SummaryThe thrombin receptor (ThrR) is a membrane-bound, G-protein-coupled receptor for the serine protease thrombin. This receptor is expressed in a wide variety of cells and tissues, and elicits a range of physiological responses associated with tissue injury, inflammation, and wound repair. To achieve a better understanding of the physiological role of the ThrR, we have employed homologous recombination to create mice with a disrupted ThrR gene. Following heterozygous (+/-) intercrosses, a total of 351 surviving offspring were genotyped. Only 7% of these offspring were identified as homozygous (-/-) for the disrupted allele, indicating a profound effect on embryonic development. Paradoxically, adult ThrR-/- mice appeared to be normal by anatomical and histological analysis, including their platelet number and function. Similarly, ThrR deficiency had no detectable effect in adult ThrR-/- mice on basal heart rate, arterial blood pressure, vasomotor responses to angiotensin II and acetylcholine, and coagulation parameters, even though the ThrR is expressed in many cardiovascular tissue types. In addition, the loss of ThrR function in the peripheral vasculature of adult ThrR-/- mice was confirmed by the absence of various standard hemodynamic effects of the ThrR-activating peptides SFLLRN-NH2 and TFLLRNPNDK-NH2 Our results indicate that ThrR deficiency has a strong impact on fetal development; however, ThrR-/- mice that proceed to full development display surprisingly little change in phenotype compared to the wild-type
Abstract-Expression of protease-activated receptor-1 (PAR-1), a cell-surface receptor for thrombin, is increased in balloon-injured rat carotid artery and human atherosclerotic tissue. To examine the role of PAR-1 in vascular injury, we compared vascular injury responses in wild-type (WT) and PAR-1-deficient (PAR-1 Ϫ/Ϫ ) mice. Arterial injury was induced by inserting a flexible guidewire into the common carotid artery and withdrawing it 6 times with rotation. Bromodeoxyuridine, delivered subcutaneously by osmotic minipump, was used to measure cellular proliferation. Mice were perfusion-fixed at 1, 2, 5, 10, and 14 days after injury. Extensive endothelial damage, mural thrombosis, platelet adherence, and medial smooth muscle cell loss and necrosis were apparent at day 1 in both WT and PAR-1 Ϫ/Ϫ mice. The incidence of thrombosis or platelet deposition in WT and PAR-1 Ϫ/Ϫ mice declined from 100% at day 1 to 25% and 21%, respectively, at 14 days. Endothelial disruption, as assessed by Evan's blue uptake, was maximum at day 1 and declined by day 14. This apparent endothelial regrowth was similar in WT and PAR-1 Ϫ/Ϫ mice. Significant medial thickening at 14 days after injury was similar in WT (from 22.8Ϯ1.7 to 30.7Ϯ1.9 m) and PAR-1 Ϫ/Ϫ (from 23.2Ϯ2.1 to 30.5Ϯ2.2 m) mice. Medial area also increased in response to injury but to a lesser extent in PAR-1 Ϫ/Ϫ mice (from 0.0250Ϯ0.0044 to 0.0312Ϯ0.0047 mm 2 ) than in WT mice (from 0.0266Ϯ0.0040 to 0.0398Ϯ0.0050 mm 2 ). Neointima was variable and occurred in 6 of 13 WT and 5 of 12 PAR-1 Ϫ/Ϫ mice. However, intimal area tended to be less in PAR-1 Ϫ/Ϫ mice (0.0016Ϯ0.0007 mm 2 ) compared with WT mice (0.0082Ϯ0.0032 mm 2 ), although this difference did not achieve statistical significance (Pϭ0.06). Cell density was significantly greater in normal carotids from PAR-1 Ϫ/Ϫ (6.4Ϯ0.5ϫ10 3 /mm 2 ) compared with WT (4.3Ϯ0.8ϫ10 3 /mm 2 ) mice and remained elevated after injury. Vessel and lumen diameters tended to increase in WT mice after injury, whereas vessel diameter was unchanged and lumen diameter actually decreased in PAR-1 Ϫ/Ϫ mice. Cell proliferation in injured carotid arteries was similar in PAR-1 Ϫ/Ϫ and WT mice. Key Words: protease-activated receptor Ⅲ knockout mouse Ⅲ thrombin Ⅲ thrombin receptor Ⅲ cell proliferation S everal lines of evidence suggest a significant role for thrombin in vascular injury responses associated with thrombosis, atherosclerosis, and mechanical arterial injury, such as that caused by balloon angioplasty or stent implantation. [1][2][3] Concentrations of thrombin at sites of vascular injury are significantly elevated. For example, 5 hours after balloon injury of the rabbit aorta, thrombin activity was 50 fmol ⅐ min Ϫ1 ⅐ cm Ϫ2 , gradually decreasing to 10 fmol ⅐ min Ϫ1 ⅐ cm Ϫ2 at 24 hours after injury. 4 These levels were sustained for at least 10 days. In human plasma, thrombin concentrations in the vicinity of a thrombus have been estimated to be as high as 140 nmol/L. 5 In addition to evidence for elevated thrombin levels at sites of vascular injury...
