Justin R. Hamilton

A protective role for protease-activated receptors in the airways.

The protection of cells in the upper intestine against digestion by pancreatic trypsin depends on the prostanoid prostaglandin E2 (PGE2) and is mediated by protease-activated receptors in the epithelium,. As the airway epithelium is morphologically similar and also expresses one of these receptors, PAR2 (ref. 3), and is a major source of PGE2 (ref. 4), we reasoned that bronchial epithelial PAR2 might also participate in prostanoid-dependent cytoprotection in the airways. Here we show that activation of PAR2, which co-localizes immunohistochemically with trypsin(ogen) in airway epithelium, causes the relaxation of airway preparations from mouse, rat, guinea-pig and humans by the release of a cyclooxygenase product from the epithelium. This physiological protective response in isolated airways also occurred in anaesthetized rats, where activation of PAR2 caused a marked and prolonged inhibition of bronchoconstriction. After desensitization of PAR2, the response to trypsin recovered rapidly by mechanisms dependent on de novo synthesis and trafficking of proteins. Our results indicate that trypsin released from the epithelium can initiate powerful bronchoprotection in the airways by activation of epithelial PAR2.

Par4 is required for platelet thrombus propagation but not fibrin generation in a mouse model of thrombosis

Thrombin, a central mediator of hemostasis and thrombosis, converts fibrinogen to fibrin and is a potent platelet activator. Activated platelets provide a surface for assembly of the tenase and prothrombinase complexes required for thrombin generation. The role of thrombin-induced platelet activation in platelet accumulation and its interplay with fibrin deposition during thrombus assembly has not been fully defined. We studied these processes during laser-induced thrombus formation by using real-time digital fluorescence microscopy in mice lacking protease-activated receptor-4 (Par4), which is necessary for thrombin responsiveness in mouse platelets. Juxtamural platelet accumulation immediately after laser injury was not different in wild-type and Par4−/− mice. However, subsequent growth of platelet thrombi was markedly diminished in Par4−/− mice. At the time of maximal thrombus size in wild type, platelet accumulation was more than 10-fold higher in wild type than in Par4−/− mice. P-selectin expression, a marker of platelet activation, was reduced and delayed in Par4−/− thrombi. In contrast to platelet activation and accumulation, the rate and amount of fibrin deposition, predominantly intramural and juxtamural in this model, were indistinguishable in Par4−/− and wild-type mice. These results suggest that platelet activation by thrombin is necessary for normal propagation of a platelet thrombus at a distance from the injured vessel wall and hence for normal thrombus growth. However, platelet activation by thrombin is unnecessary for initial and limited accumulation of platelets at or near the vessel wall, and this limited accumulation of platelets and/or platelet-independent mechanism(s) of thrombin generation are sufficient for normal fibrin deposition in this model.

Protease-Activated Receptor-2 Activation Induces Acute Lung Inflammation by Neuropeptide-Dependent Mechanisms

Protease-activated receptors (PARs) and tachykinin-immunoreactive fibers are located in the lung as sentries to respond to a variety of pathological stimuli. The effects of PAR activation on the lung have not been adequately studied. We report on the effects of instilling PAR-activating peptides (PAR-APs, including PAR1-, PAR2-, and PAR4-AP) into the lungs of ventilated or spontaneously breathing mice. PAR2-AP, but not PAR1-AP or PAR4-AP, caused a sharp increase in lung endothelial and epithelial permeability to protein, extravascular lung water, and airway tone. No responses to PAR2-AP were detected in PAR2 knockout mice. In bronchoalveolar lavage, PAR2 activation caused 8- and 5-fold increase in MIP-2 and substance P levels, respectively, and a 12-fold increase in the number of neutrophils. Ablation of sensory neurons (by capsaicin) markedly decreased the PAR2-mediated airway constriction, and virtually abolished PAR2-mediated pulmonary inflammation and edema, as did blockade of NK1 or NK2 receptors. Thus, PAR2 activation in the lung induces airway constriction, lung inflammation, and protein-rich pulmonary edema. These effects were either partly or completely neuropeptide dependent, suggesting that PAR2 can cause lung inflammation by a neurogenic mechanism.

