt, R. W. Balzary, and T. M. Cocks Protease-Activated Receptor-2 Peptides Activate Neurokinin-1 Receptors in the Mouse Isolated Trachea J. Pharmacol. Exp. Ther., May 1, 2006; 317(2): 598 - 605. [Abstract] [Full Text] [PDF]
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 M. Chignard and D. Pidard Neutrophil and Pathogen Proteinases versus Proteinase-Activated Receptor-2 Lung Epithelial Cells: More Terminators than Activators. Am. J. Respir. Cell Mol. Biol., April 1, 2006; 34(4): 394 - 398. [Abstract] [Full Text] [PDF]
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 D. Novick, M. Rubinstein, T. Azam, A. Rabinkov, C. A. Dinarello, and S.-H. Kim Proteinase 3 is an IL-32 binding protein PNAS, February 28, 2006; 103(9): 3316 - 3321. [Abstract] [Full Text] [PDF]
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T. Trian, P.-O. Girodet, O. Ousova, R. Marthan, J. M. Tunon-de-Lara, and P. Berger RNA Interference Decreases PAR-2 Expression and Function in Human Airway Smooth Muscle Cells Am. J. Respir. Cell Mol. Biol., January 1, 2006; 34(1): 49 - 55. [Abstract] [Full Text] [PDF]
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U. Johansson, C. Lawson, M. Dabare, D. Syndercombe-Court, A. C. Newland, G. L. Howells, and M. G. Macey Human peripheral blood monocytes express protease receptor-2 and respond to receptor activation by production of IL-6, IL-8, and IL-1{beta} J. Leukoc. Biol., October 1, 2005; 78(4): 967 - 975. [Abstract] [Full Text] [PDF]
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A. Hezi-Yamit, P. W. Wong, N. Bien-Ly, L. G. Komuves, K. S. S. Prasad, D. R. Phillips, and U. Sinha Synergistic induction of tissue factor by coagulation factor Xa and TNF: Evidence for involvement of negative regulatory signaling cascades PNAS, August 23, 2005; 102(34): 12077 - 12082. [Abstract] [Full Text] [PDF]
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X. Su, E. Camerer, J. R. Hamilton, S. R. Coughlin, and M. A. Matthay Protease-Activated Receptor-2 Activation Induces Acute Lung Inflammation by Neuropeptide-Dependent Mechanisms J. Immunol., August 15, 2005; 175(4): 2598 - 2605. [Abstract] [Full Text] [PDF]
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S. Dulon, D. Leduc, G. S. Cottrell, J. D'Alayer, K. K. Hansen, N. W. Bunnett, M. D. Hollenberg, D. Pidard, and M. Chignard Pseudomonas aeruginosa Elastase Disables Proteinase-Activated Receptor 2 in Respiratory Epithelial Cells Am. J. Respir. Cell Mol. Biol., May 1, 2005; 32(5): 411 - 419. [Abstract] [Full Text] [PDF]
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Y.C. G. Lee, D. A. Knight, K. B. Lane, D. S. Cheng, M. A. Koay, L. R. Teixeira, J. C. Nesbitt, R. C. Chambers, P. J. Thompson, and R. W. Light Activation of proteinase-activated receptor-2 in mesothelial cells induces pleural inflammation Am J Physiol Lung Cell Mol Physiol, April 1, 2005; 288(4): L734 - L740. [Abstract] [Full Text] [PDF]
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 Y. Hirota, Y. Osuga, T. Hirata, K. Koga, O. Yoshino, M. Harada, C. Morimoto, E. Nose, T. Yano, O. Tsutsumi, et al. Evidence for the Presence of Protease-Activated Receptor 2 and Its Possible Implication in Remodeling of Human Endometrium J. Clin. Endocrinol. Metab., March 1, 2005; 90(3): 1662 - 1669. [Abstract] [Full Text] [PDF]
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K. Page, V. S. Hughes, K. K. Odoms, K. E. Dunsmore, and M. B. Hershenson German Cockroach Proteases Regulate Interleukin-8 Expression via Nuclear Factor for Interleukin-6 in Human Bronchial Epithelial Cells Am. J. Respir. Cell Mol. Biol., March 1, 2005; 32(3): 225 - 231. [Abstract] [Full Text] [PDF]
