Effect of tiplaxtinin (PAI-039), an orally bioavailable PAI-1 antagonist, in a rat model of thrombosis.

OBJECTIVE: To assess the antithrombotic and profibrinolytic effects of tiplaxtinin (PAI-039), an orally bioavailable antagonist of PAI-1, in rat models of thrombosis. METHODS AND RESULTS: Carotid artery and vena cava vascular injury was produced by application of FeCl3 and blood flow was monitored using ultrasonic technology. To assess efficacy in a thrombosis prevention paradigm, PAI-039 was administered orally 90 min before injury (1-30 mg kg(-1)). To assess efficacy in a thrombosis treatment paradigm, vascular injury and stable thrombus formation were followed 4 h later by recovery and PAI-039 administration. PAI-039 prevented carotid artery occlusion in 20, 68 and 60% of animals pretreated with 0.3, 1.0 and 3.0 mg kg(-1), respectively. Time to occlusive thrombosis was increased from 18.2 +/- 4.6 min in controls to 32.5 +/- 8.7 (P = ns), 46.1 +/- 7.0 (P < 0.05), and 41.6 +/- 11.3 min (P < 0.05) in the respective PAI-039 treatment groups. In the vena cava protocol, PAI-039 pretreatment significantly reduced thrombus weight at PAI-039 doses of 3, 10 and 30 mg kg(-1). When PAI-039 was dosed in a treatment paradigm 4 h after stable arterial and venous thrombosis, a significant reduction in thrombus weight was observed 24 h later at PAI-039 doses of 3, 10 and 30 mg kg(-1). PAI-039 (10, 30 and 100 mg kg(-1)) had no effect on platelet aggregation in response to ADP or collagen and was not associated with increased bleeding or prolonged prothrombin time. In animals bearing no vascular injury, PAI-039 had no effect on circulating, low-levels of PAI-1 activity. In contrast, circulating PAI-1 activity increased 5-fold following the induction of vascular injury, which was completely neutralized by PAI-039. CONCLUSIONS: PAI-039 exerts antithrombotic efficacy in rat models of arterial and venous vascular injury without effecting platelet aggregation.

GW3965, a synthetic liver X receptor (LXR) agonist, reduces angiotensin II-mediated pressor responses in Sprague-Dawley rats.

BACKGROUND AND PURPOSE: Liver X receptors (LXRs) activate genes that regulate lipid and cholesterol metabolism. LXR agonists were shown recently to also increase murine renin gene expression in vivo. To further examine a link between lipid metabolism, the renin-angiotensin-aldosterone-system and blood pressure regulation, we investigated the effect of a LXR agonist (GW3965) on angiotensin II (Ang II)-mediated vasoreactivity and vascular angiotensin II receptor (ATR) gene expression. EXPERIMENTAL APPROACH: Arterial blood pressure (BP) was measured during Ang II infusions (1.5 min duration; 0.001-3 microg kg(-1)) in pentobarbital-anesthetized male Sprague-Dawley rats (n = 6-9) after oral administration of GW3965 (10 mg kg(-1), q.d.) or vehicle for 7 - 15 days. Mesenteric arteries and plasma were collected to analyze ATR gene expression and to measure plasma renin activity (PRA) and lipid profile, respectively. KEY RESULTS: Basal mean arterial pressure (MAP) was similar between groups. GW3965 dosing blunted the vasopressor effect of Ang II, which was significantly different with the 0.3 and 3 microg kg(-1) doses. No difference in heart rate, PRA or lipid profile was observed between groups. A time-course indicated that ATR type 1 and 2 gene expression of GW3965-treated vs. vehicle-treated rats decreased by 50%, reaching significance for ATR type 2, but not for ATR type 1, at time-points coinciding with BP measurements. CONCLUSIONS AND IMPLICATIONS: GW3965 decreased Ang II-mediated vasopressor responses coincident with a trend toward reduced ATR gene expression, suggesting that LXR agonists could affect vascular reactivity.

Characterization and comparative evaluation of a structurally unique PAI-1 inhibitor exhibiting oral in-vivo efficacy.