Identification of a fibrin-independent platelet contractile mechanism regulating primary hemostasis and thrombus growth

A fundamental property of platelets is their ability to transmit cytoskeletal contractile forces to extracellular matrices. While the importance of the platelet contractile mechanism in regulating fibrin clot retraction is well established, its role in regulating the primary hemostatic response, independent of blood coagulation, remains ill defined. Real-time analysis of platelet adhesion and aggregation on a collagen substrate revealed a prominent contractile phase during thrombus development, associated with a 30% to 40% reduction in thrombus volume. Thrombus contraction developed independent of thrombin and fibrin and resulted in the tight packing of aggregated platelets. Inhibition of the platelet contractile mechanism, with the myosin IIA inhibitor blebbistatin or through Rho kinase antagonism, markedly inhibited thrombus contraction, preventing the tight packing of aggregated platelets and undermining thrombus stability in vitro. Using a new intravital hemostatic model, we demonstrate that the platelet contractile mechanism is critical for maintaining the integrity of the primary hemostatic plug, independent of thrombin and fibrin generation. These studies demonstrate an important role for the platelet contractile mechanism in regulating primary hemostasis and thrombus growth. Furthermore, they provide new insight into the underlying bleeding diathesis associated with platelet contractility defects.

Atypical Protease-Activated Receptor Mediates Endothelium-Dependent Relaxation of Human Coronary Arteries

Protease-activated receptors (PARs) are a family of G protein-coupled receptors activated by a tethered ligand sequence within the amino terminal that are revealed by site-specific proteolysis. The thrombin-sensitive PAR-1 and trypsin-activated PAR-2 mediate endothelium-dependent vascular relaxation in a number of species. Because both thrombin and trypsin-like enzymes have been implicated in coronary artery disease, the purpose of this study was to investigate whether similar receptors are present in human coronary arteries. Thrombin (0.001 to 0.1 U/mL) and trypsin (0.001 to 1 U/mL) caused concentration- and endothelium-dependent relaxations of human coronary artery ring segments suspended in organ chambers for isometric tension recording and contracted with the thromboxane A2 mimetic U46619. These relaxations were dependent on the catalytic activity of each enzyme and were inhibited by the NO synthase inhibitor NG-nitro-L-arginine (100 micromol/L) and the NO scavenger oxyhemoglobin (20 micromol/L). The synthetic PAR-1 tethered ligand sequence SFLLRN-NH2 (0.01 to 10 micromol/L) also caused endothelium-dependent relaxation of U46619-contracted human coronary arteries; however, the equivalent PAR-2 ligand SLIGKV-NH2 caused almost no relaxation. In addition, desensitization to either thrombin or trypsin resulted in cross-desensitization to the other enzyme but had only a minimal affect on the response to SFLLRN-NH2. Therefore, we conclude that human coronary artery endothelial cells possess a PAR-1-like receptor that is potently activated by thrombin, trypsin, and SFLLRN-NH2 to cause NO-mediated vascular relaxation. Once cleaved, this receptor is recycled in a truncated form, able to respond to exogenous application of only its tethered ligand sequence, suggesting the presence of another endogenous activator possibly acting independently of receptor cleavage.

