Published online before print February 10, 2003, doi: 10.1161/01.CIR.0000050620.37260.75
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(Circulation. 2003;107:954.)
© 2003 American Heart Association, Inc.
Clinical Investigation and Reports |
Protease-Activated Receptor 2–Mediated Vasodilatation in Humans In Vivo
Role of Nitric Oxide and Prostanoids
From the Centre for Clinical Pharmacology & Therapeutics (J.R., R.K., P.M., P.V.) and The Wolfson Institute for Biomedical Research (R.C.), University College London, London, UK.
Correspondence to Dr Jonathan Robin, Centre for Clinical Pharmacology, BHF Laboratories, University College London, The Rayne Building, 5 University St, London WC1E 6JJ, UK. E-mail j.robin{at}ucl.ac.uk
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Abstract |
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Background— Systemic hypotension as a consequence of vascular dysfunction is a well-recognized and important feature of critical illness. Although serine protease activation has been implicated as a cause of vascular dysfunction in systemic inflammation, the mechanism is unknown. Recently, a class of receptors with an entirely novel mechanism of action, protease-activated receptors (PARs), has been identified that would explain the link between protease activation and systemic hypotension. Our aim was to test the hypothesis that in vivo activation of protease-activated receptor 2 (PAR-2) in humans would mediate vasodilatation.
Methods and Results— For these first-in-human studies, an activating peptide for the human PAR-2 receptor was synthesized and administered to healthy volunteers. Using both the dorsal hand vein technique and forearm plethysmography, we studied the effects of PAR-2 activation in human blood vessels and investigated the mechanism of vasodilation. Activation of PAR-2 receptors in vivo dilated human blood vessels in a dose-dependent manner, and the effects were reduced by inhibition of both nitric oxide and prostanoid synthesis
Conclusions— These findings demonstrate that serine protease activity can cause human vasodilation and provide a possible explanation of why serine protease activation in critical illness is associated with vascular dysfunction.
Key Words: nitric oxide • prostaglandins • receptors • vasodilation
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Introduction |
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Activation of serine proteases is a cardinal feature of the systemic inflammatory response and has been implicated as a cause of vascular collapse in sepsis and acute pancreatitis, but the mechanism by which protease activation affects blood pressure is not known. Recently, a novel mechanism has been identified that would explain the link between protease activation and vasodilatation.
Protease-activated receptors (PARs) form a class of G-protein–coupled receptors with a unique mechanism of action.1 Rather than being activated by reversible binding of a ligand, they auto-activate when part of the receptor undergoes proteolysis. Serine protease enzymes are able to activate the receptor by removal of the N-terminal sequence, unmasking a "tethered ligand," which then binds to a ligand-recognition domain on the receptor.2 A short synthetic peptide with an identical sequence to the tethered ligand (an "activating peptide") can also activate the receptor without the requirement for prior proteolysis. Use of such an activating peptide provides the opportunity to probe the vascular consequences of protease activation.3
Four PARs have been discovered, of which PAR-1 (the classic thrombin receptor) and PAR-2 (containing a trypsin-sensitive cleavage site) are the best characterized.4,5 However, despite the potential importance of these pathways in systemic inflammatory disorders in which proteases are activated, little is known of the consequences of PAR activation in human blood vessels, and there have been no studies in vivo. The aim of the present study was to explore the vascular effects of trypsin or other serine protease activity in humans by utilizing the PAR-2 activating peptide. Our hypothesis was that activation of PAR-2 would cause vasodilation. Insight into the mechanism of such an effect would be of value in understanding the pathophysiology of sepsis, acute pancreatitis, cardiopulmonary bypass, and other states associated with serine protease activation.
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Methods |
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Studies were approved by the local ethics committee and were undertaken on healthy male and female volunteers who gave written informed consent.
Peptide Synthesis
PAR-2 activating peptide, SLIGKV-NH2, and the reverse-sequence control peptide VKGILS-NH2 were synthesized in-house with manual 9-fluorenylmethoxycarbonyl (Fmoc) chemistry on a rink amide 4-Methylbenzhydrylamine polystyrene resin, according to published methodology.6 The completed peptide sequence was cleaved from the resin with 95% trifluoroacetic acid, 2.5% tri-isopropylsilane, and water, followed by precipitation and washing in tert-butyl methyl ether at -20°C. Peptide identity was confirmed by electrospray mass spectrometry and the compound purified by reverse-phase high-performance liquid chromatography. The purified compound was washed 3 times in 15% acetic acid and freeze-dried to produce a lyophilized powder. Before each study, aliquots of the lyophilized peptide were dissolved in saline (0.9% wt/vol) and sterilized by filtration.
