Pro-UK Clinical Trials | M5 Publications

ABSTRACT:Recombinant single-chain urokinase-type plasminogen activator was intravenously administered in 2 different doses in 24 patients with acute myocardial infarction and angiographically proved occlusion of the infarct-related artery. Patients with first infarction without contraindications of thrombolysis were treated within the first 4 hours after the onset of symptoms. Group A (12 patients) received 20 mg of rscu-PA as a bolus followed by 60 mg infused over 1 hour and group B received 10 mg as a bolus and 30 mg as infusion. The 2 groups showed no significant difference in age, sex, height, weight, time between onset of symptoms and start of therapy, peak values and course of infarct-related enzymes. Time to reperfusion was 43 minutes in group A versus 67 minutes in group B (p less than 0.005). The rate of reperfusion 90 minutes after start of treatment was 91% in group A and 50% in group B (p less than 0.001). Plasma levels of fibrinogen, plasminogen and alpha-2-antiplasmin did not differ significantly in both groups. Systemic lytic state (fibrinogen less than 100 mg/dl) occurred in 33% of group A and in 9% of group B. Intravenous infusion of 80 mg (but not 40 mg) of rscu-PA led to reperfusion of the occluded coronary artery in nearly all patients. Approximately one-third of the patients treated with this dose demonstrated systemic lysis.

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ABSTRACT:Recombinant single-chain urokinase-type plasminogen activator was intravenously administered in 2 different doses in 24 patients with acute myocardial infarction and angiographically proved occlusion of the infarct-related artery. Patients with first infarction without contraindications of thrombolysis were treated within the first 4 hours after the onset of symptoms. Group A (12 patients) received 20 mg of rscu-PA as a bolus followed by 60 mg infused over 1 hour and group B received 10 mg as a bolus and 30 mg as infusion. The 2 groups showed no significant difference in age, sex, height, weight, time between onset of symptoms and start of therapy, peak values and course of infarct-related enzymes. Time to reperfusion was 43 minutes in group A versus 67 minutes in group B (p less than 0.005). The rate of reperfusion 90 minutes after start of treatment was 91% in group A and 50% in group B (p less than 0.001). Plasma levels of fibrinogen, plasminogen and alpha-2-antiplasmin did not differ significantly in both groups. Systemic lytic state (fibrinogen less than 100 mg/dl) occurred in 33% of group A and in 9% of group B. Intravenous infusion of 80 mg (but not 40 mg) of rscu-PA led to reperfusion of the occluded coronary artery in nearly all patients. Approximately one-third of the patients treated with this dose demonstrated systemic lysis.

  • deBoer A, Kluft C, Gerloff J, et. al. Pharmacokinetics of Saruplase, A Recombinant Unglycosylated Human Single-Chain Urokinase-Type Plasminogen Activator and Its Effects on Fibrinolytic and Haemostatic Parameters in Health Male Subjects. Thromb Haemost 1993; 70:320-325.
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ABSTRACT:Pharmacokinetics of two doses of the recombinant single-chain urokinase-type plasminogen activator (r-scu-PA) saruplase (40 and 20 mg) and its effect on fibrinolytic and haemostatic parameters were studied in six healthy male subjects using a randomized, double-blind, placebo-controlled, cross-over study. Special precautions were taken to prevent artefactual in vitro effects on fibrinolytic activity. The clearance of saruplase ranged from 310 to 862 ml/min and the apparent volume of distribution of the central compartment was about 8 1. Both doses of saruplase caused alpha 2-antiplasmin consumption, indicating some systemic fibrinolytic activation. However, the 20 mg dose caused no detectable fibrinogen breakdown and only a small increase in total fibrin/fibrinogen degradation products (TDP) (from 0.16 microgram/ml [range 0.14 to 0.19] to 0.78 microgram/ml [range 0.56 to 1.26]), while the 40 mg dose produce a fibrinogen breakdown to an average value of 44% (range 19 to 60%) and TDP increased from 0.12 microgram/ml (range 0.11-0.12) to 2.29 micrograms/ml (range 0.45 to 5.55). The breakdown of fibrinogen was related to the quantity of saruplase converted to active two-chain u-PA (tcu-PA) in vivo (6 to 22% conversion). There were no important effects of saruplase on overall blood coagulation (activated partial thromboplastin time) and platelet function (collagen induced platelet aggregation, urinary [2,3-dinor]-thromboxane B2 excretion and plasminogen activator inhibitor 1 [PAI-1] release from platelets). Saruplase is cleared rapidly from the plasma and a variable amount is converted to tcu-PA. This two-chain form of u-PA probably causes the dose-dependent systemic fibrinolytic activation.

