- Email: [email protected]
Thrombolysis: A Critical First-Line Therapy with an Unfulfilled Potential
REVIEW
Thrombolysis: A Critical First-Line Therapy with an Unfulfilled Potential Victor Gurewich, MD Vascular Research Laboratory, Mt Auburn Hospital, Cambridge, Mass; Department of Medicine, Harvard Medical School, Cambridge, Mass.
ABSTRACT A blood clot or thrombus triggers the onset of most vascular diseases, like stroke or heart attack. Thrombolysis is the only treatment that can restore blood flow rapidly and easily. Unfortunately, the standard thrombolytic, tissue plasminogen activator (tPA), has proven inadequate and is being replaced by invasive endovascular procedures, which are time consuming and limited in their availability in relation to the scope of the problem. Historically, when tPA clinical trials began, it was not recognized sufficiently that without the other natural plasminogen activator, prourokinase (proUK), thrombolysis by tPA was seriously compromised. The reason is that the 2 activators have complementary mechanisms of action in fibrinolysis, making their combination a requirement for optimal efficacy and synergy. Biological fibrinolysis also uses both activators, explaining why such low endogenous concentrations are sufficient. A low-dose sequential combination of tPA and proUK was tested in acute myocardial infarction, where it was exceptionally effective and safe. Because native proUK at pharmacological doses was vulnerable to spontaneous conversion to urokinase, jeopardizing safety, a site-directed mutant was developed that improved proUK’s plasma stability fivefold without interfering with its mode of action. Mini-bolus tPA followed by low-dose proUK infusion is a simple, safe, effective, and promising first-line treatment of acute thrombotic disorders. Ó 2016 The Author. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). The American Journal of Medicine (2016) 129, 573-575 KEYWORDS: Fibrinolytic synergy; Mistaken assumptions; Thrombolysis; Unfulfilled potential
Cardiovascular diseases like acute myocardial infarction, ischemic stroke, or pulmonary thromboembolism, are the leading causes of death and disability worldwide, often occurring in the prime of life. Their immediate cause is an occlusive blood clot, or thrombus, and its timely removal restores blood flow in the vessel, salvages organ function, and saves lives. The only therapy capable of accomplishing this promptly is thrombolysis. Unfortunately, the thrombolytic regimen that has been in use for more than 20 years has not been sufficiently effective or safe. Funding: None. Conflict of Interest: The author is the Chief Scientific Officer of Thrombolytic Science International, LLC (TSI; Cambridge, Mass.), a company that is developing mutant proUK. Authorship: VG is the sole author of this manuscript. Requests for reprints should be addressed to Victor Gurewich, MD, Vascular Research Laboratory, Mt Auburn Hospital, 300 Mt Auburn St., Cambridge, MA 02138. E-mail address: [email protected]
As a result, thrombolysis is being replaced by cathetermediated endovascular procedures. Although these have met with some success, they are handicapped by being demanding technically, time-consuming, costly, and insufficiently accessible for the size of the problem. The standard thrombolytic regimen in use consists of high doses of the human fibrinolytic tissue plasminogen activator (tPA) alone. This contrasts with how tPA functions biologically in the fibrinolytic pathway. There, tPA induces fibrinolysis in combination with prourokinase (proUK), the other natural plasminogen activator. When these activators were tested in a low-dose sequential combination, a synergistic fibrinolytic effect was observed.1 A comparable effect was later also confirmed in vivo.2 Nevertheless, the standard tPA monotherapy regimen has endured for more than 2 decades, despite its shortcomings. As unreasonable as this may seem, the history of tPA provides some explanations. When tPA was first purified, characterized,3 and then produced by recombinant technology in
0002-9343/Ó 2016 The Author. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/). http://dx.doi.org/10.1016/j.amjmed.2015.11.033
574
The American Journal of Medicine, Vol 129, No 6, June 2016
