Systemic Vascular Effects of Thrombin and Thrombin Receptor Activating Peptide in Rats

Thrombosis Research 101 (2001) 467 ± 475

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Systemic Vascular Effects of Thrombin and Thrombin Receptor Activating Peptide in Rats Thomas Sicker, Frank Wuchold, Brigitte Kaiser and Erika Glusa Unit of Experimental Angiology, Center for Vascular Biology and Medicine, Faculty of Medicine, Friedrich Schiller University Jena, NordhaÈuser Str. 78, D-99089 Erfurt, Germany (Received 20 March 2000 by Editor D.L. Heene; revised/accepted 22 November 2000)

Abstract The proteolytic enzyme thrombin activates its receptor by cleavage of a peptide from the extracellular N-terminus. The newly generated N-terminus acts as a tethered ligand to activate the receptor. Receptor-mediated cellular effects of thrombin can be mimicked by synthetic peptides, which correspond to the amino acid sequence of the newly formed N-terminus. The aim of the present study was to investigate vascular effects of thrombin and the thrombin receptor activating peptide (TRAP: SFLLRN) in vitro and in vivo in rats. In precontracted rat aortic rings, both thrombin (0.3, 1, 3 U/ml) and TRAP (1, 3, 10, 20, 40 mM) induced endotheliumdependent relaxant responses. In anaesthetized rats, the mean arterial blood pressure (MAP) was measured continuously in the carotid artery by a pressure transducer. Thrombin and TRAP were administered as intravenous bolus injection via the femoral vein. Thrombin at doses of 3±100 U/ kg, as well as TRAP at doses of 0.1±0.6 mg/kg iv, caused a reversible decrease in MAP. Administration of TRAP at doses of 0.3 and 0.6 mg/kg led to a triphasic response in most of the animals Abbreviations: L-NAME, NG-nitro-L-arginine-methylester; MAP, mean arterial blood pressure; NO, nitric oxide; PAR, proteaseactivated receptor; TRAP, thrombin receptor activating peptide. Corresponding author: Prof. Dr. Erika Glusa, Unit of Experimental Angiology, Center for Vascular Biology and Medicine, Faculty of Medicine, Friedrich Schiller University Jena, NordhaÈuser Str. 78, D-99089 Erfurt, Germany. Fax: +49 (361) 741 1160; E-mail: .

treated (50% and 75%, respectively), i.e. a short drop of MAP was followed by an increase and finally a longer lasting decrease in MAP. Pretreatment with the nitric oxide (NO)-synthase inhibitor N G -nitro- L -arginine-methylester (L NAME) suppressed the dose-dependent vasodilator effects of thrombin. Heparin and hirudin also inhibited the hypotensive response to thrombin. The TRAP-induced triphasic reaction on MAP was not affected by the serotonin antagonists ketanserin and tropisetron, as well as the aminopeptidase inhibitor amastatin. Pretreatment with L-NAME led to an inhibition of hypotension induced by TRAP at 0.1 mg/kg, as well as of the initial transient fall in blood pressure at doses of 0.3 and 0.6 mg/kg. The studies suggest that the thrombin- and TRAPinduced vasodilation in vitro and in vivo is in part due to the release of endothelial NO. In the blood pressure response to TRAP, additional effects seem to be involved. D 2001 Elsevier Science Ltd. All rights reserved. Key Words: Thrombin; TRAP; Vascular effects; Blood pressure

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he serine protease thrombin induces a series of receptor-mediated events in various cell types, such as endothelial cells, smooth muscle cells and platelets [1±3]. Cellular responses elicited by thrombin include the production of prostacyclin and nitric oxide (NO) in endothelial cells, mitogenesis in fibroblasts, chemotaxis of monocytes and the release of platelet-

0049-3848/00/$ ± see front matter D 2001 Elsevier Science Ltd. All rights reserved. PII S0049-3848(00)00429-1