  • Koster RW, Cohen AF, Hopkins GR, et al. Pharmacokinetics and Pharmacodynamics of Saruplase, an Unglycosylated Single-Chain Urokinase-Type Plasminogen Activator, in Patients with Acute Myocardial Infarction. Thromb Haemost 1994; 71:740-744.
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ABSTRACT: We examined in patients with acute myocardial infarction (AMI) the pharmacokinetics of saruplase, an unglycosylated, single chain, urokinase-type plasminogen activator (rscu-PA) by measuring urokinase-type plasminogen activator (u-PA) antigen and total u-PA activity, its conversion to active two-chain urokinase-type plasminogen activator (tcu-PA) and evaluated its effect on haemostatic parameters. Twelve patients were studied during and after administration of 20 mg bolus plus 60 mg continuous 1 h i.v. infusion of saruplase. For u-PA antigen and total u-PA activity (expressed as protein equivalents), where 234 U corresponds to 1 microgram, respectively, steady state plasma concentrations were 2.75 +/- 8.3 and 2.50 +/- 7.0 micrograms/ml (mean +/- standard deviation) and were reached within 20 min, t1/2 lambda 1 was 9.1 +/- 1.8 and 7.8 +/- 1.3 min, t1/2 lambda 2 1.2 +/- 0.2 and 1.9 +/- 0.5 h, and the total clearance was 393 +/- 110 and 427 +/- 113 ml/min. Inactivation of saruplase in plasma was negligible. After 15 min, tcu-PA was detected in plasma. From the ratio of the areas under the curve of tcu-PA and total u-PA activities it was calculated that 28 +/- 9.3% of the saruplase dose is converted into active tcu-PA. Systemic plasminaemia occurs as shown by a decrease in alpha 2-antiplasmin and fibrinogen and an increase in fibrinogen degradation products. Thrombin-antithrombin complex formation indicated activation of the clotting system. Saruplase is eliminated rapidly from plasma in AMI patients.

  • Weaver WD, Hartmann JR, Anderson JL, et al. New Recombinant Glycosylated Prourokinase for Treatment of Patients with Acute Myocardial Infarction. J Am Coll Cardiol 1994; 24:1242-1248.
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ABSTRACT: OBJECTIVES. Three dosage regimens of a new recombinant glycosylated prourokinase (A-74187) were evaluated by measuring coronary artery patency at 90 min in patients with acute myocardial infarction. BACKGROUND. Prourokinase is a thrombolytic drug with unique pharmacologic properties that may be clinically advantageous. METHODS. Aspirin (325 mg), intravenous heparin and prourokinase (60- or 80-mg monotherapy or 60 mg "primed" with a preceding bolus dose of 250,000 IU of recombinant urokinase) were administered to 128 patients. Coronary angiography was performed at 60 min (wherever possible), 90 min (primary end point) and 24 h to determine arterial patency and reocclusion rates. Plasma was collected serially to measure fibrinogen, plasminogen, thrombin antithrombin III and fibrinopeptide A. Clinical events until hospital discharge were recorded. RESULTS. The coronary artery patency rate at 90 min was similar for all three regimens, averaging 73% (95% confidence interval [CI] 64% to 80%); Thrombolysis in Myocardial Infarction (TIMI) grade 3 flow rates averaged 52% (95% CI 42% to 61%). Arterial patency at 60 min was 62% (95% CI 50% to 73%), and reocclusion occurred in 1.4% (95% CI 0.1% to 4.1%). Prourokinase demonstrated relative fibrin specificity at all doses studied. Fibrinopeptide A and thrombin antithrombin III levels were elevated at baseline and declined rapidly during the 1st 12 h. There was no difference in the baseline values of these thrombin markers between patients with patent versus closed arteries at 90 min. There was one death; no strokes occurred. CONCLUSIONS. A-74187 prourokinase is a rapid-acting, effective fibrin-specific thrombolytic agent. Reocclusion was unusual, possibly because of aggressive anticoagulation with intravenous heparin or unique features of the drug. Full definition of the clinical effectiveness of this drug merits examination in future randomized trials evaluating clinical and angiographic effectiveness.