subject and the discontinuation of the journal Fibrinolysis, 1982,4 it was embraced with enthusiasm and high expectations which was devoted to this discipline. as a much-needed replacement for streptokinase (SK), a fibriIt is noteworthy, however, that the clinical findings nolytic discovered about 50 years earlier. This was because SK reviewed above began with a misunderstanding of tPA’s was an antigenic bacterial product that had a nonspecific, inphysiological role in thrombolysis, the principal biological direct, and inefficient mechanism of plasminogen activation defense against intravascular clot formation. It was believed that induced a bleeding diathesis. By contrast, tPA is a human that tPA was the sole activator protein with a fibrin-specific, direct, involved in this pathway, which and efficient mechanism of plasCLINICAL SIGNIFICANCE ignored the role of the other natural minogen activation. Therefore, it plasminogen activator, proUK, first was assumed with confidence that it Intravascular thrombosis is a leading identified in 1981.8,9 Because would be correspondingly more cause of mortality and morbidity. effective and safer therapeutically proUK has no fibrin-binding Thrombolytic therapy has not fulfilled its than SK. domain, but instead has a cellpromise and is being replaced by timeThese expectations were not receptor-binding domain, it was borne out by the subsequent clinbelieved that its principal fibrinoconsuming endovascular procedures. ical experience. In 3 mega-trials in lytic function was in pericellular The standard thrombolytic regimen is acute myocardial infarction5-7 proteolysis of the extravascular compromised by a misunderstanding of space.10 Subsequently, this idea was where tPA and SK were tPA’s biological role in fibrinolysis. compared, a total of 94,720 pashown to be at odds with the fibritients had to be tested before a nolytic effects seen in gene-targeted In this pathway, tPA initiates fibrinostatistically significant reduction mice deficient in the proUK cell lysis, which is then completed by the in mortality with tPA was found. receptor.11 The concept was also other natural activator, prourokinase No mortality reduction was found inconsistent with the complemen(proUK). in the first 2 trials, and in the last tary mechanism of fibrin-dependent Together, they have a synergistic trial, a small but statistically sigplasminogen activation by tPA and nificant reduction was found in proUK, which made their combined thrombolytic effect making therapy more only 1 of the 4 groups tested.7 In effects a requirement for efficacy at effective and safer. fibrin-specific doses, as well as addition, tPA was also not safer, making it synergistic.1 because significantly more patients with intracranial bleeding complications were seen Due to this property of the activators, neither tPA nor with tPA than with SK, despite tPA’s fibrin specificity. proUK alone can be sufficiently effective except at doses Paradoxical findings such as these would ordinarily be high enough to overcome their fibrin specificities, at which used to obtain new insights into mechanisms of action, but bleeding side effects also become more likely. As a result, this did not occur. Instead, when tPA received marketing when tPA was given alone, concentrations more than a approval after the last mega-trial, its use in the treatment of thousand-fold higher than physiological were required, and acute myocardial infarction became the exclusive focus of efficacy was still disappointing and bleeding complications attention. The mega-trial findings were interpreted as simply were higher than with SK.5-7 meaning that the clinical differences “between different The synergistic fibrinolytic effect of the combination also fibrinolytic agents are unlikely to be large.” may explain why even at the very low concentrations at Once it was on the market, tPA quickly became the which tPA and proUK are found normally, 2 exceptionally thrombolytic of choice, soon becoming so equated with potent inhibitors, plasminogen activator inhibitor-1 and a2thrombolysis that it was no longer specified as the activator antiplasmin, are nevertheless required to control their in publications on this subject. It was assumed that tPA was plasminogen-activating effect. The congenital absence of the plasminogen activator. As a result, when, over the suceither inhibitor is associated with a bleeding diathesis.12 ceeding years, the disappointing clinical results foretold by Clot lysis studies of synergy in vitro showed it to be optimal the mega-trials became increasingly troublesome, it was when an unequal ratio and sequential combination of the actaken as evidence of the limitations of all thrombolytic tivators was used.1 A comparable tPA and proUK