Hypersensitivity reactions associated with recombinant tissue-type plasminogen activator and urokinase.

Anaphylaxis or angioedema in response to recombinant tissue-type plasminogen activator or urokinase have been reported in only a few isolated cases. Both agents are endogenous proteins and thus considered non-antigenic. Activation of fibrinolysis may per se facilitate anaphylactoid reactions by pathophysiologic pathways that are not well understood. We report a unique case, review the literature and discuss implication for the clinician. The 25-year-old patient underwent thrombolytic treatment for extensive thrombosis of pelvic and deep lower extremity veins. The patient developed protracted anaphylactoid reactions during recombinant tissue-type plasminogen activator continuous intravenous infusion. After changing treatment to urokinase, the same symptoms recurred with more severe intensity, despite corticosteroid premedication. Symptoms resolved within hours after treatment with histamine receptor blockers. This unique observation, i.e. sequential occurrence of anaphylactoid reactions during recombinant tissue plasminogen activator and urokinase treatments, adds to existing evidence for an unspecific non-antigenic pathomechanism, and for a class effect of thrombolytics. Steroids do not prevent, but histamine receptor blockers seem to be an effective treatment of this unusual complication of thrombolytic therapy.     

Activation of prothrombin accompanying thrombolysis with recombinant tissue-type plasminogen activator.

Increases in thrombin activity in patients given fibrinolytic agents for acute myocardial infarction have been shown to be important in limiting the ultimate success of coronary thrombolysis. The present study was designed to determine whether increases in thrombin activity reflect, in part, activation of prothrombin accompanying thrombolysis. Plasma concentrations of prothrombin fragment 1.2, a polypeptide released when prothrombin is activated by factor Xa, were measured in 22 patients with acute myocardial infarction before and after treatment with 100 mg of recombinant tissue-type plasminogen activator (rt-PA). Concentrations of prothrombin fragment 1.2 increased from 0.83 +/- 1.1 nM (mean +/- SD) before rt-PA infusion to 1.5 +/- 1.5 nM 2 h after initiation of the infusion (p less than 0.05). After a 5,000-U intravenous dose of heparin given at the end of the infusion of rt-PA, concentrations of prothrombin fragment 1.2 decreased from 1.8 +/- 1.5 to 1.1 +/- 0.9 nM (n = 20, p less than 0.05), although values were still increased compared with concentrations before rt-PA. These results indicate that thrombin activity increases in patients given rt-PA at least in part because of activation of the coagulation system leading to activation of prothrombin. Thus, inhibition of the reactions involving coagulant proteins that lead to activation of prothrombin may be of value as conjunctive treatment to potentiate the efficacy of pharmacologic thrombolysis.     

Thrombolytic activity of a novel plasminogen activator, LY210825, compared with recombinant tissue-type plasminogen activator in a canine model of coronary artery thrombosis

LY210825, a recombinant tissue-type plasminogen activator (rt-PA), which contains the kringle-2 and serine protease functional domains of native tissue-type plasminogen activator, was previously produced by site-directed mutagenesis in a Syrian hamster cell line. We studied the thrombolytic potential of this molecule in a canine thrombosis model. Male hounds (16-22 kg) were anesthetized; a 2.0-cm segment of the left circumflex coronary artery (LCX) was isolated proximal to the first main branch, and the dogs were instrumented with an electromagnetic flow probe to measure coronary blood flow. An occlusive thrombus was formed after injury of the intimal surface of the LCX with an electrical current applied by a needle-tipped anode placed distal to the electromagnetic flow probe. After 1 hour of occlusion, either LY210825 or rt-PA was administered intravenously according to the following protocols: 1) a 1-hour infusion of either 0.25 mg/kg LY210825 or 0.4 mg/kg rt-PA, 2) single injections of 0.15-0.6 mg/kg LY210825, and 3) a single injection of 0.45 mg/kg LY210825 and a 3-hour infusion of 1.0 or 1.7 mg/kg rt-PA. Plasma half-lives of LY210825 and rt-PA were 58 +/- 7 and 3.3 +/- 0.3 minutes, respectively. LY210825 produced more rapid reperfusion of the LCX than did rt-PA. In the third study, 90% of the rt-PA-treated vessels reoccluded within 1 hour after cessation of drug, whereas only 25% of the LY210825-treated vessels reoccluded during a 4-hour washout period. There were significant, but relatively small, reductions produced by both plasminogen activators on plasma fibrinogen and plasminogen (25-35% decreases). Because of its longer plasma half-life, LY210825 could be administered intravenously as a single injection. In a canine model of coronary artery thrombosis, LY210825 was a more effective thrombolytic agent than was rt-PA.     