  • Michels R, Hoffmann H, Windeler J, et al. A Double-Blind Multicenter Comparison of the Efficacy and Safety of Saruplase and Urokinase in the Treatment of Acute Myocardial Infarction: Report of the SUTAMI Study Group. J Thromb Thrombolysis 1995; 2:117-124.
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ABSTRACT: Background: Urokinase or two-chain urokinase-type plasminogen activator has been shown to be effective in the treatment of acute myocardial infarction. Its parent molecule, single-chain urokinase-type plasminogen activator (scu-PA), unlike urokinase, can selectively activate fibrin-bound plasminogen. The induced clot lysis is amplified by plasmin-triggered conversion of scu-PA to urokinase and by further plasmin generation. The aim of our study was to compare the efficacy and safety of recombinant unglycosylated scu-PA, or saruplase, and urokinase at doses considered optimal in patients with acute myocardial infarction within 6 hours of onset of pain. Methods and results: In a double-blind trial 543 patients were randomized to saruplase (20 mg bolus + 60 mg/hr) or urokinase (1.5 million unit bolus + 1.5 million units/hr). Primary endpoint: The patency rates at 24-72 hours were 75.4% (95% CI 70.3-80.5%) for saruplase and 74.2% (95% CI 69.0-79.4%; P = 0.77) for urokinase. Secondary endpoint: The incidence of bleeding events in both groups was 10.7%. There were three hemorrhagic strokes in the saruplase group (ns). Other efficacy and safety evaluations: Apart from the generation of more fibrinogen degradation products under saruplase, the changes in hemostatic parameters did not differ. Hospital mortality was 4.4% for saruplase and 8.1% for urokinase. This nonsignificant difference was maintained for 1 year. Conclusion: The efficacy and safety of saruplase and urokinase in the regimens used are very similar.

  • Tebbe U, Windeler J, Boesl I, et al. Thrombolysis with Recombinant Unglycosylated Single-Chain Urokinase-Type Plasminogen Activator (Saruplase) in Acute Myocardial Infarction: Influence of Heparin on Early Patency Rate (LIMITS Study). J Am Coll Cardiol 1995; 26:365-373.
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ABSTRACT: OBJECTIVES. The Liquemin in Myocardial Infarction During Thrombolysis With Saruplase (LIMITS) study was instituted to evaluate and characterize the effect of a prethrombolytic heparin bolus (5,000 IU) on the efficacy and safety of saruplase in patients with acute myocardial infarction. BACKGROUND. Heparin has been used after thrombolytic therapy for acute myocardial infarction to prevent reocclusion of the infarct-related artery. METHODS. The study was designed as a randomized, parallel-group, double-blind, multicenter trial. Patients were treated within 6 h of onset of symptoms with either a bolus of 5,000 IU of heparin (Liquemin) (n = 56, HSH group) or placebo (n = 62, PSH group) before thrombolytic treatment with saruplase given as a 20-mg bolus followed by an infusion of 60 mg over 60 min. Thirty minutes after completion of thrombolysis, an intravenous heparin infusion was administered for 5 days. Before coronary angiography was performed at 6 to 12 h after start of lysis, an additional bolus of 5,000 IU heparin was given to all patients. End points studied were patency of the infarct-related artery, changes in the hemostatic system and bleeding complications. RESULTS. In the HSH group (heparin-saruplase-heparin), 78.6% of patients had an open infarct-related vessel (Thrombolysis in Myocardial Infarction [TIMI] flow grade 2 or 3) compared with 56.5% in the PSH group (placebo-saruplase-heparin) (intention-to-treat analysis, p = 0.01). No significant difference was observed between the two groups with regard to changes in fibrinogen and fibrin/fibrinogen degradation products. A total of eight bleeding complications (14.3%) were observed in the HSH group and five (8.1%) in the PSH group; no cerebrovascular event occurred, and no allergic reaction was reported. A total of 12 patients died during the hospital stay, 3 in the HSH group (5.4%) and 9 in the PSH group (14.5%). CONCLUSIONS. In acute myocardial infarction, the administration of a heparin bolus before thrombolytic therapy with saruplase is associated with a significantly higher patency at angiography 6 to 12 h after the start of thrombolysis without any appreciable increase in risk of bleeding.