combination therapy. Therefore, invasive endovascular procedures was evaluated in 101 patients with acute myocardial infarcbecame the alternative rather than a different thrombolytic tion.2 The treatment consisted of a mini bolus of tPA (5% of regimen. In acute myocardial infarction, percutaneous corthe monotherapy dose), followed by an infusion of a moderate onary intervention has now become the treatment of choice, dose of proUK (50% of the monotherapy dose) for 90 minutes. and endovascular procedures are being used with increasing Coronary reperfusion (Thrombolysis in Myocardial Infarction frequency for ischemic stroke as well, despite their grades 2 and 3) at 24 hours was 100%; there was only one limitations. death and no strokes. This compares with 6.3% deaths and This experience with tPA led to a conviction that 1.5% strokes in the best of the tPA mega-trials.7 thrombolytic therapy had reached its therapeutic limits. This This study design was based on the physiological fibriwas reflected by a substantial decrease in publications on the nolytic pathway in which an intravascular thrombus induces
Gurewich
Thrombolysis: A Critical First-Line Therapy
the local release of tPA from the vessel wall. The tPA binds to fibrin in the thrombus and initiates fibrinolysis.13 This creates additional plasminogen-binding sites on fibrin,14 which are 2 in number.15 Plasminogen on the first of these is activated specifically by proUK,16,17 and proUK is, in turn, activated by plasmin to urokinase (UK)18 via a hypercatalytic transitional state.19 The UK then activates the remaining fibrin-bound plasminogen. Therefore, tPA activates the first plasminogen by binding to an adjacent site on fibrin,20 and proUK/UK activate the remaining 2 plasminogens. ProUK has a double role in fibrinolysis due to its activation to UK, whereas tPA’s properties remain unchanged by its conversion to the 2-chain form. Despite the clinical success of the Pro-Urokinase and t-PA Enhancement of Thrombolysis (PATENT) Trial, it attracted little attention, reflecting the dominance of tPA monotherapy and the belief that there was little difference among thrombolytic activators. No follow-up was possible because proUK development was abandoned not long thereafter. This was because in phase-3 trials, proUK was found to be unstable in plasma at pharmacological concentrations and converted to UK.21 Being nonspecific, UK-induced systemic plasmin generation resulting in a hemophilia-like state including degradation of clotting factors I, V, and VIII. For this reason, the European Medicines Agency turned down the application for a proUK marketing license submitted by Grünenthal (Aachen, Germany). Because proUK’s instability was unrelated to its fibrinolytic mechanism of action, this problem was addressed by site-directed mutagenesis, which improved its plasma stability fivefold without interfering with its mechanism of action.22-24 By contrast, the bleeding side effects with tPA are primarily due to its lysis of hemostatic fibrin at vascular repair sites,25,26 which is due to its therapeutic fibrinolytic mechanism of action. In conclusion, by utilizing the natural, complementary, and synergistic properties of the 2 activators, the sequential combination has the potential to provide a safe first-line treatment for stroke and acute myocardial infarction, thereby reducing the delay to reperfusion by current therapies of these conditions.
References 1. Pannell R, Black J, Gurewich V. The complementary modes of action of tissue plasminogen activator (t-PA) and pro-urokinase (pro-UK) by which their synergistic effect on clot lysis may be explained. J Clin Invest. 1988;81:853-859. 2. Zarich SW, Kowalchuk GJ, Weaver WD, et al; PATENT Study Group. Sequential combination of thrombolytic therapy for acute myocardial infarction: results of the pro-urokinase and t-PA enhancement of thrombolysis (PATENT) trial. J Am Coll Cardiol. 1995;26:374-379. 3. Rijken DC, Collen D. Purification and characterization of the plasminogen activator secreted by human melanoma cells in culture. J Biol Chem. 1981;256:7035-7041. 4. Pennica D, Holmes WE, Kohr WJ, et al. Cloning and expression of human tissue-type plasminogen activator cDNA in E. coli. Nature. 1983;301:214-221. 5. Gruppo Italiano Per Lo Studio Della Sopravvivenze Nell’Infarto Miocardico. GISSI-2: a factorial randomised trial of alteplase versus streptokinase and heparin versus no heparin among 12,490 patients with acute myocardial infarction. Lancet. 1990;336(8707):65-71.