A recombinant chimeric plasminogen activator with high affinity for fibrin has increased thrombolytic potency in vitro and in vivo.

A recombinant plasminogen activator with high fibrin affinity and specificity was expressed by transfecting hybridoma cells with a plasmid that combines sequence coding for low molecular mass (32 kDa) single-chain urokinase-type plasminogen activator [scuPA(32kDa)] and anti-fibrin monoclonal antibody 59D8. The expression of the recombinant molecule [r-scuPA(32kDa)-59D8] was optimized by replacing the 3' untranslated region (initially that of high molecular mass scuPA) in the plasmid with the 3' untranslated region of either beta-globin or mouse immunoglobulin. This modification resulted in a greater than 100-fold improvement in the level of protein expression. The 103-kDa r-scuPA(32kDa)-59D8 protein displayed catalytic activity indistinguishable from that of high molecular mass scuPA and fibrin binding comparable to that of native antibody 59D8. r-scuPA(32kDa)-59D8 was 6 times more potent than high molecular mass scuPA in lysing a human plasma clot in vitro and was 20 times more potent than high molecular mass scuPA in the rabbit jugular vein model of thrombolysis. Molecules of this type may serve as prototypes for highly specific, antibody-targeted enzymes suitable for human use.     

Improved thrombolysis with a modified dose regimen of recombinant tissue-type plasminogen activator

To improve further the patency rate of infarct-related coronary arteries, the following accelerated dosage regimen of recombinant tissue-type plasminogen activator (rt-PA) was administered to 80 patients with acute myocardial infarction of less than or equal to 6 h duration: 15 mg intravenous bolus, 50 mg infusion over 30 min and 35 mg infusion over the following 60 min. After coronary angiography at 90 min coronary angioplasty was performed in 16 patients and additional thrombolysis in 3 patients. Six patients were not included in the final angiographic analysis, mostly because of borderline ST segment elevations, in order to avoid overestimation of the efficacy of this dose regimen. Four of these had a patent infarct artery; no early angiogram was performed on two. Sixty minutes after the start of infusion, 54 (74%) of 73 patients had a patent infarct-related artery (Thrombolysis in Myocardial Infarction [TIMI] grade 2 or 3) as did 67 (91%) of 74 patients at 90 min. At 24 h, 61 (92.4%) of 66 patients showed a patent infarct artery. Recurrent myocardial ischemia was noted in 12 patients, 7 (9.4%) of whom experienced reinfarction during the hospital stay. Minor local bleeding complications were observed in 14 patients (17.5%). There were four in-hospital cardiac deaths; one patient who underwent additional thrombolysis for recurrent ischemia died from bleeding complications. These results show that a rapid infusion of 100 mg of rt-PA over 90 min yields a high early patency rate of the infarct-related artery without an increase in reocclusion rate and adverse reactions.     

Clot-selective coronary thrombolysis with lowdose synergistic combinations of single-chain urokinase-type plasminogen activator and recombinant tissue-type plasminogen activator

The effect of simultaneous infusions of low-dose recombinant tissue-type plasminogen activator (t-PA) and single-chain urokinase-type plasminogen activator (scu-PA, pro-urokinase) on coronary arterial thrombolysis was investigated in 23 patients treated within 6 hours (mean 2.6 ± 1.1, range 1.2 to 5.9) of symptoms of an acute myocardial infarction. Infarct artery patency at 90 minutes was achieved in 16 (70%, 95% confidence limits of 0.47 to 0.87) of 23 patients after a 1-hour intravenous infusion of 20 and 16.3 mg of t-PA and scu-PA, respectively. At 90 minutes, the fibrinogen concentration decreased from 369 ± 207 to 316 ± 192 mg/dl (p = not significant), while plasminogen decreased to 69 ± 24% (p = 0.001) and -2-antiplasmin to 77 ± 24% (p = 0.001) of pretreatment values. Although no bleeding requiring termination of drug infusion or transfusion occurred, 1 patient with cerebrovascular amyloidosis had a fatal intracerebral hemorrhage. These findings suggest that combination therapy may allow substantial reductions in total thrombolytic doses while still achieving effective fibrin-specific coronary thrombolysis.     