Vascular expression and cellular functions of the thrombin receptor (PAR-1) and protease activated receptor 2 (PAR-2) suggest similar but distinct vascular regulatory roles. The vascular actions of PAR-1 and PAR-2 in vivo were differentiated by monitoring mean arterial pressure (MAP) and heart rate (HR) of anesthetized mice in response to intravenous SFLLRN (0.1, 0.3, and 1 mumol/kg) and SLIGRL (0.1, 0.3, and 1 mumol/kg), the respective receptor-activating sequences for PAR-1 and PAR-2, and TFLLRNPNDK (0.3, 1, and 3 mumol/kg), a synthetic peptide selective for PAR-1. All peptides dose dependently decreased MAP (order of potency: SLIGRL > SFLLRN > TFLLRNPNDK). SLIGRL induced a more prolonged hypotension with a slow return to baseline, whereas SFLLRN- and TFLLRNPNDK-induced hypotension was followed by a rapid return towards baseline and a sustained moderate hypotension. SFLLRN and TFLLRNPNDK, but not SLIGRL, decreased HR. N omega-Nitro-L-arginine methyl ester HCl (L-NAME), an inhibitor of nitric oxide synthesis, attenuated the cumulative hypotensive response to SLIGRL but had no effect on the SFLLRN and TFLLRNPNDK hypotension. However, L-NAME revealed a rebound hypertension in response to SFLLRN and TFLLRNPNDK but not SLIGRL. In conclusion, activation of either PAR-1 or PAR-2 in vivo results in hypotension. In addition, only PAR-1 activation induced hypertension following L-NAME, reflecting concurrent PAR-1-mediated vasoconstriction. Thus, these different hemodynamic responses in vivo suggest distinct physiological or pathophysiological roles for PAR-1 and PAR-2 in local vascular regulation.
Vascular expression and cellular functions of the thrombin receptor (PAR-1) and protease activated receptor 2 (PAR-2) suggest similar but distinct vascular regulatory roles. The vascular actions of PAR-1 and PAR-2 in vivo were differentiated by monitoring mean arterial pressure (MAP) and heart rate (HR) of anesthetized mice in response to intravenous SFLLRN (0.1, 0.3, and 1 mumol/kg) and SLIGRL (0.1, 0.3, and 1 mumol/kg), the respective receptor-activating sequences for PAR-1 and PAR-2, and TFLLRNPNDK (0.3, 1, and 3 mumol/kg), a synthetic peptide selective for PAR-1. All peptides dose dependently decreased MAP (order of potency: SLIGRL > SFLLRN > TFLLRNPNDK). SLIGRL induced a more prolonged hypotension with a slow return to baseline, whereas SFLLRN- and TFLLRNPNDK-induced hypotension was followed by a rapid return towards baseline and a sustained moderate hypotension. SFLLRN and TFLLRNPNDK, but not SLIGRL, decreased HR. N omega-Nitro-L-arginine methyl ester HCl (L-NAME), an inhibitor of nitric oxide synthesis, attenuated the cumulative hypotensive response to SLIGRL but had no effect on the SFLLRN and TFLLRNPNDK hypotension. However, L-NAME revealed a rebound hypertension in response to SFLLRN and TFLLRNPNDK but not SLIGRL. In conclusion, activation of either PAR-1 or PAR-2 in vivo results in hypotension. In addition, only PAR-1 activation induced hypertension following L-NAME, reflecting concurrent PAR-1-mediated vasoconstriction. Thus, these different hemodynamic responses in vivo suggest distinct physiological or pathophysiological roles for PAR-1 and PAR-2 in local vascular regulation.
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