  • Zarich SW, Kowalchuk GJ, Weaver WD, Loscalzo J, Sassaower M, Muller JE, Gurewich V. et al. Sequential Combination 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.
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ABSTRACT: OBJECTIVES. The present study was designed to test the efficacy and safety of a sequential combination of recombinant tissue-type plasminogen activator (rt-PA) and pro-urokinase in patients with acute myocardial infarction. BACKGROUND. Efforts continue to identify a thrombolytic regimen that induces rapid, complete and sustained coronary artery patency in acute myocardial infarction. The two endogenous plasminogen activators rt-PA and pro-urokinase have been shown experimentally to induce fibrinolysis by sequential and complementary mechanisms. As a result, certain combinations of these activators have been found to be synergistic in vitro and in vivo. METHODS. In a multicenter observational study with core facilities for angiographic and laboratory analysis, 101 patients with acute myocardial infarction were enrolled and given a low dose bolus of rt-PA (5 to 10 mg) followed by a 90-min infusion of pro-urokinase (40 mg/h). All patients received intravenous heparin and oral aspirin. Coronary angiography was performed in all patients at 90 min. RESULTS. Angiography at 90 min showed the infarct-related artery to be patent (Thrombolysis in Myocardial Infarction [TIMI] grade 2 or 3 flow) in 77% of patients, and 60% achieved TIMI grade 3 flow. At one center, angiography was repeated at 24 h to detect a possible reocclusion. All 28 patients with a patent infarct-related artery at 90 min had patency at 24 h (82% achieved TIMI grade 3 flow). Treatment was well tolerated, with bleeding complications essentially confined to arterial puncture site hematomas. There was only one in-hospital death. CONCLUSIONS. A sequential combination of low dose rt-PA and reduced-dose pro-urokinase produced a high TIMI 3 patency rate, was well tolerated and was associated with a low reocclusion rate.

  • Pacouret G, Barnes SJ, Hopkins G, et al. Rapid Haemodynamic Improvement Following Saruplase in Recent Massive Pulmonary Embolism. Thromb Haemost 1998; 79:264-267.
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ABSTRACT: In a single centre pilot study, saruplase (20 mg bolus plus 60 mg infusion over 1 h) was administered to twenty patients with an angiographically documented recent massive pulmonary embolism: Miller index of at least 20 and mean pulmonary artery pressure of at least 20 mmHg. The lytic ability of saruplase to cause normalization of haemodynamic parameters over the first 12 h and reperfusion of pulmonary arteries at 24 h was assessed. A decrease of 25 +/- 10% in total pulmonary resistance was evident at 30 min. Haemodynamic parameters continued to improve with total pulmonary resistance decreasing by 29 +/- 8% and 40 +/- 11% at 1 and 12 h respectively. Relative improvement in Miller index 24 +/- 6 h after saruplase treatment was 38 +/- 9%. Two patients suffered recurrent pulmonary embolism, two severe bleeding events were observed. One patient died following a haemorrhagic stroke.

