Author + information
- Received November 18, 1997
- Revision received March 13, 1998
- Accepted April 8, 1998
- Published online July 1, 1998.
- C.Michael Stein, MBBSa,* (, )
- Nancy Brown, MDa,
- Douglas E Vaughan, MDb,
- Chim C Lang, MBBSa and
- Alastair J.J Wood, MBBSa
- ↵*Address for correspondence: Dr. C. Michael Stein, Division of Clinical Pharmacology, Vanderbilt University School of Medicine, Medical Research Building, Room 560, Nashville, Tennessee 37232–6602
Objectives. This study sought to define the local regulation of vascular tissue-type plasminogen activator (t-PA) release.
Background. The vascular endothelium, through the production of t-PA and plasminogen activator inhibitor (PAI-1), is an important regulator of fibrinolysis. Plasma t-PA levels increase in response to adrenergic stimulation; however, it is unclear whether this increase is the result of systemic reflex responses or direct effects on the vascular endothelium.
Methods. Forearm blood flow dose responses were generated to low doses of agonist infused directly into the brachial artery in 15 normotensive men (mean [±SE] age 28.9 ± 2.2 years). Simultaneous arterial and venous blood samples were drawn at baseline and in response to the intraarterial administration of isoproterenol (400 ng/min), methacholine (8 μg/min) and sodium nitroprusside (SNP) (8 μg/min). PAI-1 and t-PA antigen levels were measured by enzyme-linked immunosorbent assay, and the net release across the forearm was calculated.
Results. Forearm plasma flow increased significantly from baseline (1.4 ± 0.2 ml/100 ml per min) after administration of isoproterenol, methacholine and SNP (9.7 ± 1.9, 8.7 ± 1.9 and 6.7 ± 1.1 ml/100 ml per min, respectively) (p < 0.001 by analysis of variance). Baseline net t-PA release (0.7 ± 0.3 ng/100 ml per min) increased significantly after administration of isoproterenol (26.2 ± 11.6 ng/100 ml per min, p = 0.005) and methacholine (15.3 ± 5.5 ng/100 ml per min, p = 0.001) but not after administration of SNP (1.8 ± 2.2 ng/100 ml per min, p = 0.84). There was no net release of PAI-1 across the vascular bed.
Conclusions. Marked, rapid local t-PA release occurred in response to isoproterenol, a beta-adrenoceptor agonist, and methacholine, an endothelium-dependent nitric oxide agonist, in the human forearm. This effect was selective and independent of the effects of shear stress due to increased flow because SNP induced similar increases in forearm blood flow without affecting t-PA release. Vascular t-PA release may be a potentially valuable tool for evaluating endothelial function in diseases associated with increased risk of thrombosis.
The vascular endothelium, through the production of prostacyclin, nitric oxide (NO), a tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI-1), is a key regulator of thrombosis and fibrinolysis. Reduced fibrinolytic activity is associated with an increased risk of myocardial infarction, stroke and cardiovascular mortality (1–5). Common clinical conditions, including heart failure and hypertension, which are associated with adrenergic activation and abnormal endothelium-mediated responses (6–9), are also associated with altered fibrinolysis and increased risk of cardiovascular events. Therefore, although an understanding of the homeostatic regulation of fibrinolysis under basal conditions is of interest, the regulation of fibrinolysis under conditions associated with adrenergic or endothelial activation may be of greater clinical importance. The role of adrenergic and other stimuli in the regulation of local t-PA production in humans is poorly characterized; however, several animal studies suggest that both adrenergic stimulation and endothelial NO production increase the release of t-PA (10–12). The majority of studies in humans addressing the role of adrenergic activation in the regulation of t-PA release have measured circulating t-PA concentrations after systemic infusion of an agonist and have suggested that adrenergic activation resulted in increased fibrinolysis and t-PA concentrations (13–16). Unfortunately, such studies are limited in that circulating t-PA concentrations represent the sum of t-PA release and clearance, both of which may be affected, not only by drugs, but also by changes in blood flow (13,14,17). Thus, the mechanism underlying the increased circulating t-PA concentrations noted after exercise, mental stress and administration of adrenergic agonists has remained controversial. Recently, complex modeling has been performed in an attempt to quantify the contribution of local mechanisms to the increase in fibrinolytic activity after exercise and systemic administration of adrenergic agonists (13,14). A more attractive and direct approach to the study of the local mechanisms regulating fibrinolysis involves the direct infusion of agents into a specific vascular bed, at doses that have minimal systemic effects. Blood is then sampled from the inflow and outflow of that vascular bed, thus permitting measurement of local t-PA release, free of the confounding effects of systemic reflex responses (18,19). The purpose of the present study was to measure the local production of t-PA in response to direct intrabrachial artery administration of isoproterenol, a beta-adrenergic agonist. To control for shear stress–induced increases in t-PA release, the responses to infusion of methacholine and sodium nitroprusside (SNP) at similar levels of increased blood flow were determined.