Assessment of PAR-2 Activation in Veins
Subjects were asked to lie supine in a temperature-controlled laboratory (28°C to 30°C). A congesting cuff was placed around the upper arm and inflated to 40 mm Hg. Drugs or saline were given continuously by a constant-rate infusion pump delivering 0.25 mL/min through a 23-gauge needle placed in a dorsal hand vein elevated above the level of the heart. The internal diameter of the vein was measured 10 to 15 mm downstream from the tip of the infusion needle by recording the linear displacement of a lightweight probe placed on the skin overlying the summit of the vessel when the pressure in the congesting cuff was lowered from 40 to 0 mm Hg, as described previously.3 In all studies, saline was infused for at least 30 minutes until a stable baseline vein diameter was recorded. In resting subjects, in a warm environment, superficial veins have no intrinsic tone. To observe dilator responses, veins were preconstricted with a continuous infusion of norepinephrine (31.25 to 1000 pmol/min) sufficient to reduce the diameter to <50% of its baseline value (mean preconstriction 33%). Dose-response curves to infusions of SLIGKV-NH2 were constructed (1.6, 20, and 160 nmol/min, each dose for 3 minutes). The doses and duration of infusions used to construct the dose-response curves were determined by preliminary experiments (data not shown). Each dose of SLIGKV-NH2 was separated by 5 minutes to allow the vein to return to its baseline diameter. The subsequent increase in vein size was expressed as a percentage reversal from the preconstricted diameter: Percentage reversal = (Vd-Vc)/(Vb-Vc)x100, where Vd is diameter during infusion of norepinephrine and SLIGKV-NH2, Vc is diameter during infusion of norepinephrine alone, and Vb is basal vein diameter.7
Assessment of PAR-2 Activation in Arterial Circulation
Mercury-in-silastic strain-gauge plethysmography was used to measure forearm blood flow (mL/100 mL forearm per minute) in both arms as described previously.8 The brachial artery of the nondominant arm was cannulated with a 27-gauge needle inserted under local anesthesia (2 mL of 1% lignocaine). Drugs or saline were infused continuously by a constant-rate infusion pump that delivered 0.5 mL/min. During recording periods, the hands were excluded from the circulation by inflation of wrist cuffs to 200 mm Hg. Subjects were allowed to rest for 30 minutes after cannulation before measurements were made. Baseline blood flow was determined in two 5-minute recording periods separated by 10 minutes. The ratio of flow in the infused/noninfused (control) arm was calculated for each measurement period. Vasodilator responses were expressed as the percentage increase in the ratio of forearm blood flow (infused/noninfused arm) relative to baseline flow.
Construction of Dose-Response Curves to SLIGKV-NH2
In 18 subjects, dose-response curves to SLIGKV-NH2 were constructed (16, 800, and 1600 nmol/min, each dose for 3 minutes). The doses used to construct the dose-response curves and the duration of each dose were determined by preliminary experiments (data not shown).
Pharmacological Response to Reverse-Sequence Peptide VKGILS-NH2
To demonstrate that a peptide with an identical amino acid composition but a different amino acid sequence had no effect, the above protocol was repeated in 4 subjects with the reverse-sequence peptide VKGILS-NH2.
Effect of Multiple Dose-Response Curves to SLIGKV-NH2 Within a Single Study
Previous animal studies have suggested that receptor desensitization might occur with either prolonged administration and/or large doses of SLIGKV. Because subsequent studies investigating the mechanism of vasodilation to SLIGKV-NH2 required the construction of 2 dose-response curves within the time course of a single study, it was necessary to establish that no receptor desensitization occurred after the first dose-response curve. Hence, in 6 subjects, 3 dose-response curves to SLIGKV-NH2 (16, 800, and 1600 nmol/min, each dose for 3 minutes) were constructed. Each dose-response curve was separated by 20 minutes.