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derived growth factor. The thrombin receptor or protease-activated receptor 1 (PAR-1) belongs to the family of seven transmembrane domain, Gprotein-coupled receptors [3]. Thrombin is capable of cleaving its receptor N-terminus between Arg 41 and Ser 42, thereby creating a new amino terminus, which acts as a tethered ligand to activate the receptor [3]. Peptides with sequences corresponding to the new amino terminus are capable of activating the thrombin receptor directly [1,4]. The synthetic peptides known as thrombin receptor activating peptides (TRAPs) are found to mimic receptor-mediated effects of thrombin on platelets, endothelial and other cells [1,4]. However, there are also differences between thrombin and TRAP, e.g. in their mitogenic activity [5,6]. Relaxant and contractile effects of thrombin and TRAP were investigated in various types of vessels and in different species [7±10]. The vasodilating effects of thrombin and TRAP were demonstrated to be mediated by the release of NO from the endothelium. NO activates the soluble guanylyl cyclase in smooth muscle cells and increases the cGMP level, thereby inducing relaxation of the smooth muscle [11]. The studies presented were performed to characterize and compare the ability of thrombin and TRAP to induce vascular effects in vitro and in vivo by measuring endothelium-dependent relaxant effects in precontracted aortic rings as well as the mean arterial blood pressure (MAP) in rats. Furthermore, it was investigated whether the release of NO plays a role for the vasodilating action of thrombin and TRAP and whether the proteolytic activity of thrombin is required for its vascular effects.

1. Materials and Methods 1.1. Drugs and Chemicals The following substances were used: acetylcholine hydrochloride (Dispersa, Winterthur, Switzerland); phenylephrine , serotonin and indomethacin (Serva, Heidelberg, Germany); amastatin (Sigma, Deisenhofen, Germany); calciparin (Sanofi, Munich, Germany); ICS 205-930 (tropisetron, Sandoz, Basel, Switzerland); potas-

sium chloride (Merck, Darmstadt, Germany); ketanserin, thrombin and aprotinin (Arzneimittelwerk, Dresden, Germany); NG-nitro-L-arginine-methylester (L-NAME), NG-nitro-D-arginine and L-arginine (Sigma); pentobarbitone sodium (SPOFA, Prague, Czech Republic); TRAP (SFLLNR, Bachem, Heidelberg, Germany); hirudin (recombinant hirudin HBW 023, 13300 ATU/ mg, Hoechst, Frankfurt, Germany). Composition of Krebs ± Henseleit solution (mM): NaCl 118, KCl 4.7, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25, CaCl2 2.5, glucose 11. 1.2. Animals Female Wistar rats (Charles River Wiga, Bad Sulzfeld, Germany) with a body weight between 200 and 300 g were used. After 15 min of adaptation to the room conditions of the laboratory, the rats were anaesthetized with pentobarbitone sodium (60 mg/kg ip). All protocols involving the use of animals were approved by the Animal Care and Use Committee of Thuringia, Germany. 1.3. Measurement of Tension in the Isolated Rat Aorta The rats were anaesthetized and killed by cervical dislocation and bleed to death. The thoracic aorta was quickly removed and placed in Krebs±Henseleit solution. The aorta was carefully cleaned of adhering connective tissue and cut into rings of 2±3 mm in length. Each ring was attached between L-shaped platinum hooks, placed in a 10-ml organ bath containing Krebs ± Henseleit solution (pH 7.4, 37°C) and gassed continuously with a mixture of 95% O2 and 5% CO2. Each vascular ring was connected to an isometric force transducer (Hugo-SachsElektronik, March-Hugstetten, Germany) and changes in tension were recorded continuously. A passive resting tension of 20 mN was maintained throughout the experiments. During an initial stabilization period of 40 min, the bathing medium was changed every 15 min. The rings were contracted at 30-min intervals, once with KCl (30 mM) and three times with phenylephrine (1 mM) until the effect was reproducible. Functional integrity of the endothelium was assessed by relaxation of phenylephrine-precon-

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body weight and the injection speed was 0.1 ml/10 s. 1.5. Measurement of Platelet Count

Fig. 1. Concentration ± response relationships for endothelium-dependent relaxant responses to thrombin ( ) and TRAP (&) in phenylephrine(1 mM)-precontracted rat aortic rings with intact endothelium. Means ‹ S.E.M., n = 5, * P < .05.



