Tissue-type plasminogen activator (t-PA), a thrombolytic agent with higher affinity for fibrin, is an efficient agent for coronary thrombolysis both in animal models of coronary thrombosis and in patients with evolving myocardial infarction (MI) (1-6). t-PA can occur in two different forms, single- and two-chain t-PA (7). The two-chain form is converted from the single-chain form by plasmin on the fibrin surface (8). Such a difference in structure of the t-PA molecule may induce different biological functions. Comparative properties of single- and two-chain t-PA have been evaluated widely in clinical studies (9-15). However, in animal models, such comparative studies have been performed with rabbit jugular vein (16) and canine femoral vein (17) thrombosis models, but not with a canine coronary arterial thrombosis model. Because one of the important clinical targets is acute MI, experimental studies in coronary thrombosis should be used to examine more substantially the efficacies of thrombolytic agents. Therefore, we compared the thrombolytic efficacies on the fibrinolytic system of single-chain t-PA with those of two-chain t-PA and urokinase in a canine thrombosis model.
The human t-PA was expressed in Chinese hamster ovary cells transfected with a cDNA for human t-PA derived from Bowes' melanoma cells. The single-chain t-PA used consisted of predominantly single-chain form (94%) and was purified (18) from serum-free culture medium with aprotinin. The two-chain t-PA consisted of predominantly two-chain form (96%) and was purified (18) from culture medium containing bovine serum without aprotinin. The contents of single- and two-chain form were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reduced condition as described by Laemmli (19). The specific fibrinolytic activity of the single-chain t-PA was 480,000 IU/mg protein and that of the two-chain t-PA was 460,000 IU/mg protein. Urokinase, derived from human urine (high molecular weight type, 120,000 IU/mg protein) was purchased from Japan Chemical Research (Kobe, Japan). The potencies of these plasminogen activators were determined against international standards of t-PA and urokinase by clot lysis time assay (20). In this study, the units of t-PA and urokinase used were the international units. Urokinase 1 IU was equivalent to t-PA 7.54 IU by clot lysis assay. Twenty-four male Beagle dogs (Marshall), weighing 8.5-11 kg, were housed at 23° ± 2°C with a 12-h/12-h light/dark cycle. The experiments were conducted according to the Guidelines of Experimental Animal Care issued by the Japanese Prime Minister's office.
Dogs were anesthetized with sodium pentobarbital [30 mg/kg intravenously (i.v.), Somnopentyl, Pitman-Moore], and received additional doses as required. The dogs were intubated and ventilated under positive pressure with room air supplemented with oxygen. Blood gases were monitored and maintained within physiological range. A polyethylene catheter was placed in the abdominal aorta through the left femoral artery to monitor arterial pressure. Two cannulas were placed in the bilateral femoral vein for drug infusion, for administration of 50 mg/kg/min lidocaine in saline, and for venous blood sampling. Catheters were flushed with normal saline without heparin. A left thoracotomy was performed at the fifth intercostal space, and the heart was suspended in a pericardial cradle. Another catheter was inserted in the left ventricle through the left atrial appendage to monitor left ventricular pressure (LVP). Aortic blood flow was measured with a electromagnetic flow probe (Nihon Kohden, MF-27, ID 10 mm). Pressures were measured with Gould-Statham transducers (P50). Lead II ECG, heart rate (HR), aortic pressure, and all hemodynamic variables were continuously monitored (Nihon Kohden, RM-6000) and recorded (Nihon Kohden WT-685G and Graphtec Linearcorder Mark VII, WR-3101).
Coronary thrombus formation
The left common carotid artery was isolated, and a 7F Cobra catheter (type II, USCI) was inserted selectively in the left anterior descending coronary artery (LAD) under fluoroscopic guidance. A Teflon-coated guidewire (φ0.018 inch, Cook) was inserted, and the angiographic catheter was removed. A coil (3 mm long), which was formed by wrapping a copper wire (0.3 mm diameter) in a spiral around a 23- or 25-gauge needle, was advanced into the LAD distal to the first diagonal branch over the guidewire with a 5F JB 1 catheter (Cook). The guidewire and catheter were then withdrawn slowly. An occlusive thrombus formed ≤ 10 min and was confirmed by coronary angiography. Coronary angiograms were taken with an x-ray apparatus (Circlex, Shimazu) equipped with a videographic apparatus (A-400HP, Toshiba).