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 M. Steinhoff, J. Buddenkotte, V. Shpacovitch, A. Rattenholl, C. Moormann, N. Vergnolle, T. A. Luger, and M. D. Hollenberg Proteinase-Activated Receptors: Transducers of Proteinase-Mediated Signaling in Inflammation and Immune Response Endocr. Rev., February 1, 2005; 26(1): 1 - 43. [Abstract] [Full Text] [PDF]
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A. Uehara, Y. Sugawara, T. Sasano, H. Takada, and S. Sugawara Proinflammatory Cytokines Induce Proteinase 3 as Membrane-Bound and Secretory Forms in Human Oral Epithelial Cells and Antibodies to Proteinase 3 Activate the Cells through Protease-Activated Receptor-2 J. Immunol., September 15, 2004; 173(6): 4179 - 4189. [Abstract] [Full Text] [PDF]
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V. M. Shpacovitch, G. Varga, A. Strey, M. Gunzer, F. Mooren, J. Buddenkotte, N. Vergnolle, C. P. Sommerhoff, S. Grabbe, V. Gerke, et al. Agonists of proteinase-activated receptor-2 modulate human neutrophil cytokine secretion, expression of cell adhesion molecules, and migration within 3-D collagen lattices J. Leukoc. Biol., August 1, 2004; 76(2): 388 - 398. [Abstract] [Full Text] [PDF]
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E. Camerer, A. A. Qazi, D. N. Duong, I. Cornelissen, R. Advincula, and S. R. Coughlin Platelets, protease-activated receptors, and fibrinogen in hematogenous metastasis Blood, July 15, 2004; 104(2): 397 - 401. [Abstract] [Full Text] [PDF]
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N. Mackman Role of Tissue Factor in Hemostasis, Thrombosis, and Vascular Development Arterioscler Thromb Vasc Biol, June 1, 2004; 24(6): 1015 - 1022. [Abstract] [Full Text] [PDF]
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 Y. Dai, T. Moriyama, T. Higashi, K. Togashi, K. Kobayashi, H. Yamanaka, M. Tominaga, and K. Noguchi Proteinase-Activated Receptor 2-Mediated Potentiation of Transient Receptor Potential Vanilloid Subfamily 1 Activity Reveals a Mechanism for Proteinase-Induced Inflammatory Pain J. Neurosci., May 5, 2004; 24(18): 4293 - 4299. [Abstract] [Full Text] [PDF]
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 V. S. OSSOVSKAYA and N. W. BUNNETT Protease-Activated Receptors: Contribution to Physiology and Disease Physiol Rev, April 1, 2004; 84(2): 579 - 621. [Abstract] [Full Text] [PDF]
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R. Pawlinski, B. Pedersen, G. Schabbauer, M. Tencati, T. Holscher, W. Boisvert, P. Andrade-Gordon, R. D. Frank, and N. Mackman Role of tissue factor and protease-activated receptors in a mouse model of endotoxemia Blood, February 15, 2004; 103(4): 1342 - 1347. [Abstract] [Full Text] [PDF]
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 S. SEELIGER, C. K. DERIAN, N. VERGNOLLE, N. W. BUNNETT, R. NAWROTH, M. SCHMELZ, P.-Y. VON DER WEID, J. BUDDENKOTTE, C. SUNDERKOTTER, D. METZE, et al. Proinflammatory role of proteinase-activated receptor-2 in humans and mice during cutaneous inflammation in vivo FASEB J, October 1, 2003; 17(13): 1871 - 1885. [Abstract] [Full Text] [PDF]
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S. J. Bolton, C. A. McNulty, R. J. Thomas, C. R. A. Hewitt, and A. J. Wardlaw Expression of and functional responses to protease-activated receptors on human eosinophils J. Leukoc. Biol., July 1, 2003; 74(1): 60 - 68. [Abstract] [Full Text] [PDF]
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C. D. Major, R. J. Santulli, C. K. Derian, and P. Andrade-Gordon Extracellular Mediators in Atherosclerosis and Thrombosis: Lessons From Thrombin Receptor Knockout Mice Arterioscler Thromb Vasc Biol, June 1, 2003; 23(6): 931 - 939. [Abstract] [Full Text] [PDF]