Impaired hemostasis and protection against thrombosis in protease‐activated receptor 4‐deficient mice is due to lack of thrombin signaling in platelets

Summary.  Platelets from protease‐activated receptor 4 (PAR4)‐deficient mice are unresponsive to thrombin, and Par4–/– mice have prolonged bleeding times and are protected against thrombosis. However, in addition to its role in platelets, PAR4 contributes to thrombin signaling in cells in the blood vessel wall that might participate in hemostasis and thrombosis, such as endothelial cells. To determine whether the hemostatic and thrombotic phenotypes of Par4–/– mice were due to loss of PAR4 function in hematopoietic vs. other cell types, tail bleed times and thromboplastin‐induced pulmonary embolism were examined in lethally irradiated mice reconstituted with Par4+/+ or Par4–/– bone marrow. In Par4+/+ and Par4–/– mice reconstituted with Par4+/+ marrow, the median tail bleed times were 2.0 and 1.7 min, respectively, vs. > 10 min for both Par4+/+ and Par4–/– mice reconstituted with Par4–/– marrow. In the pulmonary embolism model, Par4+/+ and Par4–/– mice reconstituted with Par4+/+ marrow survived a median of 3.7 and 2.8 min, respectively, after administration of thromboplastin, vs. > 20 min for both Par4+/+ and Par4–/– mice reconstituted with Par4–/– marrow. Further, the phenotype of mice reconstituted with Par4–/– marrow was almost as dramatic as that seen in Nf‐e2–/– mice, which lack platelets. These data strongly suggest that increased tail bleed times and protection against thrombosis in Par4–/– mice are accounted for by lack of PAR4 function in platelets, emphasize the importance of thrombin signaling in platelets among the multiple pathways and cell types that govern hemostasis and thrombosis.

Pathologic shear triggers shedding of vascular receptors: a novel mechanism for down-regulation of platelet glycoprotein VI in stenosed coronary vessels.

Ligand-induced ectodomain shedding of glycoprotein VI (GPVI) is a metalloproteinase-dependent event. We examined whether shear force, in the absence of GPVI ligand, was sufficient to induce shedding of GPVI. Human-citrated platelet-rich plasma or washed platelets were subjected to increasing shear rates in a cone-plate viscometer, and levels of intact and cleaved GPVI were examined by Western blot and ELISA. Pathophysiologic shear rates (3000-10 000 seconds(-1)) induced platelet aggregation and metalloproteinase-dependent appearance of soluble GPVI ectodomain, and GPVI platelet remnant. Shedding of GPVI continued after transient exposure to shear. Blockade of α(IIb)β(3), GPIbα, or intracellular signaling inhibited shear-induced platelet aggregation but minimally affected shear-induced shedding of GPVI. Shear-induced GPVI shedding also occurred in platelet-rich plasma or washed platelets isolated from a von Willebrand disease type 3 patient with no detectable VWF, implying that shear-induced activation of platelet metalloproteinases can occur in the absence of GPVI and GPIbα ligands. Significantly elevated levels of sGPVI were observed in 10 patients with stable angina pectoris, with well-defined single vessel coronary artery disease and mean intracoronary shear estimates at 2935 seconds(-1) (peak shear, 19 224 seconds(-1)). Loss of GPVI in platelets exposed to shear has potential implications for the stability of a forming thrombus at arterial shear rates.

Protease-activated receptor (PAR) 1 but not PAR2 or PAR4 mediates endothelium-dependent relaxation to thrombin and trypsin in human pulmonary arteries

&NA; Endothelial protease‐activated receptors (PARs) may be important sensors of vascular inflammation and injury. Activation of endothelial PAR1 and PAR2 causes nitric oxide‐mediated arterial smooth muscle relaxation in a number of species and PAR4 activation causes similar responses in isolated rat aorta. However, it is unclear whether these receptors mediate such responses in human arteries because the most potent activators of PAR1, PAR2, and PAR4, thrombin and trypsin, cause endothelium‐dependent relaxation of human coronary arteries through a common PAR1‐like receptor. This study aimed to determine whether this unique pharmacology of PARs in human coronary arteries extends to human pulmonary arteries. PAR1 and PAR2 mRNA and protein were detected in human pulmonary arteries via reverse transcription polymerase chain reaction and immunohistochemistry, respectively. PAR4 mRNA was also detected in human pulmonary arteries. Contracted human pulmonary artery ring segments suspended for isometric tension measurement relaxed in a concentration‐ and endothelium‐dependent manner to thrombin (0.001–0.1 U/ml), trypsin (0.01–1 U/ml), and the PAR1‐activating peptide, SFLLRN (0.1–10 &mgr;M). By contrast, the PAR2‐ and PAR4‐activating peptides, SLIGKV and GYPGQV, respectively, caused neither contraction nor relaxation of precontracted human pulmonary arteries. Relaxations to thrombin and trypsin cross‐desensitized, while tachyphylaxis to SFLLRN abolished subsequent relaxations to both thrombin and trypsin. We conclude that human pulmonary arteries express PAR1, PAR2, and PAR4, but that only PAR1, or a PAR1‐like receptor, is coupled to endothelium‐dependent relaxation.