575 6. 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 41,299 cases of suspected acute myocardial infarction. Lancet. 1992;339(8796):65-71. 7. The GUSTO Investigators. An international randomized trial comparing four thrombolytic strategies for acute myocardial infarction. N Engl J Med. 1993;329:673-682. 8. Husain SS, Gurewich V, Lipinski B. Purification of a new high MW single chain form of urokinase (UK) from urine. Thromb Haemost. 1981;46, 11 (abstr). 9. Husain SS, Gurewich V. Purification and partial characterization of a single chain, high molecular weight form of urokinase from human urine. Arch Biochem Biophys. 1983;220:31-38. 10. Lijnen HR, Collen D. Mechanisms of physiological fibrinolysis. Baillieres Clin Haematol. 1995;8:277-290. 11. Bugge TH, Flick MJ, Danton MJS, et al. Urokinase-type plasminogen activator is effective in fibrin clearance in the absence of its receptor or tissuetype plasminogen activator. Proc Natl Acad Sci U S A. 1996;93:5899-5904. 12. Fay W, Parker A, Condrey L, Shapiro AD. Human plasminogen activator inhibitor-1 (PA1) deficiency: characterization of a large kindred with a null mutation in the PA1-1 gene. Blood. 1997;90:204-208. 13. 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. 1982;257:2912-2919. 14. Harpel PC, Chang TS, Verderber E. Tissue plasminogen activator and urokinase mediate the binding of Glu-plasminogen to plasma fibrin I. Evidence for new binding sites in plasmin-degraded fibrin I. J Biol Chem. 1985;260, 443 (abstr). 15. Suenson E, Lutzen O, Thorsen S. Initial plasmin-degradation of fibrin as the basis of a positive feed-back mechanism in fibrinolysis. Eur J Biochem. 1984;140:513-522. 16. Liu J, Gurewich V. Fragment E-2 from fibrin substantially enhances pro-urokinase-induced glu-plasminogen activation. A kinetic study using a plasmin-resistant mutant pro-urokinase (A-158-rpro-UK). Biochemistry. 1992;31:6311-6317. 17. Fleury V, Lijnen HR, Angles-Cano E. Mechanism of the enhanced intrinsic activity of single-chain urokinase-type plasminogen activator during ongoing fibrinolysis. J Biol Chem. 1993;268:18554-18559. 18. Petersen LC. Kinetics of reciprocal pro-urokinase/plasminogen activation. Stimulation by a template formed by the urokinase receptor bound to poly (D-lysine). Eur J Biochem. 1997;245:316-323. 19. Liu J, Pannell R, Gurewich V. A transitional state of pro-urokinase which has a higher catalytic efficiency against glu-plasminogen than urokinase. J Biol Chem. 1992;267:15289-15292. 20. Voskuilen M, Vermond A, Veeneman GH, et al. Fibrinogen lysine residue Aa 157 plays a crucial role in the fibrin-induced acceleration of plasminogen activation, catalyzed by tissue-type plasminogen activator. J Biol Chem. 1987;262:5944-5946. 21. PRIMI Trial Study Group. Randomized double-blind trial of recombinant pro-urokinase against streptokinase in acute myocardial infarction. Lancet. 1989;1:863-867. 22. Liu JN, Tang W, Sun ZY, et al. A site-directed mutagenesis of prourokinase which substantially reduces its intrinsic activity. Biochemistry. 1996;35:14070-14076. 23. Liu JN, Liu JX, Liu B, et al. A prourokinase mutant which induces highly effective clot lysis without interfering with hemostasis. Circ Res. 2002;90:757-763. 24. Pannell R, Li S, Gurewich V. Highly effecvtive fibrinolysis by a sequential synergistic combination of mini-dose tPA plus low-dose mutant proUK. PLoS One. 2015;10(3):e0122018. http://dx.doi.org/ 10.1371/journal.pone.0122018. 25. Marder VJ, Sherry S. Thrombolytic therapy: current status. N Engl J Med. 1988;318:1512-1520. 26. Montoney M, Gardell SJ, Marder VJ. Comparison of the bleeding potential of vampire bat salivary plasminogen activator versus tissue plasminogen activator in an experimental rabbit model. Circulation. 1995;91:1540-1544.