  • Tebbe U, Michels R, Adgey J, et al. Randomized, Double-Blind Study Comparing Saruplase with Streptokinase Therapy in Acute Myocardial Infarction: the COMPASS Equivalence Trial. J Am Coll Cardiol 1998; 31:487-493.
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ABSTRACT: OBJECTIVES: This study sought to demonstrate the equivalence of saruplase and streptokinase in terms of 30-day mortality. BACKGROUND: The use of thrombolytic agents in the treatment of acute myocardial infarction is well established and has been shown to substantially reduce post-myocardial infarction mortality. METHODS: Three thousand eighty-nine patients with symptoms compatible with those of acute myocardial infarction for < 6 h entered the study at a total of 104 centers and were randomized to receive streptokinase (1.5-MU infusion over 60 min) or saruplase (20-mg bolus and 60-mg infusion over 60 min). In the saruplase group, a bolus of heparin (5,000 IU) was administered before saruplase, and a corresponding blinded double-dummy placebo bolus was administered before streptokinase. All patients received intravenous heparin infusions for > or = 24 h starting 30 min after the end of the thrombolytic infusions; the infusions were titrated to maintain an activated partial thromboplastin time at 1.5 to 2.5 times that of normal. RESULTS: Death of any cause up to 30 days after randomization occurred in 88 (5.7%) of 1,542 patients randomized to receive saruplase and 104 (6.7%) of 1,547 patients randomized to receive streptokinase (odds ratio 0.84, p < 0.01 for equivalence). Hemorrhagic strokes occurred more often in patients receiving saruplase (0.9% vs. 0.3%), whereas thromboembolic strokes were more prevalent in the streptokinase-treated patients (0.5% vs. 1.0%). The rate of bleeding was similar in the two treatment groups (10.4% vs. 10.9%). Hypotension and cardiogenic shock occurred less frequently in the saruplase group. Reinfarction rates were similar. CONCLUSIONS: Saruplase is a clinically safe and effective thrombolytic medication. This profile ranks saruplase favorably among the currently available thrombolytic agents.

  • Furlan A, Higashida R, Wechsler L, et al. Intra-Arterial Prourokinase for Acute Ischemic Stroke. The PROACT II Study: A Randomized Controlled Trial. J Am Med Assoc 1999; 282:2003-2011.
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ABSTRACT: CONTEXT: Intravenous tissue-type plasminogen activator can be beneficial to some patients when given within 3 hours of stroke onset, but many patients present later after stroke onset and alternative treatments are needed. OBJECTIVE: To determine the clinical efficacy and safety of intra-arterial (IA) recombinant prourokinase (r-proUK) in patients with acute stroke of less than 6 hours' duration caused by middle cerebral artery (MCA) occlusion. DESIGN: PROACT II (Prolyse in Acute Cerebral Thromboembolism II), a randomized, controlled, multicenter, open-label clinical trial with blinded follow-up conducted between February 1996 and August 1998. SETTING: Fifty-four centers in the United States and Canada. PATIENTS: A total of 180 patients with acute ischemic stroke of less than 6 hours' duration caused by angiographically proven occlusion of the MCA and without hemorrhage or major early infarction signs on computed tomographic scan. INTERVENTION: Patients were randomized to receive 9 mg of IA r-proUK plus heparin (n = 121) or heparin only (n = 59). MAIN OUTCOME MEASURES: The primary outcome, analyzed by intention-to-treat, was based on the proportion of patients with slight or no neurological disability at 90 days as defined by a modified Rankin score of 2 or less. Secondary outcomes included MCA recanalization, the frequency of intracranial hemorrhage with neurological deterioration, and mortality. RESULTS: For the primary analysis, 40% of r-proUK patients and 25% of control patients had a modified Rankin score of 2 or less (P = .04). Mortality was 25% for the r-proUK group and 27% for the control group. The recanalization rate was 66% for the r-proUK group and 18% for the control group (P<.001). Intracranial hemorrhage with neurological deterioration within 24 hours occurred in 10% of r-proUK patients and 2% of control patients (P = .06). CONCLUSION: Despite an increased frequency of early symptomatic intracranial hemorrhage, treatment with IA r-proUK within 6 hours of the onset of acute ischemic stroke caused by MCA occlusion significantly improved clinical outcome at 90 days.