Treatment of prosthetic tricuspid valve thrombosis with recombinant tissue-type plasminogen activator

Recombinant tissue-type plasminogen activator (rt-PA) was admministeredintravenously to a 64-years old female with thrombotic malfunctionof a Björk–Shiley prosthetic tricuspid valve. 150mg of single-chain rt-PA was infused over 8 hours followed byan additional dose of 50 mg over the next 8 hours. At the endof the first infusion, restoration of normal valve functionwas demonstrated by fluoroscopic and echo-Doppler examinations.Mild systemic activation of the fibrinolytic system occurred,with a decrease of fibrinogen and ₂-antiplasmin to 53% and 33%,respectively, of the preinfusion value at the nadir.     

Treatment of prosthetic tricuspid valve thrombosis with recombinant tissue-type plasminogen activator

Recombinant tissue-type plasminogen activator (rt-PA) was admministeredintravenously to a 64-years old female with thrombotic malfunctionof a Björk–Shiley prosthetic tricuspid valve. 150mg of single-chain rt-PA was infused over 8 hours followed byan additional dose of 50 mg over the next 8 hours. At the endof the first infusion, restoration of normal valve functionwas demonstrated by fluoroscopic and echo-Doppler examinations.Mild systemic activation of the fibrinolytic system occurred,with a decrease of fibrinogen and ₂-antiplasmin to 53% and 33%,respectively, of the preinfusion value at the nadir.     

Coronary thrombolysis with recombinant human tissue-type plasminogen activator: a prospective, randomized, placebo-controlled trial

Forty-five patients with acute transmural myocardial infarction and angiographically confirmed complete coronary occlusion were prospectively randomized, two for one, to treatment of acute coronary thrombosis with intravenous recombinant human tissue-type plasminogen activator (rt-PA) or placebo. Each of five additional consecutive patients was treated with a high dose of rt-PA for 2 hr. Twenty-five of 33 patients (75%) receiving 0.5 to 0.75 mg/kg of rt-PA over 30 to 120 min had angiographically proven recanalization within 90 min of initiation of therapy. Only one of 14 patients given placebo had spontaneous recanalization within 45 min (p less than .001). Thirteen placebo-treated patients were crossed over to the intracoronary rt-PA group. Nine (69%) exhibited subsequent recanalization within 45 min. Levels of circulating fibrinogen decreased after treatment with rt-PA by an average of only 8% of baseline values. None of the patients manifested a depletion of fibrinogen level to below 100 mg/dl. Six patients who were completely unresponsive to rt-PA were subsequently treated with intracoronary streptokinase and none responded. Thus, either intravenous or intracoronary rt-PA induced coronary thrombolysis without eliciting clinically significant fibrinogenolysis in patients with evolving myocardial infarction due to thrombotic coronary occlusion.     

Platelet activation and subsequent inhibition by plasmin and recombinant tissue-type plasminogen activator.

The success of plasminogen activators in recanalizing occluded coronary arteries may be influenced by their effect on blood platelets; however, some previous studies have shown platelet activation by plasmin and thrombolytic agents while others have shown an inhibitory effect. Moreover, it has not been determined whether these effects reflect an alteration of intracellular signal transduction, fibrinogenolysis, degradation of adhesive protein receptors, or a combination of these events. To distinguish among these possibilities, the increase of cytoplasmic [Ca2+] [( Ca2+]i), which is an intracellular marker of platelet activation that precedes fibrinogen binding to the surface of activated platelets, was measured along with aggregation and release of 5-hydroxytryptamine (5-HT) in washed human platelets incubated with plasmin or recombinant tissue-type plasminogen activator (rt-PA). Plasmin (0.1 to 1.0 CU/mL) induced a prompt, concentration-dependent [Ca2+]i increase when added to platelets, but subsequently inhibited the [Ca2+]i increase in response to thrombin or the endoperoxide analog U44069. Platelet aggregation accompanied the [Ca2+]i increase if the platelets were stirred, while the aggregation of platelets unstirred during plasmin incubation was inhibited upon agonist addition and resumption of stirring. The release of 5-HT paralleled the [Ca2+]i increase induced by plasmin and was also inhibited after the subsequent addition of a second agonist. The effects of rt-PA, added with plasminogen (100 micrograms/mL), were similar to those of plasmin, and could be accounted for by the concentration of plasmin generated. The ADP scavengers apyrase and CP/CK each prevented the [Ca2+]i increase, and aggregation caused by plasmin or rt-PA, and also prevented their inhibitory effects on thrombin-induced activation. Thus, plasmin and rt-PA initially activate platelets, inducing a [Ca2+]i increase, and, if the platelets are stirred, aggregation. Such activation is followed by subsequent inhibition of cellular activation by a second agonist; the inhibitory effect is in proportion to the degree of initial activation, and ADP is an important cofactor in both processes. These platelet effects occur at rt-PA concentrations achievable clinically, and may affect the success of therapy with thrombolytic and adjunctive agents.     