Thrombolysis: A Critical First-Line Therapy with an Unfulfilled Potential Victor Gurewich, MD Vascular Research Laboratory, Mt Auburn Hospital, Cambridge, Mass; Department of Medicine, Harvard Medical School, Cambridge, Mass.
ABSTRACT A blood clot or thrombus triggers the onset of most vascular diseases, like stroke or heart attack. Thrombolysis is the only treatment that can restore blood flow rapidly and easily. Unfortunately, the standard thrombolytic, tissue plasminogen activator (tPA), has proven inadequate and is being replaced by invasive endovascular procedures, which are time consuming and limited in their availability in relation to the scope of the problem. Historically, when tPA clinical trials began, it was not recognized sufficiently that without the other natural plasminogen activator, prourokinase (proUK), thrombolysis by tPA was seriously compromised. The reason is that the 2 activators have complementary mechanisms of action in fibrinolysis, making their combination a requirement for optimal efficacy and synergy. Biological fibrinolysis also uses both activators, explaining why such low endogenous concentrations are sufficient. A low-dose sequential combination of tPA and proUK was tested in acute myocardial infarction, where it was exceptionally effective and safe. Because native proUK at pharmacological doses was vulnerable to spontaneous conversion to urokinase, jeopardizing safety, a site-directed mutant was developed that improved proUK’s plasma stability fivefold without interfering with its mode of action. Mini-bolus tPA followed by low-dose proUK infusion is a simple, safe, effective, and promising first-line treatment of acute thrombotic disorders. Ó 2016 The Author. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). The American Journal of Medicine (2016) 129, 573-575 KEYWORDS: Fibrinolytic synergy; Mistaken assumptions; Thrombolysis; Unfulfilled potential
Cardiovascular diseases like acute myocardial infarction, ischemic stroke, or pulmonary thromboembolism, are the leading causes of death and disability worldwide, often occurring in the prime of life. Their immediate cause is an occlusive blood clot, or thrombus, and its timely removal restores blood flow in the vessel, salvages organ function, and saves lives. The only therapy capable of accomplishing this promptly is thrombolysis. Unfortunately, the thrombolytic regimen that has been in use for more than 20 years has not been sufficiently effective or safe. Funding: None. Conflict of Interest: The author is the Chief Scientific Officer of Thrombolytic Science International, LLC (TSI; Cambridge, Mass.), a company that is developing mutant proUK. Authorship: VG is the sole author of this manuscript. Requests for reprints should be addressed to Victor Gurewich, MD, Vascular Research Laboratory, Mt Auburn Hospital, 300 Mt Auburn St., Cambridge, MA 02138. E-mail address: [email protected]
As a result, thrombolysis is being replaced by cathetermediated endovascular procedures. Although these have met with some success, they are handicapped by being demanding technically, time-consuming, costly, and insufficiently accessible for the size of the problem. The standard thrombolytic regimen in use consists of high doses of the human fibrinolytic tissue plasminogen activator (tPA) alone. This contrasts with how tPA functions biologically in the fibrinolytic pathway. There, tPA induces fibrinolysis in combination with prourokinase (proUK), the other natural plasminogen activator. When these activators were tested in a low-dose sequential combination, a synergistic fibrinolytic effect was observed.1 A comparable effect was later also confirmed in vivo.2 Nevertheless, the standard tPA monotherapy regimen has endured for more than 2 decades, despite its shortcomings. As unreasonable as this may seem, the history of tPA provides some explanations. When tPA was first purified, characterized,3 and then produced by recombinant technology in
0002-9343/Ó 2016 The Author. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/). http://dx.doi.org/10.1016/j.amjmed.2015.11.033
574
The American Journal of Medicine, Vol 129, No 6, June 2016