M5 Publications | Pro-UK Clinical Trials

  • Liu JN, Tang W, Sun ZY, Kung, W, Sarmientos, P, Pannell, R, Gurewich, V. A site-directed mutagenesis of pro-urokinase which substantially reduces its intrinsic activity. Biochemistry 1996; 35:14070-14076.
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ABSTRACT: Single-chain urokinase-type plasminogen activator or pro-urokinase is a zymogen with an intrinsic catalytic activity which is greater than that of most other zymogens. To study the structural basis for this activity, a three-dimensional homology model was calculated using the crystallographic structure of chymotrypsinogen, and the structure-function relationship was studied using site-directed mutagenesis and kinetic analysis. This model revealed a unique Lys300 in pro-urokinase which could form a weak interaction with Asp355, adjacent to the active site Ser356. It was postulated that this lysine, by its epsilon-amino group, may serve to pull Ser356 close to the active position, thereby inducing the higher intrinsic activity of pro-urokinase. This was consistent with the published finding that a homologous lysine (Lys416) in single chain tissue plasminogen activator when mutated to serine induced some reduction in activity. To test this hypothesis, a site-directed mutant with a neutral residue (Lys300-->Ala) was produced and characterized. The Ala300-pro-urokinase had a 40-fold lower amidolytic activity than that of pro-urokinase. It was also stable in plasma at much higher concentrations than pro-urokinase, reflecting much attenuated plasminogen activation. Plasmin activatability was comparable to that of pro-urokinase, but the resultant two-chain derivative (Ala300-urokinase) had a lower enzymatic activity (approximately 33% that of urokinase) due to a reduction of kcat. Interestingly, the KM of two-chain Ala300-urokinase against plasminogen was 5.8-fold lower than that of urokinase, being similar to that of pro-urokinase which has a KM about 5-fold lower than urokinase. In conclusion, the hypothesis that Lys300 is a key structural determinant of the high intrinsic activity of pro-urokinase was confirmed by these studies. This residue also appears to be important for the full expression of the enzymatic activity of urokinase.

  • Sun Z, Jiang Y, Ma Z, Wu H, Liu BF, Xu Y, Chen Y, Gurewich V, Liu JN. Identification of a Flexible Loop (297-313) of Urokinase-Type Plasminogen Activator, Which Helps Determine Its Catalytic Activity. J Biol chem. 1997; 272:23818-23823.
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ABSTRACT: Pro-urokinase has a much higher intrinsic catalytic activity than other zymogens of the serine protease family. Lys300(c143) in an apparent "flexible loop" region (297-313) was previously shown to be an important determinant of this intrinsic catalytic activity. This was related to the loop allowing the positive charge of Lys300(c143) to transiently interact with Asp355(c194), thereby inducing an active conformation of the protease domain (Liu, J. N., Tang, W., Sun, Z., Kung, W., Pannell, R., Sarmientos, P., and Gurewich, V. (1996) Biochemistry 35, 14070-14076). To further test this hypothesis, the charge at position 300(c143) and the flexibility of the loop were altered using site-directed mutagenesis designed according to a computer model to affect the interaction between Lys300(c143) and Asp355(c194). When the charge at Lys300(c143) but not Lys313(c156) was reduced, a significant reduction in the intrinsic catalytic activity occurred. Similarly, when the flexibility (wobbliness) of the loop was enhanced reducing the size of side chain, the intrinsic catalytic activity was also reduced. By contrast, when the loop was made less flexible, the intrinsic catalytic activity was increased. These findings were consistent with the hypothesis. The effects of these mutations on two-chain activity were less and often discordant with the intrinsic catalytic activity, indicating that they can be modulated independently. This structure-function disparity can be exploited to create a more zymogenic pro-urokinase (lower intrinsic catalytic activity) with a high catalytic activity, as exemplified by two of the mutants. The changes in intrinsic catalytic activity and two-chain activity induced by the mutations were due to changes in kcat rather than Km. Some significant structure-function differences between pro-urokinase and its highly homologous counterpart, tissue plasminogen activator, were also found.

