Arterial thrombin activity after angioplasty in an atherosclerotic rabbit model: time course and effect of hirudin.

BACKGROUND: A 2-hour infusion of the direct thrombin inhibitor hirudin at the time of balloon angioplasty limits restenosis in the focally atherosclerotic rabbit. Although short-term administration of hirudin may have a prolonged biological effect, the effect of hirudin on vessel thrombin activity has not been previously studied in an animal model of angioplasty. We hypothesized that a short intravenous infusion of hirudin would result in prolonged inhibition of arterial wall-associated thrombin activity (ATA) after angioplasty. METHODS AND RESULTS: Sixty-one rabbits received recombinant hirudin (r-hirudin)(1 mg/kg bolus plus 1 mg x kg(-1) x h(-1)x2hours) or bolus heparin (controls, 150 U/kg) intravenously at the time of femoral balloon angioplasty. ATA was measured through exposure of arterial segments ex vivo to fibrinogen and conducting an assay for fibrinopeptide A (FPA). ATA was low in nonballooned, atherosclerotic vessels (FPA=0.5+/-0.3 ng x mL(-1) x mg(-1)) but increased significantly at 24 hours after angioplasty in the heparin group (3.7+/-0.9 ng x mL(-1) x mg(-1), P<.01 versus baseline, n=9) but not in the hirudin group (FPA = 1.4+/-0.3; P=NS versus baseline, P<.02 versus heparin controls, n=8). The time course of ATA after angioplasty was assessed in 44 rabbits. Thrombin activity peaked at 48 hours and declined to baseline at 72 hours and 7 days. FPA values between the heparin and r-hirudin groups were similar at these later time points. CONCLUSIONS: A 2-hour intravenous infusion of r-hirudin suppressed ATA measured 24 hours after angioplasty in the focally atherosclerotic rabbit. This prolonged biological effect may account, in part, for the reduction in restenosis seen in this model.

Effectiveness of hirulog in reducing restenosis after balloon angioplasty of atherosclerotic femoral arteries in rabbits.

BACKGROUND: Thrombin may play an important role in restenosis after balloon angioplasty (BA). Angiographic and pathologic restenosis have been shown to be reduced after BA in an atherosclerotic rabbit model using recombinant desulfatohirudin, a selective and direct thrombin inhibitor. We hypothesized that potent and specific thrombin inhibition with the synthetic peptide hirulog given intravenously at the time of angioplasty would reduce restenosis in rabbits, confirming a specific role of thrombin in restenosis. METHODS AND RESULTS: Focal femoral atherosclerosis was induced in 27 rabbits by air desiccation endothelial injury followed by a 2% cholesterol diet for 1 month. Rabbits received either heparin (150 units/kg bolus, n = 14) or hirulog (5 mg/kg bolus followed by 5 mg/kg/h for 2 h, n = 13) at the time of BA (2.5-mm balloon with three 60-second, 10-atm inflations 60 s apart). Angiograms performed before and after BA and before sacrifice were analyzed quantitatively. Rabbits were sacrificed 28 days after BA for quantitative histopathologic analysis. Minimum luminal diameter (mm) did not differ between treatment groups before (1.1 +/- 0.2 vs. 1.2 +/- 0.1 mm) or after (1.5 +/- 0.2 vs. 1.6 +/- 0.1) BA in arteries from heparin-versus hirulog-treated rabbits, respectively. At 28 days, however, minimum luminal diameter was significantly less (1.0 +/- 0.4 vs. 1.5 +/- 0.2, p = 0.0001) and percent stenosis was greater (0.46 +/- 0.25 vs. 0.22 +/- 0.08, p = 0.0002) in arteries from heparin- versus hirulog-treated rabbits, respectively. Similarly, quantitative histopathology showed less cross-sectional area narrowing by plaque in the hirulog group (56 +/- 24 vs. 42 +/- 21%, p = 0.04). CONCLUSION: A 2-hour infusion of hirulog at the time of angioplasty improved late angiographic luminal dimensions and reduced cross-sectional area narrowing by plaque in rabbits compared with heparin controls. Together with previous studies, this confirms a specific role for thrombin in restenosis after angioplasty.

Effect of thrombin inhibition with desulfatohirudin on early kinetics of cellular proliferation after balloon angioplasty in atherosclerotic rabbits.