subject and the discontinuation of the journal Fibrinolysis, 1982,4 it was embraced with enthusiasm and high expectations which was devoted to this discipline. as a much-needed replacement for streptokinase (SK), a fibriIt is noteworthy, however, that the clinical findings nolytic discovered about 50 years earlier. This was because SK reviewed above began with a misunderstanding of tPA’s was an antigenic bacterial product that had a nonspecific, inphysiological role in thrombolysis, the principal biological direct, and inefficient mechanism of plasminogen activation defense against intravascular clot formation. It was believed that induced a bleeding diathesis. By contrast, tPA is a human that tPA was the sole activator protein with a fibrin-specific, direct, involved in this pathway, which and efficient mechanism of plasCLINICAL SIGNIFICANCE ignored the role of the other natural minogen activation. Therefore, it plasminogen activator, proUK, first was assumed with confidence that it Intravascular thrombosis is a leading identified in 1981.8,9 Because would be correspondingly more cause of mortality and morbidity. effective and safer therapeutically proUK has no fibrin-binding Thrombolytic therapy has not fulfilled its than SK. domain, but instead has a cellpromise and is being replaced by timeThese expectations were not receptor-binding domain, it was borne out by the subsequent clinbelieved that its principal fibrinoconsuming endovascular procedures. ical experience. In 3 mega-trials in lytic function was in pericellular The standard thrombolytic regimen is acute myocardial infarction5-7 proteolysis of the extravascular compromised by a misunderstanding of space.10 Subsequently, this idea was where tPA and SK were tPA’s biological role in fibrinolysis. compared, a total of 94,720 pashown to be at odds with the fibritients had to be tested before a nolytic effects seen in gene-targeted In this pathway, tPA initiates fibrinostatistically significant reduction mice deficient in the proUK cell lysis, which is then completed by the in mortality with tPA was found. receptor.11 The concept was also other natural activator, prourokinase No mortality reduction was found inconsistent with the complemen(proUK). in the first 2 trials, and in the last tary mechanism of fibrin-dependent Together, they have a synergistic trial, a small but statistically sigplasminogen activation by tPA and nificant reduction was found in proUK, which made their combined thrombolytic effect making therapy more only 1 of the 4 groups tested.7 In effects a requirement for efficacy at effective and safer. fibrin-specific doses, as well as addition, tPA was also not safer, making it synergistic.1 because significantly more patients with intracranial bleeding complications were seen Due to this property of the activators, neither tPA nor with tPA than with SK, despite tPA’s fibrin specificity. proUK alone can be sufficiently effective except at doses Paradoxical findings such as these would ordinarily be high enough to overcome their fibrin specificities, at which used to obtain new insights into mechanisms of action, but bleeding side effects also become more likely. As a result, this did not occur. Instead, when tPA received marketing when tPA was given alone, concentrations more than a approval after the last mega-trial, its use in the treatment of thousand-fold higher than physiological were required, and acute myocardial infarction became the exclusive focus of efficacy was still disappointing and bleeding complications attention. The mega-trial findings were interpreted as simply were higher than with SK.5-7 meaning that the clinical differences “between different The synergistic fibrinolytic effect of the combination also fibrinolytic agents are unlikely to be large.” may explain why even at the very low concentrations at Once it was on the market, tPA quickly became the which tPA and proUK are found normally, 2 exceptionally thrombolytic of choice, soon becoming so equated with potent inhibitors, plasminogen activator inhibitor-1 and a2thrombolysis that it was no longer specified as the activator antiplasmin, are nevertheless required to control their in publications on this subject. It was assumed that tPA was plasminogen-activating effect. The congenital absence of the plasminogen activator. As a result, when, over the suceither inhibitor is associated with a bleeding diathesis.12 ceeding years, the disappointing clinical results foretold by Clot lysis studies of synergy in vitro showed it to be optimal the mega-trials became increasingly troublesome, it was when an unequal ratio and sequential combination of the actaken as evidence of the limitations of all thrombolytic tivators was used.1 A comparable tPA and proUK combination therapy. Therefore, invasive endovascular procedures was