The dogs with total occlusion of coronary artery confirmed by coronary angiogram at 60 min after the thrombus formation were used for the thrombolysis experiments. The 24 dogs were divided into four groups randomly (6 dogs in each group). Single-chain t-PA (5,000 IU/kg/min), two-chain t-PA (5,000 IU/kg/min), urokinase (equivalent activity in vitro 663 IU/kg/min), or vehicle (physiological saline, pH 3, contained 0.1% human serum albumin) was administered intravenously at the rate of 50 ml/h for 1 h. Continuous infusion of thrombolytic agents was maintained by an infusion pump (Nipro) in all experiments. Thrombolysis was confirmed in each dog by coronary angiography performed every 5 min.
Venous blood samples were taken for determination of levels of fibrinogen, plasminogen, and α2-plasmin inhibitor and of concentration of t-PA related antigen and of that of urokinase-related antigen. The blood samples collected in sodium citrate and aprotinin (Trasyrole, Bayer, final concentration 1,250 KIU/ml) were used for the determination of fibrinogen, and those collected in sodium citrate were used for the remaining fibrinolytic factors. The blood was taken before coil insertion and just before and every 15 min after the onset of drug infusion. Blood samples were cooled immediately on ice, and plasma fraction was separated by centrifugation. Fibrinogen levels were determined by a thrombin time method of Clauss (21) at the end of the experiments, and other aliquots were stored frozen at -20°C until analyzed. α2-Plasmin inhibitor and plasminogen were determined by chromogenic substrate assay (22). Circulating levels of t-PA antigen and urokinase antigen were measured by enzyme-linked immunosorbent assay (23,24).
All data are mean ± SEM. Statistical analysis of the data for comparison of the four treatment groups was performed by one-way analysis of variance; p < 0.05 was considered significant.
Angiographic results confirmed the stability of thrombotic occlusion during the 1-h pretreatment period. Furthermore, repeated coronary angiography showed no evidence of antegrade flow in the LAD for the entire experimental period in all dogs in the vehicle group. Except for 1 dog in the single-chain t-PA group, all animals that were infused with thrombolytic agents exhibited LAD recanalization angiographically. A typical coronary angiogram is shown in Fig. 1.
The time to reperfusion was defined as the time at which the angiogram showed complete antegrade filling of LAD. The reperfusion time in dogs receiving thrombolytic agents was significantly shorter than that of the control dogs receiving vechicle (Table 1). One dog that received single-chain t-PA administration did not show reperfusion during the experimental period, but other dogs with thrombolytic agents exhibited clear recanalization, which was identified by angiogram, However, the reperfusion time was not significantly different in the three thrombolytic agents used in the present study. Reocclusion occurred in 1 dog in the two-chain t-PA group. Reperfusion had little effect on hemodynamic parameters, including aortic pressure, HR, aortic flow, LV systolic pressure, LV end-diastolic pressure (LVEDP) and LV dP/dt in each group (Fig. 2).
Plasma t-PA antigen levels increased rapidly and reached approximate plateau (0.4 μg/ml) within the first 15 min of intravenous t-PA infusion in both the single- and the two-chain t-PA groups and were not detected in the vehicle group (Fig. 3). Plasma urokinase antigen levels exhibited a tendency similar to that of t-PA antigen levels, but were lower (0.2 μg/ml). Figures 4-6 summarize the results of fibrinogen, plasminogen, and α2-plasmin inhibitor levels. Neither single nor two-chain t-PA caused significant depletion of fibrinogen (81.6 ± 4.6 and 86.1 ± 3.0%, respectively), whereas urokinase caused marked decrease of fibrinogen (43.5 ± 15.5%, p < 0.01, as compared with vehicle group) and 3 of the dogs had levels of <20% of preinfusion values. Plasma plasminogen levels were significantly decreased only in the urokinase group, similar to plasma fibrinogen (50.6 ± 15.1%). Plasma α2-plasmin inhibitor levels were significantly decreased in the urokinase and single-chain t-PA groups (43.5 ± 17.2 and 69.3 ± 3.2%, respectively).