tracted vessels in response to acetylcholine (1 and 10 mM). To test the vascular effects of thrombin and TRAP, rings were precontracted with phenylephrine (1 mM). After the maximum of contraction has been reached, thrombin or TRAP was added to the organ bath. Furthermore, the vascular effects of thrombin and TRAP were tested after 10 min preincubation with various inhibitors. The relaxation was expressed as the percentage of phenylephrine-induced contraction before the addition of the agonists. 1.4. Measurement of Blood Pressure and Heart Rate in Rats After anaesthesia, a median cervical incision was made, the trachea was cannulated and the rats were ventilated with room air at 53 breaths/min and a tidal volume of 2.3 ml using a rodent respirator (Hugo-Sachs-Elektronik). The rectal temperature was measured and adjusted to 37°C. A catheter was filled with heparinized saline solution (250 U/ml) and inserted into the carotid artery. Animals were allowed to stabilize for 20 min before starting measurement of MAP and heart rate. MAP was measured continuously in the carotid artery by a pressure transducer (Hugo-Sachs-Elektronik), which was connected to a Graphtec Linearcorder (Graphtec, Tokyo, Japan). Subdermal needle electrodes were inserted for recording lead II electrocardiogram (ECG). For the administration of drugs, a catheter was placed into the femoral vein. The injection volume was 0.1 ml/100 g

Blood was diluted with Thrombo Plus (Sarstedt, Nystedt, Germany) and filled in a Buerker counting chamber. After a period of 20 min, the platelets were counted by means of a phasecontrast microscope. 1.6. Statistical Analysis The results are given as means ‹ S.E.M.; for statistical analysis, the Wilcoxon test for pair differences was used. Values were considered statistically significant at P < .05.

2. Results 2.1. Effects of Thrombin and Trap on Precontracted Rat Aortic Rings In rat aortic rings with intact endothelium, phenylephrine (1 mM) caused a contractile force of 9.7 ‹ 0.4 mN (n = 44), which was equivalent to about 87% of the maximum force evoked by phenylephrine. Acetylcholine (10 mM) relaxed the precontracted rings by 86.2 ‹ 0.8% (n = 84).

Fig. 2. Influence of L-NAME (100 mM) and indomethacin (3 mM) on the thrombin (3 U/ml) and TRAP-induced (20 mM) relaxation of phenylephrine-precontracted rat aortic rings with intact endothelium. Influence of amastatin (40 mM) on TRAP-mediated vascular relaxation. Means ‹ S.E.M., n = 5 ± 8, * P < .05.

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sence of indomethacin (Fig. 2) and, furthermore, it was also completely inhibited by pretreatment with L-NAME. To exclude a proteolytic degradation of TRAP, the vessels were preincubated with the aminopeptidase inhibitor amastatin (40 mM) 10 min before the addition of TRAP. Amastatin did not affect the TRAP-induced relaxation (Fig. 2). 2.2. Effect of Thrombin on the MAP in Rats Fig. 3. Dose-dependent decrease in the MAP after intravenous bolus injection of thrombin in anaesthetized rats. Thrombin doses (3, 10, 30, 100 U/kg) were administered via the femoral vein. Means ‹ S.E.M., n = 5, * P < .05.

In precontracted aortic rings with intact endothelium, thrombin at concentrations of 0.3, 1 and 3 U/ml (corresponding to 3, 10 and 30 nM) caused a concentration-dependent transient relaxation (Fig. 1). The thrombin-induced relaxation appeared within 20 s and the initial contraction was reached after 10 min. TRAP at concentrations of 1, 3, 10, 20 and 40 mM elicited transient relaxant responses (Fig. 1). There were no significant differences in the time course and in the extent of relaxation between TRAP and thrombin. On the molar basis, TRAP was less potent than thrombin by more than three orders of magnitude. To study whether the vascular effects of thrombin are mediated by prostaglandins, the vessels were pretreated with indomethacin (3 mM) for 10 min. In the presence of indomethacin, the thrombin-induced relaxation was significantly increased (Fig. 2). To demonstrate whether the thrombin-mediated relaxation is based on the release of NO from endothelium, the relaxant effect of thrombin was investigated in the presence of L-NAME (100 mM), an inhibitor of the NO-synthase. When L-NAME was added to the organ bath 10 min before thrombin, relaxation induced by 3 U/ml thrombin was inhibited by 96% (Fig. 2). The same is true for the relaxant response to acetylcholine 10 mM, which was inhibited by L-NAME by 89%. As seen with thrombin, relaxation caused by TRAP (20 mM) was also increased in the pre-