Fifteen healthy, normotensive, nonsmoking male volunteers (mean [±SE] age 28.9 ± 2.2 years) were studied. Nine subjects were black, and six were white. No subject had clinically significant abnormalities on history, physical examination or routine laboratory tests, including complete blood count, prothrombin and partial thromboplastin times; renal and liver function tests; and an electrocardiogram (ECG). Subjects did not take any medications for at least 2 weeks before each study day and abstained from caffeine and alcohol for 5 days before each study day. All subjects provided written informed consent, and the study protocol was approved by the Vanderbilt Committee for the Protection of Human Subjects. These subjects were participating in studies examining ethnic differences in vascular flow responses, and those data have been published elsewhere (20,21).
All experiments were performed in the morning, in the same temperature-controlled room, with the subjects resting supine in bed. An intravenous cannula was placed in an antecubital vein of both arms. After subdermal administration of 1% lidocaine, an 18-gauge polyurethane catheter (Cook) was inserted into the brachial artery of the nondominant arm, allowing direct intraarterial administration of drugs. Arterial catheter patency before the infusion of isoproterenol was maintained with an infusion of normal saline at a rate of 30 ml/h and before the infusion of methacholine or SNP, with a 40-ml/h infusion of 5% dextrose in water. By altering the concentration of the drug infusion, the total flow rate through the canula was maintained constant at 30 ml/h during intraarterial administration of isoproterenol and at 40 ml/h during the administration of methacholine and SNP. Arterial blood pressure was measured by means of a pressure transducer (Hewlett-Packard), and heart rate was recorded from a continuous ECG monitor. A trace dose of [3H]norepinephrine was infused intravenously into the arm contralateral to the arterial line for the determination of norepinephrine kinetics, and those data have been reported elsewhere (20).
After the arterial line and intravenous catheters had been placed, subjects rested quietly for 60 min. Baseline measurements were determined, and isoproterenol 10 to 400 ng/min, in incremental doses, was infused intraarterially using a Harvard infusion pump (Harvard Apparatus). Each dose of isoproterenol was infused for 7 min, with blood flow recordings performed during the last 2 min. After completion of the isoproterenol dose response, 5% dextrose in water was infused at a rate of 40 ml/h for a washout period of 30 to 40 min until forearm blood flow had returned to baseline. Methacholine (0.25 to 8 μg/min) and SNP (0.25 to 8 μg/min), in random order, were infused intraarterially in incremental doses. Each dose was infused for 5 min, with measurements of forearm blood flow made during the last 2 min of each infusion. A 30-min washout period between the infusion of the two drugs allowed blood flow to return to baseline before infusion of the second drug.
Blood was drawn simultaneously from the brachial artery and antecubital vein from the arm into which drugs were infused at baseline and after the infusion of the highest doses of isoproterenol (400 ng/min), methacholine (8 μg/min) and SNP (8 μg/min). In one subject, for technical reasons, the SNP response was determined using the second highest dose (4 μg/min). Samples were drawn into cooled tubes with ethyleneglycoltetraacetic acid (EGTA) and reduced glutathione (Amersham Corporation), placed on ice and centrifuged at 4°C, separated and immediately frozen.