In the present study, thrombolytic effects of single- and two-chain t-PA were evaluated in a canine coronary artery thrombosis model. The occlusive thrombus in coronary artery was produced by a copper coil. At 1 h after the thrombus formation, thrombolytic agents were administered by intravenous constant infusion. To allow evaluation of the effects of t-PA on the thrombolytic efficacies, heparin was not infused. The administered dosage was determined to induce a reperfusion rate of >80% by the infusion of two-chain t-PA in the same canine coronary artery model. To compare both forms of t-PA with urokinase, the administered dosage of urokinase was set to show the activity equivalent to that of two-chain t-PA by clot lysis time method (20). Thrombolytic efficacies assessed by the reperfusion rate and the time to reperfusion were not different among the three groups with thrombolytic treatment. Single-chain t-PA yielded plasma t-PA antigen levels similar to those obtained with two-chain t-PA. Although urokinase, which has no fibrin specificity, caused marked fibrinogen depletion and systemic fibrinolytic activation, both single- and two-chain t-PA had little effect on the fibrinolytic system.
Clinical studies of two forms of t-PA (9-15), indicate that single-chain t-PA is less potent than two-chain t-PA in terms of both coronary reperfusion and systemic fibrinogenolysis. As for the cause, single-chain t-PA is assumed to yield plasma levels ≈35% lower than those obtained with two-chain t-PA and single-chain t-PA is assumed to have rapid clearance. However, our results demonstrate that single-chain t-PA yielded plasma levels similar to those obtained with two-chain t-PA. Although the reason for this discrepancy is not known, the species differences may be one cause. Indeed, the disposition rates of single- and two-chain forms of t-PA in rabbits are identical (25).
The differences in structure between single- and two-chain t-PA may be trivial. Moreover, t-PA derived from different sources may vary in the extent of glycosylation as well as in carbohydrate structure. Glycosylated single-chain urokinase is more effective than nonglycosylated single-chain urokinase (26). Although urokinase and t-PA used in the present study were of the glycosylated form, the fine structure of carbohydrate chain has not yet been determined.
Steady-state t-PA antigen levels were achieved ≈15 min after onset of infusion. These results are consistent with previous observations in dogs (27,28). They also demonstrated that at steady state active t-PA accounted for 40-60% of the total t-PA antigen. Neither single- nor two-chain t-PA induced systemic fibrinogen breakdown at the dosage used in the present study (Fig. 4). α2-Plasmin inhibitor was significantly decreased in the single-chain t-PA group as compared with vehicle. However, the comparison of α2-plasmin inhibitor between single- and two-chain t-PA was not significant. t-PA induces thrombolysis with minimal systemic lytic effect, attributed to its high affinity for fibrin (29) and enhancement of its activity in the presence of fibrin (30). In the present study, the clot-selective effect of t-PA was reproduced. In contrast to both forms of t-PA, urokinase, which has no fibrin specificity (29), induced systemic fibrinolytic state, accompanied by the consumption of plasminogen, α2-plasmin inhibitor, and fibrinogen (31). Thus, single- and two-chain t-PA were demonstrated to be safe thrombolytic agents as compared with urokinase. Our results indicate that single-chain t-PA possesses thrombolytic efficacy equipotent to that of two-chain t-PA in an animal model of coronary thrombosis.
1. The TIMI Study Group. The thrombolysis
in myocardial infarction (TIMI) trial: phase 1 findings. N Engl J Med
2. Verstraete M, Bernard R, Bory M, et al. Randomised trial of intravenous recombinant human tissue-type plasminogen activator versus intravenous streptokinase in acute myocardial infarction. Lancet
3. Verstraete M, Bliefeld W, Brower RW, et al. Doubleblinded randomised trial of intravenous tissue plasminogen activator versus placebo in acute myocardial infarction. Lancet
4. Gold HK, Fallon JT, Yasuda T, et al. Coronary thrombolysis
with recombinant human tissue-type plasminogen activator. Circulation
5. Bergmann SR, Fox KAA, Terpogossian MM, Sobel BE, Collen D. Clot-sensitive coronary thrombolysis
with tissue-type plasminogen activator. Science
6. Flameng W, Van De Werf F, Vanharcke J, Verstraete M, Collen D. Coronary thrombolysis