  • Sun Z, Liu BF, Chen Y, Gurewich V, Zhu D, Liu JN. Analysis of the Forces Which Stabilizes the Active Conformation of Urokinase-Type Plasminogen Activator. Biochemistry 1998; 37:2935-2940.
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ABSTRACT: It was recently proposed that hydrophobic interactions control the active conformation of serine proteases in the trypsin family (Hedstrom et al. (1996) Biochemistry 35, 4515-23) rather than a charge interaction with Asp next to the active site Ser, as formerly believed. In the present study, certain site-directed mutants of the serine protease zymogen pro-urokinase (pro-UK) and its two-chain enzymatic derivative urokinase (UK) were characterized. The results provide information on the structure-function of the catalytic domain of pro-UK/UK, which is relevant to this controversy. Mutations at Asp355(c194), which eliminated its charge, induced a 6250-fold reduction in the catalytic activity of UK. By contrast, reducing the hydrophobicity at the neoterminal Ile159(c16) of UK had relatively little effect. However, when both the hydrophobicity and the size of the side chain were reduced by a glycine substitution at this position, a major reduction (9090-fold) in the catalytic efficiency of UK occurred. This effect was related to the smaller side chain increasing the cavity and the flexibility of the N-terminus and thereby interfering with its charge interaction with Asp355(c194). A similar mechanism, rather than a change in hydrophobicity, is believed also to explain the reduction in the stabilization energy of the activation domain observed in a trypsin mutant by Hedstrom et al. (1996). Although hydrophobic interaction facilitated the charge interaction with Asp355(c194), the latter was the primary force which stabilized the active conformation of UK. The charge interaction with Asp355(c194) was also found to be the principal determinant of the intrinsic catalytic activity of single-chain pro-UK. Additionally, the findings confirmed that the KM of pro-UK for its natural substrate was significantly lower than that of UK. Since this same phenomenon was also seen with each of the mutants, the substrate binding pocket of these single-chain zymogens was better formed than that of their two-chain, enzymatic derivatives.

  • Liu JN, Liu JX, Liu B, Sun Z, Zhang P, Zhang J, Gurewich V. A Prourokinase Mutant Which Induces Highly Effective Clot Lysis Without Interfering With Hemostasis. Circ Res 2002; 90:757-763.
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ABSTRACT: Prourokinase (proUK) is a zymogenic plasminogen activator that at pharmacological doses is prone to nonspecific activation to urokinase. This has handicapped therapeutic exploitation of its fibrin-specific physiological properties. To attenuate this susceptibility without compromising specific activation of proUK on a fibrin clot, a Lys300-->His mutation (M5) was developed. M5 had a lower intrinsic activity and, therefore, remained stable in plasma at a 4-fold higher concentration than did proUK. M5 had a higher 2-chain activity and induced more rapid plasminogen activation and fibrin-specific clot lysis in vitro. Sixteen dogs embolized with radiolabeled clots were infused with saline, proUK, tissue plasminogen activator, or M5. The lower intrinsic activity allowed a higher infusion rate with M5, which induced the most rapid and efficient clot lysis (50% clot lysis by approximately 600 microg/kg M5 versus approximately 1200 microg/kg proUK). In association with this, M5 caused neither a significant increase in the primary bleeding time nor secondary bleeding (total blood loss). By contrast, these measurements increased 4-fold and 5-fold, respectively, with proUK and >5-fold and 8-fold, respectively, with tissue plasminogen activator. Clot lysis by M5 and hemostasis were further evaluated in 6 rhesus monkeys. M5 again induced rapid clot lysis without a significant increase in the primary bleeding time, and secondary bleeding did not occur. In conclusion, a site-directed mutation designed to improve the stability of proUK in blood at therapeutic concentrations induced superior clot lysis in vitro and in vivo without causing significant interference with hemostasis.