Plasminogen activator inhibitor-1 (PAI-1) is the major physiological inhibitor of both tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). Elevated levels of PAI-1 are associated with thrombosis and vascular disease, suggesting that high plasma PAI-1 may promote a hypercoagulable state by disrupting the natural balance between fibrinolysis and coagulation. In this study, we identify WAY-140312 as a structurally novel small molecule inactivator of PAI-1, compare its inhibitory activity with other previously identified small molecule inhibitors, and investigate the mechanism of inactivation of PAI-1 in the presence of both tPA and uPA. In an immunofunctional assay, WAY-140312 inhibited PAI-1 with an estimated inhibitory concentration (IC(50)) of 11.7 micro m, which was the lowest value obtained of the four different PAI-1 inactivators tested. Surface activity profiling indicated that the critical micelle concentration for WAY-140312 was 95.8 micro m, and that each inhibitor exhibited unique physical chemical properties. Using a sensitive direct activity assay, the IC(50) for WAY-140312 was similar when either tPA or uPA was used as the target protease. Immunoblot analysis demonstrated that WAY-140312 near the IC(50) inhibited the complex formation between either tPA or uPA and PAI-1. After oral administration, WAY-140312 exhibited 29% bioavailability with a plasma half-life of approximately 1 h. In an in-vivo model of vascular injury, a 10 mg kg(-1) oral dose of WAY-140312 was associated with improvement in arterial blood flow and reduction in venous thrombosis. Thus, WAY-140312 represents a structurally novel small molecule inhibitor of PAI-1, and is the first such molecule to exhibit efficacy in animal models of vascular disease following oral administration.

Conversion of atrial fibrillation by the experimental antiarrhythmic drug tedisamil in two canine models.

INTRODUCTION: Tedisamil is an experimental bradycardic agent possessing action potential-prolonging effects. It has been proven effective in terminating ventricular arrhythmias in several animal models and atrial flutter in a conscious dog model. There are no reports to date evaluating tedisamil's efficacy in terminating atrial fibrillation (AF). METHODS AND RESULTS: Two different canine models of AF were used. One group of dogs (n = 6) was subjected to 28 days of chronic fibrillatory pacing at 50 Hz using an implantable neural stimulator. Sustained AF was achieved in all dogs within 14 days of initiating pacing. A second set of dogs (n = 5) had AF induced via bilateral vagal stimulation. Tedisamil 1 mg/kg was 100% effective in terminating AF in both models. Cardioversion was associated with a statistically significant prolongation of the fibrillatory cycle length immediately before return to normal sinus rhythm in both models. A dose-response trial was performed in the vagal AF group as well as in a second group of three dogs that underwent chronic fibrillatory pacing. The efficacy of tedisamil was dose dependent, with limited efficacy at 0.1 and 0.3 mg/kg intravenously in both models. Tedisamil was able to prevent reinduction of sustained AF 30 minutes after administration of 1 mg/kg in the chronic pacing model in all dogs. Side effects included minor hypersalivation in most dogs receiving the 1 mg/kg dose. No ventricular ectopy or arrhythmias were observed. CONCLUSION: Tedisamil is effective for conversion of sustained AF to normal sinus rhythm in two different models of AF.

Effects of selective cyclooxygenase-2 inhibition on vascular responses and thrombosis in canine coronary arteries.

BACKGROUND: Prostanoid synthesis via the action of cyclooxygenase-2 (COX-2) is a component of the inflammatory response. Prostacyclin, a product of COX-2 in vascular endothelium, has important physiological roles, such as increasing blood flow to injured tissues, reducing leukocyte adherence, and inhibiting platelet aggregation. We examined the possibility that selective COX-2 inhibition could suppress the protective effects of prostacyclin, resulting in an alteration of the hemostatic balance and vascular tone. METHODS AND RESULTS: Circumflex coronary artery thrombosis was induced in dogs by vascular electrolytic injury. Orally administered celecoxib (COX-2 inhibition) or high-dose aspirin (HDA) (COX-1 and COX-2 inhibition) did not alter time to occlusive thrombus formation compared with controls (celecoxib 77.7+/-7.2 minutes, HDA 72.0+/-18.5 minutes, control 93.0+/-21.8 minutes). Oral HDA with an endothelial recovery period (HDA-ER) (COX-1 inhibition) produced a significant increase in time to vessel occlusion (257.0+/-41.6 minutes). The observed increase in time to occlusion was abolished when celecoxib was administered to animals dosed with HDA-ER (80.7+/-20.6 minutes). The vasomotor effect of endothelium-derived prostacyclin was examined by monitoring coronary flow during intracoronary administration of arachidonic acid or acetylcholine. In celecoxib-treated animals, vasodilation in response to arachidonic acid was reduced significantly compared with controls. CONCLUSIONS: The results indicate important physiological roles for COX-2-derived prostacyclin and raise concerns regarding an increased risk of acute vascular events in patients receiving COX-2 inhibitors. The risk may be increased in individuals with underlying inflammatory disorders, including coronary artery disease.