In vivo, the vascular effects of thrombin and TRAP were investigated by measuring MAP in anaesthetized rats. The intravenous bolus injection of thrombin at doses of 3, 10, 30 and 100 U/ kg caused an immediate, gradual, reversible and dose-dependent decrease in MAP. The baseline level of blood pressure was reached after 2±4 min. The decrease in MAP at a dose of 30 U/kg was about 40 mm Hg (Fig. 3). After administration of thrombin no significant changes in the heart rate were found. The platelets were counted before as well as 1 and 5 min after bolus injection of thrombin. The platelet counts did not differ significantly before and after application of thrombin at doses of 10, 30 and 100 U/kg iv (Table 1). 2.3. Pharmacological Influence on Thrombin-Induced Changes in MAP To assess whether the thrombin-induced hypotension is due to the release of endothelial NO, the animals were pretreated with L-NAME (100 Table 1. Platelet counts after intravenous bolus injection of thrombin in anaesthetized rats

Thrombin dose 10 U/kg 30 U/kg 100 U/kg

Time after injection

Platelet count  103/Ml (means ‹ S.E.M., n = 5)

Control 1 min 5 min Control 1 min 5 min Control 1 min 5 min

952 ‹ 6.0 960 ‹ 9.5 959 ‹ 11.3 951 ‹ 7.0 926 ‹ 16.7 973 ‹ 15.0 963 ‹ 12.7 977 ‹ 6.9 933 ‹ 14.7

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TRAP at doses of 0.3 and 0.6 mg/kg iv in most of the animals treated (50% and 75%, respectively) a triphasic response was seen. A short drop (RI) of MAP was followed by an increase (K) and finally a longer lasting decrease (RII) in MAP. For a better understanding, the two effects of TRAP on the MAP were classified in type A (decrease in MAP like thrombin) and type B (decrease in MAP like serotonin, i.e. a triphasic response). The changes in MAP in type A and B after intravenous bolus injection of TRAP are summarized in Table 2. Fig. 4. Time-dependent effect of pretreatment with LNAME (100 mg/kg iv) on thrombin (30 U/kg iv)induced decrease in MAP in anaesthetized rats. Means ‹ S.E.M., n = 6, * P < .05.

mg/kg iv). After injection of L-NAME, a significant increase in MAP by 26 ‹ 1.3 mm Hg (n = 5) was found. Thrombin at a dose of 30 U/kg iv was injected 15, 25, 40 and 60 min after the administration of L-NAME. Up to 40 min after the injection of L-NAME, a significant inhibition of the vasodepressor response to thrombin was found (Fig. 4). In contrast to the in vitro studies, pretreatment of the animals with indomethacin (1 mg/kg iv) had no influence on thrombininduced effects on MAP, i.e. up to 60 min after the injection of indomethacin the decrease in MAP after bolus injection of thrombin was not changed. The thrombin-induced fall in MAP was dependent on the proteolytic activity of the enzyme. Pretreatment with hirudin, the most potent and selective thrombin inhibitor known, at a dose of 1 mg/kg iv prevented the decrease in MAP after thrombin injection even at the high dose of 100 U/kg. Heparin at a dose of 25 U/kg iv inhibited the thrombin (30 U/kg)-induced drop in blood pressure by 90% ( P < .001) (Fig. 5). The proteinase inhibitor aprotinin at a dose of 100 mg/kg iv did not influence the vasodilator effect of thrombin (Fig. 5).