Heterogeneous mechanisms of endothelium‐dependent relaxation for thrombin and peptide activators of protease‐activated receptor‐1 in porcine isolated coronary artery

Mechanisms of protease‐activated receptor‐1 (PAR1)‐ and PAR2‐induced relaxation were investigated in pre‐contracted porcine coronary artery ring preparations. Thrombin (0.01–0.3 u ml−1) and the PAR1‐activating peptide SFLLRN (0.1–10 μM) caused concentration‐ and endothelium‐dependent relaxation. pEC50s (−log u ml−1 for enzymes, −log M for peptides) and maximum relaxations (Rmax, %) for thrombin were 1.8±0.1 and 93.5±2.8% respectively, and for SFLLRN 6.8±0.1 and 90.8±1.3%. Similar concentration‐ and endothelium‐dependent relaxations occurred with trypsin (pEC50 2.3±0.2; Rmax 94.1±1.9%) and the PAR2‐activating peptide SLIGRL (pEC50 6.5±0.2; Rmax 92.4±1.6%). Relaxations to thrombin, SFLLRN, trypsin and SLIGRL were significantly inhibited (P<0.05) to similar extents by the nitric oxide (NO) synthase inhibitor NG‐nitro‐L‐arginine (L‐NOARG; 100 μM) and the NO scavenger oxyhaemoglobin (20 μM), both separately and in combination. In the presence of the L‐type voltage‐operated calcium channel (L‐VOCC) inhibitor nifedipine (0.3 μM), K+ (67 mM) abolished the L‐NOARG‐resistant relaxations to thrombin, SFLLRN, trypsin and SLIGRL. However, nifedipine alone significantly (P<0.05) reduced the pEC50 (1.5±0.1) and Rmax (77.5±7.0%) for thrombin but had no effect on relaxations to SFLLRN, trypsin or SLIGRL. Furthermore, L‐NOARG‐resistant relaxations to thrombin were abolished by nifedipine, whereas relaxations to SFLLRN, trypsin or SLIGRL were not further inhibited by combined treatment with nifedipine and L‐NOARG, than they were with L‐NOARG treatment alone. Similar selective inhibition of the L‐NOARG‐resistant relaxation to thrombin, but not SFLLRN, occurred with verapamil (1 μM) and diltiazem (3 μM). Our results suggest heterogeneous mechanisms in the NO‐independent relaxation to thrombin and peptide activators of PAR1 in the porcine coronary artery.

Protease-activated receptors as targets for antiplatelet therapy

Arterial thrombosis, manifesting as acute myocardial infarction or ischaemic stroke, is the single most common cause of morbidity and mortality in industrialised societies. Platelets are a pre-requisite for the formation of arterial thrombi and, as a consequence, novel antiplatelet agents are sought to meet the significant clinical need for a potent, safe, and orally available therapy for the management of cardiovascular disease. Platelet thrombin receptors, termed protease-activated receptors (PARs), represent one promising candidate for the development of such therapy. This review outlines the role of platelet PARs in haemostasis and thrombosis and discusses the preclinical and clinical evidence supporting the potential of PAR antagonists as novel antiplatelet therapy.