Forearm blood flow was measured, using mercury in Silastic strain gauge plethysmography (22), in the arm into which vasodilators were infused intraarterially, as previously described (20,21). The wrist was supported in a sling to raise the level of the forearm to above that of the atrium. The hand was excluded from the measurement of blood flow by inflation of a pediatric sphygmomanometer cuff to 200 mm Hg around the wrist before and during measurement of forearm blood flow. The volume of the forearm, excluding the hand and wrist, was measured by water displacement.
The arterial concentrations of t-PA and PAI-1 antigen, determined as described below, were subtracted from their respective venous concentrations at each sampling point to determine the arteriovenous concentration gradient (V–A). A positive arteriovenous gradient indicates net release, whereas a negative one would reflect net uptake in the forearm. The net release was calculated as the arteriovenous concentration gradient multiplied by the forearm plasma flow. Forearm plasma flow was derived from the hematocrit and the forearm blood flow.
Antigen levels of t-PA and PAI-1 were measured using commercially available enzyme-linked immunosorbent assays (Biopool AB, Umea, Sweden), as previously described (23).
Results are expressed as mean value ± SE, and were analyzed by repeated measures analysis of variance (ANOVA) and with post hoc testing performed using the t test for paired samples or, for data that were not normally distributed, the Wilcoxon matched pairs signed-rank test with the Bonferroni correction for multiple comparisons (SPSS for Windows Release 6). Because the primary, predefined outcome of interest in the study was net t-PA release across the forearm, this was the outcome subjected to post hoc testing. A two-tailed p value <0.05 was the criterion for statistical significance.
Subjects were healthy and did not have risk factors for endothelial dysfunction, except for two subjects whose serum cholesterol concentrations were 256 and 265 mg/dl. The demographic data of the subjects studied are shown in Table 1.
Forearm plasma flow increased significantly from baseline (1.4 ± 0.2 ml/100 ml per min) after the administration of agonists, resulting in responses to isoproterenol (400 ng/min), methacholine (8 μg/min) and SNP (8 μg/min) of 9.7 ± 1.9, 8.7 ± 1.9 and 6.7 ± 1.1 ml/100 ml per min, respectively (p < 0.001 by ANOVA), representing a 7.3-, 6.5- and 5.1-fold increase in forearm plasma flow in response to isoproterenol, methacholine and SNP, respectively (not significantly different: p = 0.14 by ANOVA). Venous concentrations of t-PA antigen increased from a baseline value of 3.0 ± 0.5 ng/ml to 4.9 ± 0.5 ng/ml and 4.4 ± 0.8 ng/ml after infusion of isoproterenol and methacholine, respectively, but not after SNP (3.2 ± 0.6 ng/ml) (Table 2). Arterial t-PA antigen concentrations during the infusion of isoproterenol, methacholine and SNP were similar. Thus the arteriovenous concentration gradient of t-PA antigen and the net release of t-PA antigen increased significantly after the infusion of isoproterenol and methacholine but not after SNP. The net t-PA antigen release increased from 0.7 ± 0.3 ng/100 ml per min at baseline to 26.2 ± 11.6 ng/100 ml per min (p = 0.005) and 15.3 ± 5.5 ng/100 ml per min (p = 0.001) after infusion of isoproterenol and methacholine, respectively, but was not significantly altered by SNP (1.8 ± 2.2 ng/100 ml per min, p = 0.84) (Table 2, Fig. 1). ⇓ This response represents a 38-, 22- and 2-fold increase in net t-PA release in response to isoproterenol, methacholine and SNP, respectively.
Arterial and venous PAI-1 antigen concentrations at baseline were similar (13.5 and 13.6 ng/ml), with no significant arteriovenous difference and no net release across the forearm. The infusion of isoproterenol, methacholine and SNP resulted in no significant arteriovenous difference of PAI-1 antigen (−1.19 ± 0.79, −0.65 ± 0.98 and −0.09 ± 1.1 ng/ml, respectively, p = 0.8 by ANOVA).
Clinically insignificant changes in heart rate or blood pressure occurred only after the highest dose of isoproterenol (400 ng/min), which produced a minor increase in heart rate from 60.3 ± 2.6 to 64.1 ± 2.4 beats/min and systolic blood pressure from 120.7 ± 2.2 to 124.5 ± 2.0 mm Hg and decreased diastolic blood pressure from 70.6 ± 1.5 to 65.7 ± 1.6 mm Hg.