BACKGROUND: Thrombin may have a pivotal role in restenosis after angioplasty. Hirudin, a potent thrombin inhibitor, reduces luminal narrowing by plaque after angioplasty in a rabbit model of atherosclerosis. Because cellular proliferation is believed to be an important mechanism for restenosis and thrombin has been shown to be a potent smooth muscle cell mitogen in vitro, we hypothesized that the mechanism of the effect of hirudin on limiting luminal narrowing by plaque occurs via inhibition of cellular proliferation. METHODS AND RESULTS: Femoral atherosclerosis was induced in 108 rabbits, and balloon angioplasty was performed. At angioplasty, group 1 rabbits (n=38) were treated with a 2-hour infusion of hirudin, and group 2 rabbits (n=41) were treated with heparin. Group 3 rabbits (n=29) were treated with hirudin (n=15) or heparin (n=14) and killed at 7 or 28 days to determine cross-sectional area narrowing by plaque and cellular proliferation with the use of bromodeoxyuridine labeling. At 29, 71, or 167 hours after angioplasty, group 1 and 2 rabbits were injected with 3H-thymidine and killed 1 hour later, and labeling indexes were determined. A significant increase in the index of 3H-thymidine-labeled nuclei was observed in the intima of "ballooned" arteries compared with "nonballooned" atherosclerotic arteries at both 30 hours (0.06+/-0.05 versus 0.01+/-0.01, P<.01) and 72 hours (0.10+/-0.06 versus 0.004+/-0.004, P<.01). By 7 days, the index of labeled cells was similar to baseline (0.04+/-0.03 versus 0.01+/-0.01, P=.12). Hirudin had no effect on the 3H-thymidine labeling indexes at any of the time points studied despite the fact that hirudin treatment in group 3 rabbits resulted in less cross-sectional area narrowing by plaque at both 7 and 28 days after angioplasty (41+/-16 versus 24+/-12 at 7 days and 60+/-21 versus 44+/-17 at 28 days, heparin versus hirudin; P<.03). CONCLUSIONS: Balloon angioplasty resulted in a marked increase in cellular proliferation that peaked at 72 hours. A 2-hour infusion of hirudin failed to reduce early 3H-thymidine labeling, suggesting that inhibition of cell proliferation within the first 7 days after angioplasty is not the predominant mechanism by which hirudin exerts its effect on limiting luminal narrowing by plaque 28 days after balloon angioplasty in this animal model.

Attenuation of the reflex pressor response to muscular contraction by an antagonist to somatostatin.

Although group III and IV fibers are known to compose the afferent pathway of the reflex arc causing the pressor response to static muscular contraction, little is known about the neurotransmitters released by these muscle afferents. Somatostatin might be one of these neurotransmitters because this peptide is found in the terminals of fine afferent fibers ending in the dorsal horn of the lumbar spinal cord. Therefore, in chloralose-anesthetized cats, the reflex pressor response to static contraction was examined before and after subarachnoid injections onto the lumbosacral cord of a peptide antagonist to somatostatin. We found that before giving the antagonist, the pressor response to contraction of the triceps surae muscles in 12 cats averaged 33 +/- 4 mm Hg, while 37 +/- 7 minutes after giving the antagonist, the pressor response averaged only 18 +/- 3 mm Hg (p less than 0.001). In contrast, the antagonist to somatostatin had no effect on either the pressor response to electrical stimulation of the cut central end of the sciatic nerve or the pressor response to stimulation of the posterior diencephalon. Furthermore, subarachnoid injection of a peptide antagonist to luteinizing hormone-releasing hormone had no effect on the reflex pressor response to static contraction. Our findings are consistent with the hypothesis that somatostatin plays a role in the spinal transmission of the contraction-induced pressor reflex arising from hind limb skeletal muscle.

Inhibition of aortic chemoreceptor discharge by pressor response to muscular contraction.

We have examined the effect of static contraction of the hindlimb muscles on the discharge of aortic chemoreceptors in chloralose-anesthetized cats. The responses of the chemoreceptors to contraction were dependent on the arterial pressure response to this maneuver. When contraction reflexly evoked a pressor response of at least 20 mmHg, the discharge of 26 chemoreceptors was reduced from control levels by 53% (P less than 0.01). The contraction-induced inhibition of chemoreceptor discharge was prevented by phentolamine, an alpha-adrenergic antagonist that also attenuated the contraction-induced pressor response. In addition, the inhibition evoked by contraction was simulated by injection of phenylephrine and inflation of an aortic balloon, both of which evoked pressor responses. However, when contraction failed to significantly change arterial pressure, the discharge of 20 aortic chemoreceptors was not significantly changed from control levels. We conclude that the reflex pressor response to static contraction inhibits the discharge of aortic chemoreceptors. This inhibition of discharge needs to be considered when interpreting the effects of aortic barodenervation on the cardiovascular responses to exercise.

Stimulation of groups III and IV phrenic afferents reflexly decreases total lung resistance in dogs.

Little is known about the reflex effect on airway caliber evoked by stimulation of phrenic afferents. Therefore, in chloralose-anesthetized, paralyzed dogs, we recorded airflow, airway pressure, arterial pressure, and heart rate while electrically stimulating a phrenic nerve. Total lung resistance was calculated breath by breath. The phrenic nerve was stimulated at 3, 5, 20, 70, 140, and 200 times motor threshold and the compound action potential was recorded. Stimulation of the phrenic nerve at three and five times threshold, which activated groups I, II, and a few group III fibers, had no effect on any of the variables measured. Stimulation at 20 times threshold, which activated many group III fibers and groups I and II fibers, reflexly decreased resistance. Stimulation at 70, 140, and 200 times threshold, which activated groups I-IV fibers, evoked progressively greater decreases in lung resistance. The reflex bronchodilation evoked by phrenic nerve stimulation was unaffected by propranolol or phentolamine but was abolished by atropine. We conclude that activation of groups III and IV phrenic nerve afferents reflexly decreased total lung resistance by withdrawing cholinergic tone to airway smooth muscle.