  • Gurewich V, Pannell R, Simmons-Byrd A, Badylak S. Thrombolysis Versus Bleeding from Hemostatic Sites by a Prourokinase Mutant Compared with Tissue Plasminogen Activator. J Thromb Haemost 2006; 4:1559-1565.
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ABSTRACT: BACKGROUND: A single site mutant (M5) of prourokinase (proUK) was developed to make proUK less vulnerable to spontaneous activation in plasma. This was a problem that seriously compromised proUK in clinical trials, as it precluded proUK-mediated fibrinolysis at therapeutic concentrations. METHODS AND RESULTS: After completing dose-finding studies, 12 anesthetized dogs with femoral artery thrombosis were given either M5 (2.0 mg kg(-1)) or tissue plasminogen activator (t-PA) (1.4 mg kg(-1)) by i.v. infusion over 60 min (20% administered as a bolus). Two pairs of standardized injuries were inflicted at which hemostasis was completed prior to drug administration. Blood loss was quantified by measuring the hemoglobin in blood absorbed from these sites. Thrombolysis was evaluated at 90 min and was comparably effective by both activators. Rethrombosis developed in one t-PA dog. The principal difference found was that blood loss was 10-fold higher with t-PA (mean approximately 40 mL) than with M5 (mean approximately 4 mL) (P = 0.026) and occurred at more multiple sites (mean 2.7 vs. 1.2). This effect was postulated to be related to differences in the mechanism of plasminogen activation by t-PA and M5 in which the latter is promoted by degraded rather than intact (hemostatic) fibrin. In addition, two-chain M5 was efficiently inactivated by plasma C1 inactivator, an exceptional property which helped contain its non-specific proteolytic effect. CONCLUSIONS: Intravascular thrombolysis by M5 was accompanied by significantly less bleeding from hemostatic sites than by t-PA. This was attributed to the proUK paradigm of fibrinolysis being retained at therapeutic concentrations by the mutation.

  • Pannell R, Kung W, Gurewich V. C1-Inhibitor Prevents Non-Specific Plasminogen Activation by a Prourokinase Mutant Without Impeding Fibrin-Specific Fibrinolysis. J Thromb Haemost 2007; 5:1047-1054.
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ABSTRACT: BACKGROUND: Prourokinase (prouPA) is unstable in plasma at therapeutic concentrations. A mutant form, M5, made more stable by reducing its intrinsic activity was therefore developed. Activation to two-chain M5 (tcM5) induced a higher catalytic activity than two-chain urokinase plasminogen activator (tcuPA), implicating an active site functional difference. Consistent with this, an unusual tcM5 complex with plasma C1-inhibitor was recently described in dog and human plasma. The effect of C1-inhibitor on fibrinolysis and fibrinogenolysis by M5 is the subject of this study. METHODS AND RESULTS: Zymograms of tcM5 and tcuPA incubated in plasma revealed prominent tcM5-C1-inhibitor complexes, which formed within 5 min. The inhibition rate by purified human C1-inhibitor (250 microg mL(-1)) was about 7-fold faster for tcM5 than it was for tcuPA (10 microg mL(-1)). The effect of the inhibitor on the stability of M5 and prouPA was determined by incubating them in plasma at high concentrations (10-20 microg mL(-1)) +/- C1-inhibitor supplementation. Above 10 microg mL(-1), depletion of all plasma plasminogen occurred, indicating plasmin generation and tcM5/tcuPA formation. With supplemental C1-inhibitor, M5 stability was restored but not prouPA stability. Clot lysis by M5 +/- supplemental C1-inhibitor showed no attenuation of the rate of fibrinolysis, whereas fibrinogenolysis was prevented by C1-inhibitor. Moreover, because of higher dose-tolerance, the rate of fibrin-specific lysis reached that achievable by non-specific fibrinolysis without inhibitor. CONCLUSIONS: Plasma C1-inhibitor stabilized M5 in its proenzyme configuration in plasma by inhibiting tcM5 and thereby non-specific plasminogen activation. At the same time, fibrin-specific plasminogen activation remained unimpaired. This unusual dissociation of effects has significant implications for improving the safety and efficacy of fibrinolysis.

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