2.5. Pharmacological Influence on TRAP-induced Changes in MAP The pharmacological influence on TRAP-induced effects on MAP was stressed on type B. The triphasic blood pressure response to TRAP was similar to that induced by serotonin. However, pretreatment with the serotonin antagonists ICS 205-930 (200 mg/kg iv) or ketanserin (100 mg/kg iv) given 2 min before TRAP did not influence TRAP-induced changes in MAP. The same was found when the rats were pretreated with amastatin (1 mg/kg iv). The decrease in blood pressure induced by TRAP at a dose of 0.1 mg/kg was prevented by L-NAME at a dose of 200 mg/ kg. However, when TRAP was given at the higher doses of 0.3 and 0.6 mg/kg, L-NAME only

2.4. Effect of TRAP on MAP in rats TRAP was administered intravenously at doses of 0.1, 0.3 and 0.6 mg/kg. TRAP elicited different, dose-dependent hemodynamic effects. Like thrombin, at a dose of 0.1 mg/kg, TRAP induced a reversible transient decrease in MAP. Using

Fig. 5. Effect of heparin (25 U/kg), hirudin (1 mg/kg), indomethacin (1 mg/kg) and aprotinin (100 mg/kg) on thrombin (30 U/kg)-induced decrease in MAP in anaesthetized rats. The inhibitors were administered intravenously 2 min before thrombin injection. Means ‹ S.E.M., n = 5 ± 8, * P < .05.

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Table 2. Changes in MAP after intravenous bolus injection of TRAP in anaesthetized rats TRAP (mg/kg)

Type A (n)

0.3

8

0.6

8

Type B (n) 8 8 8 24 24 24

inhibited the initial transient fall in blood pressure. The second and third phases were not significantly affected.

3. Discussion The present study demonstrates vascular effects of thrombin and TRAP in rats in vitro and in vivo. In precontracted rat aortic rings with intact endothelium, thrombin and TRAP elicited a relaxant response via release of endothelial NO. This effect of thrombin and TRAP on the rat aorta was also described by other authors [12]. In contrast to the relaxant effects in the aorta, thrombin caused contractions of porcine pulmonary arteries [10], rabbits femoral arteries [13] and porcine aortic rings [14]. The differences of the vascular effects of thrombin might be related to species-dependent expressions of thrombin receptors on endothelium and smooth muscle cells [14]. The responses of isolated vessels to TRAP are also species-dependent. TRAP is able to induce either contraction or relaxation of guinea pig aorta, canine coronary arteries, porcine pulmonary arteries, porcine coronary arteries and canine saphenous veins [7,10,15]. When compared to thrombin, TRAPinduced vascular effects required much higher concentrations. The reason might be that one molecule of thrombin may split off the N-terminus from several receptors. After proteolytic cleavage by thrombin the newly generated extracellular ligand is tethered and cannot diffuse away. In contrast, the agonist peptide TRAP is diffused into the incubation medium and, thus, more molecules might be required to

Changes in MAP (mm Hg) (means ‹ S.E.M.)

Time-dependent changes in MAP (min) (means ‹ S.E.M.)

Decrease, R = 21 ‹ 3.9 * Decrease, RI = 14 ‹ 2.1 * Increase, K = 8 ‹ 2.8 * Decrease, RII = 5 ‹ 1.8 * Decrease, R = 25 ‹ 3.3 * Decrease, RI = 19 ‹ 3.5 * Increase, K = 20 ‹ 2.6 * Decrease, RII = 10 ‹ 1.6 *

0.7 ‹ 0.07 0.4 ‹ 0.03 0.9 ‹ 0.14 3.5 ‹ 1.0 1.7 ‹ 0.28 0.2 ‹ 0.03 0.8 ‹ 0.07 3.5 ‹ 1.25

induce the same effect as thrombin. Moreover, the affinity of TRAP for the receptor could be lower than that of thrombin [10]. An accelerated degradation of the peptide in the organ bath can be excluded because the TRAP-induced relaxation was not affected by the aminopeptidase inhibitor amastatin. In previous studies on isolated rat aortic rings, it was shown that the thrombin-induced relaxation was associated with an increase in the level of cGMP, which causes relaxation of the vascular smooth muscle via protein phosphorylation and dephosphorylation of the myosin light chain [8]. The results presented showed that a pretreatment with L -NAME inhibited the vascular effects of both thrombin and TRAP. In earlier studies, it was demonstrated that the relaxant action of thrombin on pig pulmonary arteries was abolished after mechanical removal of the endothelium and the vasoconstriction was enhanced [10]. Whereas in deendothelialized rat vessels, thrombin did not induce a vasoconstriction, TRAP was found to cause a contraction of endothelium-denuded rat aorta rings in a concentration-dependent manner [8]. According to the results obtained by Tesfamariam et al. [7], prostaglandins seem not to be involved in the vascular action of thrombin or TRAP because preincubation with indomethacin, a cyclooxygenase inhibitor, had no inhibitory effect on the relaxation caused by either thrombin or TRAP. In the present studies, indomethacin even increased the relaxation induced by thrombin and TRAP. The mechanism is not yet clear, but it could be possible that thrombin and TRAP trigger the release of vasoconstrictor prostaglandins [7]. The synth-

