The use of thrombin inhibitors to examine the role of thrombin as a mediator of (a) reocclusion after successful thrombolysis, and (b) smooth muscle hyperplasia after vessel wall injury in animal model systems
Date of Award
Doctor of Philosophy (PhD)
Using antithrombin III-dependent and independent thrombin inhibitors, we evaluated the role of thrombin in (a) reocclusion after successful thrombolysis, and (b) intimal thickening and stenosis induced by injury to a vessel wall. To accomplish this, two animal models were developed in the rat. The first was a model of tissue plasminogen activator (tPA) induced-thrombolysis, whereas the second was a carotid model of injury-induced stenosis.
In the rat model of tPA-induced thrombolysis, we compared the effects of heparin, hirudin, hirulog (a synthetic hirudin-derived peptide), and D-Phe-Pro-ArgCH₂Cl (PPACK) in doses that produced a 4-fold prolongation of the baseline activated partial thromboplastin time (APTT) with saline, in terms of their ability to accelerate thrombolysis and to prevent reocclusion. A thrombus rich in red cells and fibrin was formed in the distal aorta by a combination of vessel injury and stasis. Thrombolysis was induced with tPA (1 mg/kg bolus, followed by 1 mg/kg/hr over 30 min.) and the rats were randomized to receive a concomitant 80 min. infusion of a thrombin inhibitor or saline. Blood flow and pre- and post-stenotic blood pressures were monitored continuously, and the time to clot lysis, the duration of vessel patency, and the number of reocclusions were determined. Compared to saline, heparin had no significant effect on these variables. In contrast, hirudin, hirulog and PPACK significantly (p<0.01) increased the percentage of time that the vessel remained patent from 63.9 ± 7.7 to 90.7 ± 2.2, 94.0 ± 0.9, and 94.7 ± 1.0% respectively, by significantly (p<0.01) decreasing the number of reocclusions. The superiority of the ATIII-independent inhibitors over heparin supports the hypothesis that the limited effectiveness of heparin in this setting reflects its inability to inactivate clot-bound thrombin. Compared to saline, hirulog and PPACK also significantly (p<0.02) accelerated the time to thrombolysis from 10.5 ± 2.3 to 4.4 ± 0.6, and 4.2 ± 0.8 min., respectively, whereas heparin and hirudin did not. The ability of the lower molecular weight inhibitors of thrombin to accelerate lysis may reflect their greater accessibility to clot-bound thrombin. Hirudin, standard heparin, and low molecular weight heparin were compared with saline in terms of their ability to prevent injury-induced stenosis in the rat common carotid artery. After balloon injury, animals were randomized to receive a one week course of subcutaneous treatment with a thrombin inhibitor or saline. All of the thrombin inhibitors were given in concentrations that produced a continuous 6-fold prolongation of the baseline thrombin clotting times, and 2-fold prolongation of the APTT. Two weeks after injury, the animals were euthanized and the extent of stenosis was assessed by a combination of quantitative angiography, resin casting, and planimetry. Compared to saline, only low molecular weight heparin significantly (p<0.01) inhibited injury-induced stenosis as assessed by angiography (from 22.9 ± 3.0% to 8.8 ± 7.8%), resin casting (from 46.7 ± 4.8% to 22.5 ± 8.3%), and by planimetry (from 21.8 ± 3.4% to 11.3 ± 2.1%). (Abstract shortened by UMI.)
Klement, Petr, "The use of thrombin inhibitors to examine the role of thrombin as a mediator of (a) reocclusion after successful thrombolysis, and (b) smooth muscle hyperplasia after vessel wall injury in animal model systems" (1994). Open Access Dissertations and Theses. Paper 4046.