Effect of Inhibition of Nitric Oxide Synthesis
In 6 subjects, after construction of a baseline dose-response curve, the NO clamp technique was used.9 N-monomethyl-L-arginine (L-NMMA; Clinalfa AG) 4 µmol/min was infused to inhibit vascular NO production. After 10 minutes, nitroglycerin (NTG; 0.5 to 2 nmol/min) was coinfused to restore flow to baseline. After 15 minutes of a constant dose of NTG, a further dose-response curve to SLIGKV-NH2 (16, 800, and 1600 nmol/min, each dose for 3 minutes) was constructed.
Effect of Inhibition of Vascular Prostanoid Synthesis
In separate experiments in 6 subjects, after construction of a baseline dose-response curve, 1 g of aspirin (Aspergic, Synthelabo) was administered intravenously over 10 minutes. After a further period of 20 minutes, another dose-response curve to SLIGKV-NH2 (16, 800, and 1600 nmol/min, each dose for 3 minutes) was constructed.
Statistical Analysis
All data are expressed as mean±SEM and compared as stated. P<0.05 was considered statistically significant. Where responses are expressed as an area under the curve (AUC), this was calculated by the simple trapezoidal rule.
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Results |
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Safety and Tolerability
In all subjects, infusion of SLIGKV-NH2 was well tolerated, and no ill effects were detected.
SLIGKV-NH2 in Veins and Arterial Circulation
In the dorsal hand vein, SLIGKV-NH2 caused a dose-dependent dilatation, such that the percentage reversal of preconstriction was 16±1.3 (1.6 nmol/min), 31±10 (20 nmol/min), and 51±8 (160 nmol/min; P=0.006, 1-way ANOVA; P=0.004, posttest for linear trend; Figure 1A). In forearm resistance vessels, SLIGKV-NH2 caused a dose-dependent dilatation, such that the percentage change in forearm flow was 28±17 (160 nmol/min), 68±30 (800 nmol/min), and 98±32 (1600 nmol/min; P<0.0001, repeated-measures ANOVA; Figure 1B).
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) dilated human forearm resistance vessels in dose-dependent manner (n=18, P<0.0001). Reverse-sequence peptide VKGILS-NH2 (
) had no effect (n=4, P=1.0). Data are expressed as mean±SEM and compared by repeated-measures ANOVA. C, In 6 subjects, no desensitization was observed in 3 consecutive dose-response curves to SLIGKV-NH2, each separated by 20 minutes (n=6, P=0.18). First (
Pharmacological Response to Reverse-Sequence Peptide VKGILS-NH2
In forearm resistance vessels, VKGILS-NH2 had no effect, such that the percentage change in forearm flow was -6±6 (160 nmol/min), 8±8 (800 nmol/min), and 3±3 (1600 nmol/min; P=1.0, repeated-measures ANOVA; Figure 1B).
Repeated Doses of SLIGKV-NH2
No desensitization was observed in 3 consecutive dose-response curves to SLIGKV-NH2. The percentage changes in forearm flow for the first curve were 38±12 (160 nmol/min), 76±17 (800 nmol/min), and 122±33 (1600 nmol/min), and for the third curve, they were 19±8 (160 nmol/min), 85±29 (800 nmol/min), and 118±44 (1600 nmol/min; P=0.18, repeated-measures ANOVA; Figure 1C). When analyzed by the AUC method, there was a 3% reduction in AUC between the first and third curves that also was not statistically significant (P=0.88, paired t test).
Inhibition of Nitric Oxide Synthesis
As expected, L-NMMA reduced basal blood flow in the infused arm by 26±4% (P=0.006, paired t test); this was returned to baseline by NTG (mean dose 0.5 nmol/min) such that after the NO clamp had been established, there was no significant difference in forearm flow compared with baseline (change in forearm flow ratio 5±8%; P=0.66, paired t test). In the presence of an NO clamp, dose-dependent dilation to SLIGKV-NH2 was reduced by 69% (AUC method, P=0.05, paired t test; Figure 2).
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Inhibition of Prostaglandin Synthesis
Systemic aspirin administration did not significantly change baseline blood flow (change in absolute flow -6±11% for the infused arm; P=0.66, paired t test). In forearm resistance vessels, systemic aspirin significantly reduced dose-dependent dilation to SLIGKV-NH2 by 31% (AUC method, P=0.03, paired t test; Figure 3).
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Discussion |
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The results of this study demonstrate that PAR-2 activation by SLIGKV-NH2 causes arterial and venous dilation in humans and that the effects are reduced by inhibition of nitric oxide (NO) or prostaglandin synthesis. Because PAR-2 is activated by trypsin and other serine proteases, these results provide an explanation of why serine protease activation causes vasodilation. They also contribute to an understanding of the mechanism of inflammation-induced NO and prostanoid release in humans.