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A. Uehara, K. Muramoto, H. Takada, and S. Sugawara Neutrophil Serine Proteinases Activate Human Nonepithelial Cells to Produce Inflammatory Cytokines Through Protease-Activated Receptor 2 J. Immunol., June 1, 2003; 170(11): 5690 - 5696. [Abstract] [Full Text] [PDF]
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W. C. Aird The role of the endothelium in severe sepsis and multiple organ dysfunction syndrome Blood, May 15, 2003; 101(10): 3765 - 3777. [Abstract] [Full Text] [PDF]
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S. Dulon, C. Cande, N. W. Bunnett, M. D. Hollenberg, M. Chignard, and D. Pidard Proteinase-Activated Receptor-2 and Human Lung Epithelial Cells: Disarming by Neutrophil Serine Proteinases Am. J. Respir. Cell Mol. Biol., March 1, 2003; 28(3): 339 - 346. [Abstract] [Full Text] [PDF]
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F. Schmidlin, S. Amadesi, K. Dabbagh, D. E. Lewis, P. Knott, N. W. Bunnett, P. R. Gater, P. Geppetti, C. Bertrand, and M. E. Stevens Protease-Activated Receptor 2 Mediates Eosinophil Infiltration and Hyperreactivity in Allergic Inflammation of the Airway J. Immunol., November 1, 2002; 169(9): 5315 - 5321. [Abstract] [Full Text] [PDF]
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V. Temkin, B. Kantor, V. Weg, M.-L. Hartman, and F. Levi-Schaffer Tryptase Activates the Mitogen-Activated Protein Kinase/Activator Protein-1 Pathway in Human Peripheral Blood Eosinophils, Causing Cytokine Production and Release J. Immunol., September 1, 2002; 169(5): 2662 - 2669. [Abstract] [Full Text] [PDF]
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A. F. Milia, M. B. Salis, T. Stacca, A. Pinna, P. Madeddu, M. Trevisani, P. Geppetti, and C. Emanueli Protease-Activated Receptor-2 Stimulates Angiogenesis and Accelerates Hemodynamic Recovery in a Mouse Model of Hindlimb Ischemia Circ. Res., August 23, 2002; 91(4): 346 - 352. [Abstract] [Full Text] [PDF]
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E. Camerer, H. Kataoka, M. Kahn, K. Lease, and S. R. Coughlin Genetic Evidence That Protease-activated Receptors Mediate Factor Xa Signaling in Endothelial Cells J. Biol. Chem., May 3, 2002; 277(18): 16081 - 16087. [Abstract] [Full Text] [PDF]
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 J. E Cuffe, M. Bertog, S. Velazquez-Rocha, O. Dery, N. Bunnett, and C. Korbmacher Basolateral PAR-2 receptors mediate KCl secretion and inhibition of Na+ absorption in the mouse distal colon J. Physiol., February 15, 2002; 539(1): 209 - 222. [Abstract] [Full Text] [PDF]
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 S.R. COUGHLIN Protease-activated Receptors in the Cardiovascular System Cold Spring Harb Symp Quant Biol, January 1, 2002; 67(0): 197 - 208. [Abstract] [PDF]
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 S. R. Macfarlane, M. J. Seatter, T. Kanke, G. D. Hunter, and R. Plevin Proteinase-Activated Receptors Pharmacol. Rev., June 1, 2001; 53(2): 245 - 282. [Abstract] [Full Text] [PDF]
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J. R. Hamilton, A. G. Frauman, and T. M. Cocks Increased Expression of Protease-Activated Receptor-2 (PAR2) and PAR4 in Human Coronary Artery by Inflammatory Stimuli Unveils Endothelium-Dependent Relaxations to PAR2 and PAR4 Agonists Circ. Res., July 6, 2001; 89(1): 92 - 98. [Abstract] [Full Text] [PDF]
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