Tedisamil and dofetilide-induced torsades de pointes, rate and potassium dependence.

1. Tedisamil is a bradycardiac agent that prolongs the QT interval of the ECG and prevents cardiac arrhythmias. Given this profile, tedisamil might be expected to have proarrhythmic actions similar to Class III antiarrhythmic drugs. To address this question, the actions of dofetilide and tedisamil were examined in rabbit isolated hearts in which bradycardia was induced by AV ablation. 2. The QT interval was prolonged in a reverse rate-dependent fashion by dofetilide (3 and 30 nM) and tedisamil (0.3 and 3 microM). 3. Torsades de pointes was observed in 1/7 hearts treated with 3 nM dofetilide and 0/7 hearts treated with 0.3 microM tedisamil. The incidence of torsades de pointes was increased to 5/7 in hearts treated with 30 nM dofetilide and to 7/7 in hearts treated with 3 microM tedisamil (both P < 0.05 vs control). 4. The actions of 30 nM dofetilide and 3 microM tedisamil were also examined in hearts paced at 50, 100, 200 and 50 beats min(-1) successively. Both drugs caused torsades de pointes in 5/5 hearts paced at 50 beats min(-1); however, the incidence was reduced to 0/5 during pacing at 200 beats min(-1). Thus, drug-induced proarrhythmia was bradycardia-dependent. 5. Drug-induced prolongation of the interval between the peak and end of the T-wave (QTa-e) was reverse rate-dependent and was associated with the occurrence of torsades de pointes (r = 0.91, P < 0.01). 6. The results suggest that tedisamil, like dofetilide, presents a risk for development of torsades de pointes.

Effect of NO donors on protein phosphorylation in intact vascular and nonvascular smooth muscles.

It is generally well accepted that nitrovasodilator-induced relaxation of vascular smooth muscle involves elevation of cGMP and activation of a specific cGMP-dependent protein kinase [protein kinase G (PKG)]. However, the protein targets of PKG and the underlying mechanisms by which this kinase leads to a relaxant response have not been elucidated. Several types of smooth muscle, including rat myometrium and vas deferens, are not relaxed by sodium nitroprusside, even at concentrations that produce marked elevation of cGMP and activation of PKG. The main objective of our studies was to compare PKG-mediated protein phosphorylation in intact rat aorta, rat myometrium, and rat vas deferens using two-dimensional gel electrophoresis. In intact rat aorta, seven PKG substrates were detected during relaxation of the tissue. None of the PKG substrates identified in the rat aorta appeared to be phosphorylated in the myometrium or vas deferens after administration of various cGMP-elevating agents. Thus the failure of the rat myometrium and rat vas deferens to relax in the face of cGMP elevation and PKG activation may be due to a lack of PKG substrate phosphorylation.

An updated two-dimensional gel electrophoresis technique for the detection of drug-induced changes in protein phosphorylation in intact smooth muscle.

Two-dimensional gel electrophoresis is widely used in many areas of scientific research. The necessity for greater resolution and more sensitive protein detection with this technique have resulted in a steadily changing methodology. Complete descriptions of some aspects of two-dimensional gel electrophoresis are available in the earlier literature. However, simplified methods incorporating recent advances specifically designed to use two-dimensional gel electrophoresis for the measurement of protein phosphorylation in intact tissue are lacking. This report describes, in detail, each of the steps involved in carrying out such measurements including intact tissue labeling with 32P, homogenization and protein sample preparation, two-dimensional gel electrophoresis using isoelectric focusing followed by vertical second-dimension SDS-PAGE, staining, autoradiography, and quantitative analysis of changes in phosphorylation of specific proteins. This method incorporates a number of modifications taken from other published sources and includes several novel changes developed in our laboratory. To illustrate the utility of this technique we have included a set of results analyzing the phosphorylation patterns induced by the addition of a nitric oxide donor, sodium nitroprusside, to intact strips of rat aorta. We were able to demonstrate SNP-induced phosphorylation of a number of proteins, several of which have not been previously described in earlier reports in which the patterns of PKG-mediated phosphorylation were investigated.

Evidence that spontaneous contractile activity in the rat myometrium is not inhibited by NO-mediated increases in tissue levels of cyclic GMP.

