THROMBOLYSIS Synergism of thrombolytic agents in vivo

The existence of significant synergism between tissue-type plasminogen activator (t-PA) and single-chain urokinase-type plasminogen activator (scu-PA), and between t-PA and urokinase in thrombolysis in vivo is described. In a quantitative preparation of thrombolysis, consisting of rabbits in which a blood clot was induced in the jugular vein with `251-labeled fibrin, intravenous infusion over 4 hr of t-PA, scu-PA, or urokinase in amounts of 0.5, 1.0, or 2.0 mg/kg body weight resulted in significant thrombolysis (30% to 60%). The simultaneous infusion of t-PA and scu-PA or of t-PA and urokinase had a significantly greater (p < .001) thrombolytic effect than could be anticipated on the basis of the added effects of each agents alone. However, no synergism was observed between scu-PA and urokinase. The observed a2-antiplasmin consumption and fibrinogen breakdown after urokinase at higher doses did not occur with the equivalent thrombolytic combinations of t-PA and urokinase. The combined use of synergic thrombolytic agents in patients may permit a significant reduction in total administered doses, probably with elimination of the systemic activation of the fibrinolytic system and the concomitant fibrinogen breakdown that is unavoidable with the currently used thrombolytic doses of each agent. Circulation 74, No. 4, 838-842, 1986. ONE APPROACH to the treatment of thromboembolic disease is the use of pharmacologic agents that activate the fibrinolytic system in blood. During activation of this system, the inactive zymogen plasminogen is converted to the active enzyme plasmin, a proteolytic enzyme with relatively low fibrin specificity. When plasmin circulates freely in the blood, it degrades a number of proteins, including fibrinogen and the blood coagulation factors V and VIII. Plasma does, however, contain a fast-acting plasmin inhibitor, a2-antiplasmin, which reacts extremely rapidly with free plasmin but only slowly with plasmin generated on the fibrin surface. Clot-specific fibrinolysis therefore requires generation of plasmin at the fibrin surface, out of reach of a2-antiplasmin.' One promising approach to obtaining specific thrombolysis is the use of thrombolytic agents, the action of which is stimulated by the presence of fibrin. Indeed, streptokinase and urokinase, which have no From the Center for Thrombosis and Vascular Research, University of Leuven, Belgium. Address for correspondence: D. Collen, M.D., Ph.D., Center for Thrombosis and Vascular Research, K. U. Leuven Campus Gasthuisberg, Gebouw Onderwijs en Navorsing, Herestraat 49, B-3000 Leuven, Belgium. Received April 1, 1986; revision accepted June 5, 1986 Dr. Stump is the recipient of Clinical Investigator Award HL 01176 of the National Heart. Lung, and Blood Institute. specific affinity for fibrin, activate circulating and fibrin-bound plasminogen equally well. Plasmin formed in the circulation is immediately neutralized by a2antiplasmin, and once the inhibitor is exhausted, several plasma proteins are then degraded by plasmin (fibrinogen, factor V, factor VIII). An overt systemic fibrinolytic state and very low fibrinogen levels may occasionally lead to major bleeding, although this is not a frequent complication when thrombolytic agents are only used over short time periods. Two plasminogen activators with demonstrated fibrin specificity are tissue-type plasminogen activator (t-PA), 1 2.* and single-chain urokinase-type plasminogen activator (scu-PA).3` Although both agents have shown promise in preliminary clinical studies for efficacious fibrin-specific thrombolysis, a certain degree of systemic fibrinolytic activation has been evident, as manifested by decreased fibrinogen and a2-antiplasmin levels in some patients. To optimize thrombolytic therapy in terms of both efficacy and safety some modifications may therefore still be required. *The nomenclature tissue-type plasminogen activator (t-PA, either in its single-chain form or two-chain form), single-chain urokinase-type plasminogen activator (scu-PA), and urokinase used is as adopted by the Intemnational Committee on Thrombosis and Haemostasis (Thromb Haemost 54: 893, 1985). CIRCULATION 838 by gest on O cber 9, 2017 http://ciajournals.org/ D ow nladed from LABORATORY INVESTIGATION-THROMBOLYSIS It has been