and infarct size reduction after intravenous infusion of recombinant tissue-type plasminogen activator in non-human primates. J Clin Invest
7. Ranby M, Bergsdorf N, Nilsson T. Enzymatic properties of the single- and two-chain form of tissue plasminogen activator. Thromb Res
8. Rijken DC, Hoylaerts M, Collen D. Fibrinolytic properties of single-chain and two-chain human extrinsic (tissue-type) plasminogen activator. J Biol Chem
9. Harry D, Garabedion SM, Herman K, Gould MD, Robert C, Leinbach MD. Comparative properties of two clinical preparations of recombinant human tissue-type plasminogen activator in patients with acute myocardial infarction. J Am Coll Cardiol
10. Topol EJ, Morris DC, Smalling RW. A multicenter, randomized, placebo-controlled trial of a new from of recombinant tissue-type plasminogen activator (activase) in acute myocardial infarction. J Am Coll Cardiol
11. Mueller HS, Rao AK, Forman SA, TIMI Investigators. Thrombolysis
in myocardial infarction (TIMI): comparative studies of coronary reperfusion and systemic fibrinogenolysis with two-forms of recombinant tissue-type plasminogen activator. J Am Coll Cardiol
12. Garabedian HD, Gold HK, Leinbach RC, et al. Labolatory monitoring of hemostasis during thrombolytic therapy with recombinant human tissue-type plasminogen activator. Thromb Res
13. Gruppo Italiano per lo Studio della Sopravvivenza nell'Infarto Miocardico. GISSI-2: a factorial randomized trial of alterplase versus streptokinase and heparin versus no heparin among 12490 patients with acute myocardial infarction. Lancet
14. ISIS-3 (Third International Study of Infarct Survival) Collaborative Group. ISIS-3: a randomised comparison of streptokinase vs tissue plasminogen activator vs anistreplase and of aspirin plus heparin vs aspirin alone among 41299 cases of suspected acute myocardial infarction. Lancet
15. The GUSTO investigators. An international randomized trial comparing four thrombolytic strategies for acute myocardial infarction. N Engl J Med
16. Collen D, Stassen JM, Verstraete M. Thrombolysis
with human extrinsic (tissue-type) plasminogen activator in rabbits with experimental jugular vein thrombosis. J Clin Invest
17. Korninger C, Matsuo O, Suy R, Stassen JM, Collen D. Thrombolysis
with human extrinsic (tissue-type) plasminogen activator in dogs with femoral vein thrombosis. J Clin Invest
18. Kaufman RJ, Wasley JC, Spiliotes AJ, et al. Coamplification and coexpression of human tissue-type plasminogen activator and murine dihydrofolate reductase sequences in chinese hamster ovary cells. Mol Cell Biol
19. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4
20. Gaffney PJ, Curtis AD. A collaborative study of a proposed international standard for tissue plasminogen activator (t-PA). Thromb Haemost
21. Clauss VA. Gerinnungsphysiologische Schnellmethode zur bestimnung des Fibrinogens. Acta Haematol
22. Friberger P. Methods for the determination of plasmin, antiplasmin and plasminogen by means of the substrate S-2251. Haemostasis
23. Bergsdorf N, Nilsson T, Wallen P. An enzyme linked immunoabsorbent assay for determination of tissue plasminogen activator applied to patients with thromboembolic disease. Thromb Haemost
24. Stump DC, Thienpont M, Collen D. Urokinase-related proteins in human urine. J Biol Chem
25. Korninger C, Stassen JM, Collen D. Turnover of human extrinsic (tissue-type) plasminogen activator in rabbits. Thromb Haemost
26. Lenich C, Pannel R, Henkin J, Gurewich V. The influence of glycosylation on the catalytic and fibrinolytic properties of prourokinase. Thromb Haemost
27. Van De Werf F, Bergmann SR, Fox KAA, et al. Coronary thrombolysis
with intravenously administered human tissue-type plasminogen activator produced by recombinant DNA technology. Circulation
28. Fong KL, Carl S, Mico BA, et al. Dose-dependent pharmacokinetics of recombinant tissue plasminogen activator in anesthetized dogs following intravenous infusion. Drug Metab Dispos
29. Rijken DC, Collen D. Purification and characterization of the plasminogen activator secreted by human melanoma cells in culture. J Biol Chem
30. Hoylaerts M, Rijken DC, Lijnen HR, Collen D. Kinetics of the activation of plasminogen by human tissue plasminogen activator. Role of fibrin. J Biol Chem
31. Matsuo O, Rijken DC, Collen D. Comparison of the relative fibrinogenolytic, fibrinolytic and thrombolytic properties of tissue plasminogen activator and urokinase in vitro. Thromb Haemost