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esis of those prostaglandins might be inhibited by indomethacin. The results obtained with thrombin and TRAP in vitro were confirmed by their vascular effects in vivo measuring the MAP in anesthetized rats. The intravenous bolus injection of thrombin caused a reversible and dose-dependent decrease in MAP. A hypotensive effect after injection of thrombin was also observed in dogs [16,20]. In accordance with other studies [17,20], our investigations also showed that the effect of thrombin is not associated with an activation of platelets and a decrease in plasma concentrations of fibrinogen. Both heparin, an inhibitor of thrombin, which acts indirectly via antithrombin III, and hirudin, which directly inactivates thrombin, inhibited the hypotensive action of the enzyme indicating that the proteolytic activity of thrombin is required for its vascular effect. In contrast to the findings in vitro, indomethacin did not affect the thrombin-induced changes in MAP. Therefore, the release of prostaglandins is not responsible for the vasodilator effect of thrombin. In comparison to thrombin, TRAP showed different effects on blood pressure. At lower doses, it produced a reversible decrease in MAP like thrombin, but at higher doses, it induced a triphasic response with a short drop of MAP followed by an increase and then a longer lasting decrease in MAP. Similar changes in MAP after injection of TRAP were described in mice, dogs and rats [18±21]. The triphasic reaction of TRAP on MAP was similar to that induced by serotonin. However, pretreatment with serotonin antagonists like ICS 205-930 (tropisetron) and ketanserin did not affect the triphasic reaction. Thus, this action of TRAP seems to be based on additional effects on membranes in vivo by an as yet unidentified mechanism. The activation of platelets in rats can be excluded because TRAP does not activate rat platelets [22]. Cheung et al. [19] demonstrated a dosedependent decrease in MAP in anaesthetized mice after bolus injection of TRAP. They could also show an initial transient fall in blood pressure which was followed by an increase in MAP. In contrast to our findings, the second hypertensive response was obviously not so clear. It is known that TRAP can activate both PAR-1 and PAR-2 [23]. According to the literature, L-NAME

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induces an increase in MAP accompanied by bradycardia [24] and inhibits the hypotension induced by thrombin. These data indicate that the formation of NO plays an important role in the regulation of blood flow and control of blood pressure. The decrease in blood pressure after intravenous injection of TRAP at 0.1 mg/kg was prevented by L-NAME, whereas at higher doses, L-NAME only inhibited the initial transient fall of blood pressure. Emilsson et al. [25] and Chintala et al. [21] found in rats that the hypotensive response to TRAP was not affected by L-NAME, which confirms our results and indicates that PAR-1-mediated hypotension in vivo is only in part based on the release of NO. In rats, the influence of TRAP on prostaglandins could be excluded by pretreatment with indomethacin [21]. In our studies, the second depressor response to TRAP was also not inhibited by indomethacin. Therefore, it is assumed that NO-independent mechanisms might be responsible for this action such as autonomic reflexes [26], modulation of the renin ± angiotensin ± aldosterone system or a direct effect on the vascular smooth muscle [21]. The study presented demonstrates vascular effects of the proteolytic enzyme thrombin, which is known to induce various receptormediated cellular effects, as well as of the TRAP, which directly activates the receptor. Proteolytically active thrombin at low doses induces vasodilatation due to the release of NO from endothelium. Like thrombin, TRAP causes an endothelium-dependent vasodilatation that leads to a decrease in MAP, but, at higher doses, the blood pressure reaction shows a triphasic course. Unlike thrombin, the vascular effect of TRAP seems to be only in part mediated by a mechanism that involves the receptor-mediated release of NO. The authors thank Mrs. Weiû for her expert technical assistance. This study was supported by a grant of the Deutsche Forschungsgemeinschaft (DFG Gl 178/1-3).

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