1. There is conflicting evidence in the literature concerning the role of cyclic GMP in the regulation of myometrial contractility and the importance of hormonal status on the uterine response to cyclic GMP-elevating agents. The objective of the present study was to investigate further the importance of cyclic GMP in the control of uterine contractility, by monitoring the effects of cyclic GMP-elevating agents on spontaneous contractions and cyclic GMP levels in myometrial strips from pregnant rats and from ovariectomized rats under the influence of oestrogen and/or progesterone. 2. Sodium nitroprusside (SNP) 5 mM, atrial natriuretic peptide (ANP) 100 nM, L-arginine 1 mM and 8-bromo-cyclic GMP 100 mM had no relaxant effect on the spontaneous contractions of myometria from pregnant rats or from ovariectomized rats under the influence of oestrogen or progesterone. 3. Tissue levels of cyclic GMP were significantly elevated by SNP in all treatment groups, including pregnant animals. For example, in ovariectomized, progesterone-treated rats, SNP raised cyclic GMP levels approximately 8 fold from a basal level of 2.9 +/- 0.4 pmol mg(-1) protein to 24.8 +/- 4.0 pmol mg(-1) protein. ANP increased cyclic GMP levels approximately 2 fold in all treatment groups, except in the pregnant animals. L-Arginine elevated cyclic GMP significantly only in ovariectomized, vehicle-treated myometria. 4. The activity of cyclic GMP-dependent protein kinase (PKG) was significantly increased (3 fold) in myometria exposed to SNP (5 mM). Thus, the inability of SNP to relax uterine preparations was not due to a failure of SNP-elevated cyclic GMP to activate PKG. 5. The more potent NO donor, S-nitroso-N-acetylpenicillamine (SNAP), at a concentration of 100 microM was able to inhibit spontaneous contractions significantly in myometrial preparations from both non-ovariectomized and ovariectomized rats treated with oestrogen or progesterone. 6. Tissue levels of cyclic GMP were markedly increased by SNAP at concentrations of 10, 30 and 100 microM. At 100 microM, cyclic GMP levels increased from 1.9 +/- 0.2 pmol mg(-1) protein to 74.0 +/- 18.0 pmol mg(-1) protein. However, complete or partial blockade of SNAP-induced increases in cyclic GMP levels by the soluble guanylyl cyclase inhibitor, ODQ (25 microM), had no effect on the relaxant response to SNAP. Thus, the relaxant effect of SNAP in this tissue appears to be mediated via a mechanism independent of cyclic GMP. 7. Taken as a whole, the results of the present study indicate that cyclic GMP does not play an important role in the control of contractility in the rat uterus.

Activation of guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase in rat vas deferens and distal colon is not accompanied by inhibition of contraction.

There is good evidence that in vascular smooth muscle, the relaxant effects of sodium nitroprusside (SNP) are mediated by increases in cGMP levels and activation of cGMP-dependent protein kinase (PKG). However, in rat vas deferens and rat distal colon, cGMP-elevating agents such as SNP and atrial natriuretic factor (ANF) have been shown to elevate cGMP without inducing relaxation. The lack of relaxation might be explained by either lack of activation of PKG by these agents or low levels of PKG in these tissues. The object of the present study was to investigate these possibilities by simultaneously monitoring cGMP levels, PKG activity and contractility in isolated strips of rat vas deferens, rat proximal colon and distal colon exposed to high concentrations of SNP or ANF. Verification of the specificity of the assay for PKG was obtained using MonoQ chromatography to resolve soluble smooth muscle extracts, followed by immunoblotting with a PKG-specific antibody to identify the kinase. In rat vas deferens, 5 mM SNP increased cGMP levels (14-fold) and PKG activity ratios (3.4-fold) but did not inhibit phenylephrine-induced contractions. In both rat proximal and rat distal colon, 100 nM ANF significantly elevated cGMP levels and PKG activity ratios, but only in the proximal colon was inhibition of spontaneous contractions observed. Total PKG activity was much lower (approximately 16 pmol PO4/min/mg protein) in rat vas deferens, which was not relaxed by SNP, than in rabbit aorta (approximately 148 pmol PO4/min/mg), which was relaxed. However, in the rat proximal colon, despite low PKG levels (approximately 11 pmole/min/mg), ANF did inhibit contractions. Thus the inability of the cGMP-elevating agents SNP and ANF to inhibit contractions in rat vas deferens and rat distal colon cannot be explained by either of the possibilities suggested above.