No studies on PAR-2 have previously been performed in humans in vivo, and to the best of our knowledge, only 2 human arterial preparations have been studied. In contrast to the present study, PAR-2 activation in human coronary10 or pulmonary artery11 segments failed to produce any significant vascular responses under basal conditions. This apparent discrepancy with the present results might be explained by differences in receptor expression, either between organs or between conduit vessels and the resistance arteries and the veins that we studied. However, basal expression of PAR-2 was demonstrated in both coronary and pulmonary artery preparations, which suggests that the absence of PAR-2 responses in those studies was due to functional uncoupling of the receptor from dilator mechanisms. A possible explanation is that both studies used explanted vessels from transplant patients, who were necessarily both chronically sick and had received extensive pharmacological therapy. The present results clearly show that PAR-2 activation causes vasodilation in healthy humans in vivo, and in this respect, the results are similar to those seen in experimental animals.12,13
Interestingly, in studies of isolated human vessels, pretreatment with either interleukin-1 (IL-1) or tumor necrosis factor-
(TNF-) upregulated PAR-2 expression and, in the coronary artery, revealed endothelium-dependent vasodilatation to PAR-2–activating peptides.14 This response was abolished by either removal of the endothelium or combined treatment with N-nitro-L-arginine (an NO synthase [NOS] inhibitor) and indomethacin.
The results we report are consistent with the dilator effects seen in animal tissues in vivo and show that PAR-2 is expressed basally and can be activated in healthy humans in arterial and venous beds. The dose ratio required to dilate the vein and the forearm suggest a mixed arteriovenous effect. Although responses to SLIGKV-NH2 were consistent within individuals, we observed considerable heterogeneity between individuals (range of percentage change in forearm blood flow for SLIGKV-NH2 1600 nmol/min, -22% to 211%). From the results, we cannot tell whether the source of the variability is genetic or environmental. However, a polymorphic form of human PAR-2 with a single amino acid substitution in the ligand-recognition domain of the receptor has been characterized (PAR-2F240S) with allelic frequencies of 0.916 and 0.084 for the common and rare alleles, respectively.15 In vitro, PAR-2 receptors with this variation display reduced sensitivity to trypsin and differential responses to PAR-activating peptides. Although the allele frequency of this polymorphism makes it unlikely that it would account for all of the heterogeneity observed in subjects in the present study, unrecognized polymorphisms might also affect the dilator response. Whatever the mechanism, substantial interindividual differences in the responses to the activating peptide suggest that the same would be true for vascular responses to protease activation. This may be of clinical importance, because it is well recognized that it is difficult to predict which individuals will show cardiovascular collapse in response to similar inflammatory stimuli. We cannot tell whether some of the variability seen in the present study was due to changes in basal inflammatory state between individuals, and this would be interesting to test in future studies.
After administration of L-NMMA, a competitive inhibitor of NOS, the dilator response to SLIGKV-NH2 was almost abolished. The dose of L-NMMA used has been demonstrated to inhibit endothelial NO production.16 Because resistance vessels produce NO continuously, infusion of L-NMMA produces vasoconstriction and thereby reduces resting forearm blood flow. By using the NO clamp technique, using the NO donor NTG to restore baseline flow and NO levels during infusion of L-NMMA, we were able to ensure that our results were not confounded by the reduction in baseline flow caused by L-NMMA.9 Because the study was undertaken in healthy volunteers, it is most likely that the isoform of NOS that was activated was constitutively expressed endothelial NOS (eNOS), an isoform that has increasingly been implicated in the NO-mediated dilation that occurs in humans in response to sepsis or systemic inflammation (in contrast to the predominant role of inducible NOS in rodent models).17 In addition to the effects of NOS inhibition, blockade of cyclo-oxygenase with aspirin attenuated the dilator response to SLIGKV-NH2. This is unusual, because aspirin rarely alters dilator responses to other endothelium-dependent agonists in the forearm of healthy individuals.18 Thus, both prostanoids and NO mediate PAR-2 vasodilation in humans, and the results suggest that combined NOS and cyclo-oxygenase inhibition might abolish the response to SLIGKV.