recently shown that the mechanisms of action of t-PA and scu-PA are quite distinct. t-PA binds directly to the fibrin clot, where its activation of plasminogen is markedly enhanced.7 scu-PA, on the other hand, is capable of very efficient direct activation of plasminogen both in the presence and absence of fibrin. In plasma this activation process is strongly inhibited and is reversed only when fibrin is present, a process that occurs without direct binding of scu-PA to fibrin.' Because of the differing mechanisms of these agents, the possibility of synergistic action has been considered and investigated in vitro. With recombinant scu-PA and t-PA, only additive clot lysis effects could be demonstrated.8 More detailed studies using natural scu-PA from a human lung adenocarcinoma cell line9 have confirmed these earlier observations.* However, because of the more pronounced fibrin-specific effects of scu-PA seen in vivo,'0 and because of the recognition of additional factors in vivo that regulate the thrombolytic action of t-PA,'` we have explored further the possibility of synergism in a wellcharacterized animal preparation of jugular vein thrombosis.12 Materials and methods Thrombolytic agents. t-PA was purified from the conditioned medium of a melanoma cell line in the absence of the plasmin inhibitor aprotinin13' 14 and scu-PA was purified from the conditioned medium of a lung adenocarcinoma cell line,9 as described elsewhere. t-PA was in the two-chain form with a specific activity of 500,000 IU/mg when calibrated against the first International Reference Preparation for t-PA. 15 scu-PA was present as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with an apparent Mr of 54,000 under reducing conditions. Two-chain urokinase-type plasminogen activator was removed by benzamidine-Sepharose absorption to less than 1% as measured by activity with the chromogenic substrate Pyro-Glu-Gly-Arg-p-nitroanilide (S-2444). The preparation so treated had a specific activity in plasminogen-enriched bovine fibrin plates of 80,000 lU/mg. Both t-PA and scuPA were essentially free of contaminating proteins. Urinary urokinase (Winkinase), specific activity 100,000 IU/mg, was a gift from Dr. E. Murano, Bureau of Biologics, Bethesda, MD. Rabbit preparation of jugular vein thrombosis. Experimental thrombolysis in rabbits with jugular vein thrombosis was carried out as previously described. 12 In essence, an external jugular vein was exposed and cleared over a distance of 4 cm. Small side branches were ligated, a cannula was introduced in the main side branch (facial vein), and a woolen thread was introduced in the lumen. A 4 cm long vein segment was isolated between two vessel clamps and the volume of the segment was determined by saline injection via the catheter. The vein segment was then rinsed with a thrombin solution (100 NIH U/ml) and filled with a mixture of '251-labeled fibrinogen and rabbit blood, avoiding injection of air bubbles. In all instances a clot *Collen D et al: Unpublished observations. Vol. 74, No. 4, October 1986 formed quickly and was allowed to age for 30 min. The 125I content of the clot was determined from an isotope balance. 12 Thrombolysis was performed by intravenous infusion of solvent (controls), t-PA, scu-PA, or urokinase or simultaneous infusions of t-PA and scu-PA, of t-PA and urokinase, or of scuPA and urokinase in a total volume of 20 ml, by a constant-rate infusion pump. The infusions were given via a contralateral marginal ear vein over 4 hr. Thirty minutes after the end of the infusion the thrombosed segment of the jugular vein was removed after careful suturing of both ends, and the residual radioactive material was measured. The extent of thrombolysis at completion of the infusion was calculated as the difference between the radioactivity originally incorporated in the clot and that remaining in the vein segment, and was expressed in percent of the original radioactivity. Three experiments were performed with each dose and combination of thrombolytic agents, and the results were expressed as mean + SEM. Two milliliter blood samples were drawn on citrate (final concentration O.OlM) before the start of the infusion and at hourly intervals for 270 min. These plasma samples were used for measurement of radioactivity, fibrinogen, and a2-antiplasmin levels as described. 12