Although vasoactive drugs and increased blood flow are widely appreciated to influence systemic t-PA levels, the local factors that regulate t-PA release are less well understood. In the present study we found that local intrabrachial artery infusion of isoproterenol and methacholine resulted in a rapid, large and selective increase in t-PA release across the forearm. These effects were independent of blood flow because SNP, which increased forearm blood flow to a similar extent, had no such effect. This conclusion is further supported by the observation that the proportional increases in t-PA release induced by isoproterenol and methacholine were much greater than their respective proportional increases in blood flow.
Isoproterenol-stimulated t-PA release
The findings of increased t-PA antigen release in response to a beta-adrenergic agonist extend the findings of previous studies demonstrating increased plasma t-PA antigen concentrations after systemic infusion of isoproterenol (14) and increased forearm t-PA antigen release during mental stress (18), which induces systemic adrenergic activation. Epinephrine, which is released in response to stress, is the endogenous ligand for the beta-adrenergic receptor but is a mixed adrenergic agonist with both alpha- and beta-adrenergic effects. Previous studies (16) have suggested that both alpha- and beta-adrenergic agonists increase fibrinolysis. Thus, the specific role of the beta-adrenergic receptor in the regulation of t-PA antigen release is uncertain. In the present study, the beta-adrenergic receptor agonist isoproterenol was found to increase acute t-PA secretion in the forearm. This finding indicates that beta-adrenergic receptors contribute to the regulation of t-PA antigen release locally in the vasculature and may thus contribute to the regulation of fibrinolysis under conditions associated with increased adrenergic stimulation. Because there appear to be regional variations in the production of t-PA in different vascular beds (24) the proportional increase of t-PA in response to β-adrenergic receptor stimulation in vascular beds other than the forearm may differ.
The dose of isoproterenol infused intraarterially resulted in systemic changes that were of minimal physiologic importance. Heart rate increased by an average of 4 beats/min, and blood pressure changed by ∼5 mm Hg after the intraarterial infusion of isoproterenol (400 ng/min). These minor autonomic changes would not be expected to result in the large increase in t-PA antigen across the forearm. That arterial t-PA antigen concentrations did not increase after isoproterenol, whereas venous concentrations did, confirms that systemic reflex responses did not contribute to the large increase in t-PA antigen release across the forearm that occurred after infusion of isoproterenol.
Methacholine-stimulated t-PA release
The findings of a 22-fold increase in t-PA antigen release after methacholine (8 μg/min) and no significant increase after SNP infusion are consistent with the findings of Jern et al. (19), who reported a 12-fold increase in forearm t-PA release after the infusion of methacholine (4 μg/min) and also found no net release of t-PA antigen after the infusion of SNP. Methacholine, an endothelium-dependent vasodilator, is thought to act on endothelial muscarinic receptors, resulting in the activation of NO synthase, with production of NO from l-arginine (25). SNP, in contrast, is thought to release NO directly (25). We used methacholine and SNP not only to localize the site of NO responses but also to provide control data that would allow comparison of the isoproterenol responses with those of other agonists that increased forearm blood flow to a similar extent. These comparisons are particularly important because shear stress has been reported to increase t-PA release (26). However, because SNP had no effect on t-PA antigen release in the present study, increased blood flow and consequent shear stress do not explain the t-PA antigen responses to isoproterenol and methacholine. In addition, because methacholine resulted in a marked release of t-PA antigen but SNP did not, it is likely that the effects of NO on t-PA production are endothelium dependent or that the effects of methacholine are independent of NO, perhaps mediated directly through other mechanisms such as effects on muscarinic receptors. However, the observation that intraarterial administration of substance P, which results in endothelium-dependent NO-mediated vasodilation, also increases t-PA release in the forearm (27) suggests that an endothelium-dependent NO-mediated effect is a possible explanation for the increase in t-PA release after methacholine.