evaluated in 101 patients with acute myocardial infarcbecame the alternative rather than a different thrombolytic tion.2 The treatment consisted of a mini bolus of tPA (5% of regimen. In acute myocardial infarction, percutaneous corthe monotherapy dose), followed by an infusion of a moderate onary intervention has now become the treatment of choice, dose of proUK (50% of the monotherapy dose) for 90 minutes. and endovascular procedures are being used with increasing Coronary reperfusion (Thrombolysis in Myocardial Infarction frequency for ischemic stroke as well, despite their grades 2 and 3) at 24 hours was 100%; there was only one limitations. death and no strokes. This compares with 6.3% deaths and This experience with tPA led to a conviction that 1.5% strokes in the best of the tPA mega-trials.7 thrombolytic therapy had reached its therapeutic limits. This This study design was based on the physiological fibriwas reflected by a substantial decrease in publications on the nolytic pathway in which an intravascular thrombus induces
Gurewich
Thrombolysis: A Critical First-Line Therapy
the local release of tPA from the vessel wall. The tPA binds to fibrin in the thrombus and initiates fibrinolysis.13 This creates additional plasminogen-binding sites on fibrin,14 which are 2 in number.15 Plasminogen on the first of these is activated specifically by proUK,16,17 and proUK is, in turn, activated by plasmin to urokinase (UK)18 via a hypercatalytic transitional state.19 The UK then activates the remaining fibrin-bound plasminogen. Therefore, tPA activates the first plasminogen by binding to an adjacent site on fibrin,20 and proUK/UK activate the remaining 2 plasminogens. ProUK has a double role in fibrinolysis due to its activation to UK, whereas tPA’s properties remain unchanged by its conversion to the 2-chain form. Despite the clinical success of the Pro-Urokinase and t-PA Enhancement of Thrombolysis (PATENT) Trial, it attracted little attention, reflecting the dominance of tPA monotherapy and the belief that there was little difference among thrombolytic activators. No follow-up was possible because proUK development was abandoned not long thereafter. This was because in phase-3 trials, proUK was found to be unstable in plasma at pharmacological concentrations and converted to UK.21 Being nonspecific, UK-induced systemic plasmin generation resulting in a hemophilia-like state including degradation of clotting factors I, V, and VIII. For this reason, the European Medicines Agency turned down the application for a proUK marketing license submitted by Grünenthal (Aachen, Germany). Because proUK’s instability was unrelated to its fibrinolytic mechanism of action, this problem was addressed by site-directed mutagenesis, which improved its plasma stability fivefold without interfering with its mechanism of action.22-24 By contrast, the bleeding side effects with tPA are primarily due to its lysis of hemostatic fibrin at vascular repair sites,25,26 which is due to its therapeutic fibrinolytic mechanism of action. In conclusion, by utilizing the natural, complementary, and synergistic properties of the 2 activators, the sequential combination has the potential to provide a safe first-line treatment for stroke and acute myocardial infarction, thereby reducing the delay to reperfusion by current therapies of these conditions.
References 1. Pannell R, Black J, Gurewich V. The complementary modes of action of tissue plasminogen activator (t-PA) and pro-urokinase (pro-UK) by which their synergistic effect on clot lysis may be explained. J Clin Invest. 1988;81:853-859. 2. Zarich SW, Kowalchuk GJ, Weaver WD, et al; PATENT Study Group. Sequential combination of thrombolytic therapy for acute myocardial infarction: results of the pro-urokinase and t-PA enhancement of thrombolysis (PATENT) trial. J Am Coll Cardiol. 1995;26:374-379. 3. Rijken DC, Collen D. Purification and characterization of the plasminogen activator secreted by human melanoma cells in culture. J Biol Chem. 1981;256:7035-7041. 4. Pennica D, Holmes WE, Kohr WJ, et al. Cloning and expression of human tissue-type plasminogen activator cDNA in E. coli. Nature. 1983;301:214-221. 5. Gruppo Italiano Per Lo Studio Della Sopravvivenze Nell’Infarto Miocardico. GISSI-2: a factorial randomised trial of alteplase versus streptokinase and heparin versus no heparin among 12,490 patients with acute myocardial infarction. Lancet. 1990;336(8707):65-71.
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