In animals, studies on mechanisms of vasodilation after PAR-2 activation have demonstrated NO-dependent and NO-independent mechanisms.19 In some studies, prostanoids have been implicated as an NO-independent mechanism,20,21 and in rat microvasculature22–24 and mouse mesenteric arterioles,21 an endothelium-derived hyperpolarizing factor–like response may contribute. It is unclear how much of the variability relates to species differences and how much to variation between vascular beds. Responses may also vary according to whether trypsin or an activating peptide is used as a pharmacological probe.20 The present study clearly shows that in humans, there is a major role for NO in PAR-2–mediated vasodilation, at least in the forearm arterial bed.
Several potential endogenous activators of PAR-2 have been identified. Experimentally, PAR-2 may be activated by trypsin25 (which may be extrapancreatic in origin26), mast cell tryptase,27 and coagulation factors VIIa28 and Xa.28,29 Moreover, the activator need not necessarily be endogenously derived, as evidenced by the recent demonstration of PAR-2 activation by a bacterial protease.30 For the present experiments, we chose to activate PAR-2 using SLIGKV-NH2 rather than a serine protease such as a trypsin. Although this decision was principally based on ethical considerations, SLIGKV-NH2 has other advantages. Whereas trypsin at high concentrations may also activate other members of the PAR family, SLIGKV-NH2 is a specific activator of PAR-2. Because antagonists capable of preventing SLIGKV-NH2–mediated activation of PAR-2 were not available, we were not able to demonstrate that vasodilation could be inhibited by PAR-2 receptor blockade. However, studies in animals and in vitro have shown that responses to SLIGKV-NH2 are specific and selective for PAR-2, and scrambled peptides are devoid of effect.31 Using the reverse-sequence peptide VKGILS-NH2 as a control, we were able to demonstrate that a peptide with an identical amino acid composition but a different sequence to SLIGKV-NH2 had no effect.
Clinical Implications
What are the potential clinical implications of this study, which identifies a mechanism that links serine protease activation, NO release, prostanoid production, and vasodilatation in humans? PAR-2 activation by circulating, locally generated, or bacterial serine proteases might contribute to the vascular dysfunction observed in human systemic inflammatory responses and sepsis and provides a novel therapeutic target. Interestingly, vascular PAR-2 expression is upregulated by TNF-, IL-1, and lipopolysaccharide,14,32 which suggests that PAR-2 dilator responses may be further enhanced through receptor upregulation in systemic inflammation. Indeed, in a rat model, pretreatment with lipopolysaccharide substantially enhanced the sensitivity to the hypotensive response elicited by systemic administration of PAR-2–activating peptide.20 Septic patients have a significant elevation in both circulating immunoreactive trypsin and endogenous trypsin inhibitors,33 and immunoreactive trypsin levels have also been shown to correlate with risk of complications such as adult respiratory distress syndrome.34 Circulating levels of trypsin are also elevated in the systemic inflammatory response observed in human acute pancreatitis, and both the overall severity of the illness35 and the development of associated acute lung injury correlate with trypsin activity in a rat model of the disease.36
The present study clearly shows that PAR-2 activation that would accompany trypsin (or other serine protease) activity will cause arterial and venous dilation, and the magnitude of the effect will vary between individuals. It identifies PAR-2 activation as a novel potential pathophysiological mechanism of vasodilatation in humans.
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Acknowledgments |
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This work was supported by grants from the Special Trustees of the Middlesex Hospital and The Wellcome Trust. Dr Kharbanda was funded by a British Heart Foundation fellowship.
Received June 21, 2002; revision received November 5, 2002; accepted November 6, 2002.
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 A. K Chan, N. Vergnolle, M. D Hollenberg, and P.-Y. von der Weid Proteinase-activated receptor 2 activation modulates guinea-pig mesenteric lymphatic vessel pacemaker potential and contractile activity J. Physiol., October 15, 2004; 560(2): 563 - 576. [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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P. E. Szmitko, C.-H. Wang, R. D. Weisel, G. A. Jeffries, T. J. Anderson, and S. Verma Biomarkers of Vascular Disease Linking Inflammation to Endothelial Activation: Part II Circulation, October 28, 2003; 108(17): 2041 - 2048. [Full Text] [PDF]
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D. K.W. Chew, J. M. Orshal, and R. A. Khalil Elastase-Induced Suppression of Endothelin-Mediated Ca2+ Entry Mechanisms of Vascular Contraction Hypertension, October 1, 2003; 42(4): 818 - 824. [Abstract] [Full Text] [PDF]
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