Endothelial fibrinolytic function
Active t-PA in plasma is rapidly inhibited by PAI-1, with a second-order rate constant of 107 mol/liter per s (28). Thus, the effect of a local increase in t-PA release could potentially be offset by a simultaneous increase in PAI-1 release. However, we found no evidence of agonist-stimulated PAI-1 release in the forearm. Jern et al. (18,19) also reported no net release of PAI-1 from the forearm during mental stress or after the infusion of methacholine or SNP.
Both constitutive and regulated secretion of t-PA has been shown (29) to occur in human endothelial cells studied in vitro. The rapidity with which t-PA increased in response to stimuli such as thrombin in vitro suggests that release of stored t-PA, rather than increased synthesis, accounts for much of the increase. A similar mechanism is likely to explain the increase in t-PA in response to isoproterenol and methacholine noted in the present study. Local stimulated release of t-PA may play an important role in fibrinolysis because the t-PA released by endothelial cells is enzymatically active, and high local concentrations are achieved (29). Recent studies (30) indicate that t-PA is present in catecholamine storage vesicles and is coreleased with catecholamines. Thus, a reservoir of t-PA in catecholamine storage vesicles would be consistent with the observations that stimuli that activate the adrenergic nervous system, such as exercise and mental stress, also result in an increase in t-PA concentrations. We previously showed (31) that isoproterenol, acting through presynaptic beta-adrenergic receptors, is a potent stimulus to the local release of norepinephrine in the human forearm. Thus, we speculate that some of the increase in t-PA release after isoproterenol represents t-PA coreleased with norepinephrine from catecholamine storage vesicles in the sympathetic nerve terminal.
The present study demonstrates a role for the beta-adrenergic receptor in the regulation of local t-PA release, and future studies will be required to define the relative importance of beta1- and beta2-adrenoceptors. Also, because the increase in forearm blood flow in response to both mental stress and intraarterial isoproterenol may be partially mediated through endothelial NO production (32), it will be of interest to define the interrelation between the adrenergic nervous system and NO in the regulation of fibrinolysis by the vascular endothelium.
The major findings of the present study demonstrating that local t-PA release is regulated rapidly, substantially and in a selective fashion by isoproterenol and methacholine may be particularly pertinent to conditions such as hypertension, heart failure and hypercholesterolemia in which blunted endothelium-dependent NO responses, measured as NO-mediated vasodilation, occur (6–9). Furthermore, in hypertension both endothelium-dependent NO-mediated vasodilation and beta-adrenergic receptor-mediated vasodilation are blunted (6,7,33,34). Therefore in clinical settings that are associated with impaired endothelium-mediated vasodilation, if a deficient NO-mediated or a deficient adrenoceptor-mediated t-PA release, or both, is also present, it may contribute to the increased risk of myocardial infarction and stroke in these patients. Thus, studies comparing the regulation of vascular t-PA release in healthy subjects and in patients with diseases such as hypertension or hypercholesterolemia will be of interest.
Marked, rapid local t-PA release occurred in response to isoproterenol, a beta-adrenoceptor agonist, and methacholine, an endothelium-dependent NO agonist, in the human forearm. This effect was selective and independent of the effects of shear stress due to increased flow because SNP induced similar increases in forearm blood flow without affecting t-PA release. We speculate that vascular t-PA release may be a potentially valuable tool for evaluating endothelial function in diseases associated with increased risk of thrombosis.
☆ This study was supported by a Grant-in-Aid from the American Heart Association, Dallas, Texas and by USPHS Grants GM 46622, HL 56251, HL 51387 and GM 5M01-RR00095 from the National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland. Dr. Stein is the recipient of a Pharmaceutical Manufacturers of America (Washington, D.C.) Faculty Development Award in Clinical Pharmacology. Dr. Lang was supported by a Merck Sharp and Dohme (Rahway, New Jersey) International Fellowship in Clinical Pharmacology. Dr. Vaughan is the recipient of a Clinical Investigator Award from the Department of Veterans Affairs Research Service, Washington, D.C.
- analysis of variance
- electrocardiogram, electrocardiographic
- nitric oxide
- plasminogen activator inhibitor
- sodium nitroprusside
- tissue-type plasminogen activator
- arteriovenous concentration gradient
- Received November 18, 1997.
- Revision received March 13, 1998.
- Accepted April 8, 1998.
- American College of Cardiology
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