Requirement of heparin for arterial and venous thrombolysis with recombinant tissue-type plasminogen activator

The effect of concomitant intravenous (IV) heparin (200 U/kg bolus, followed by 100 U/kg/h) on the efficacy of arterial and venous thrombolysis with IV recombinant tissue-type plasminogen activator (rt-PA; 0.5 mg/kg over 1 hour) was investigated in a combined femoral arterial and venous thrombosis model in the dog. The arterial model consisted of a high-grade stenosis, endothelial damage, and a thrombotic occlusion, and the venous model consisted of a 125I-fibrin-labeled blood clot. After a dose-finding pilot study in four dogs, a randomized, prospective, and blind study was performed in 20 animals pretreated with 2.8 mg/kg IV acetyl salicylic acid (ASA). The combination of rt-PA and heparin (group I, n = 10) induced early (less than 30 minutes) arterial reperfusion in seven dogs, late (greater than 30 minutes) reflow in two dogs, and persistent occlusion in one dog. rt-PA alone (group II, n = 10) was associated with early reperfusion in one dog, late reflow in three dogs, and persistent occlusion in six dogs (P = .018). Reocclusion occurred in five of nine reperfused dogs of group I and in one of four reperfused dogs of group II (P = not significant). Venous clot lysis amounted to 81% +/- 4% (mean +/- SEM) for group I and to 49% +/- 7% for group II (P less than .001). Template bleeding times increased moderately, but significantly, in group I (from 2.2 +/- 0.2 minutes at baseline to 7.0 +/- 1.4 minutes at 30 minutes, P = .006), but only marginally in group II (from 2.2 +/- 0.2 minutes to 3.6 +/- 0.7 minutes, P = .09). No systemic fibrinogen depletion was observed. Thus, the concommitant use of heparin with rt-PA accelerates arterial reperfusion and enhances venous thrombolysis in dogs pretreated with ASA. These results, obtained in a randomized prospective study design, add to a growing body of experimental and clinical evidence, indicating that thrombolytic therapy with rt-PA requires concomitant adjunctive IV heparin for optimal efficacy, even in the face of treatment with ASA.     

Intravenous recombinant tissue-type plasminogen activator in patients with unstable angina pectoris. Results of a placebo-controlled, randomized trial

Because thrombus formation may contribute to coronary obstruction in patients with unstable angina pectoris, we performed a pilot investigation to determine whether thrombolytic therapy can relieve coronary narrowing in this acute ischemic syndrome. Sixty-seven patients with rest angina and angiographic evidence of coronary stenosis were randomly assigned to receive either low-dose intravenous recombinant tissue-type plasminogen activator (rt-PA) (0.75 mg/kg over 1 hour), high-dose intravenous rt-PA (0.75 mg/kg over 1 hour; total dose, 100 mg over 6 hours), or intravenous placebo followed by repeat coronary angiography at 24-48 hours to assess change in the severity of coronary narrowing. Each patient also received oral aspirin and intravenous heparin. Mean values of coronary stenosis severity (percent of diameter reduction) declined to a similar extent in each group: placebo, 75 +/- 14% to 72 +/- 14% (p = 0.07); low-dose rt-PA, 75 +/- 16% to 71 +/- 18% (p = 0.03), and high-dose rt-PA, 82 +/- 11% to 77 +/- 17% (p = 0.18), with only the low-dose rt-PA group achieving statistical significance. Resolution of intracoronary filling defects, increase in antegrade flow grade, or both also occurred equally among the three groups. There was considerable variation in individual patient response. Between 29% and 50% of patients within each group demonstrated a decrease in stenosis severity, whereas 50% to 57% noted either improvement in antegrade flow or resolution of intracoronary thrombus. There was no difference in incidence of major bleeding events among the three groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

重组葡激酶与重组组织型纤溶酶原激活剂治疗急性心肌梗死的随机多中心临床试验

目的研究新型溶栓剂——重组葡激酶（r-Sak）治疗急性心肌梗死（AMI）的冠状动脉通畅率、临床疗效及安全性。方法本研究为多中心、随机、平行对照临床试验,入选发病12h内、年龄≤70岁、ST段抬高的AMI患者,随机分为r-Sak组104例,给予r-Sak 3mg静注,12mg于30min内静脉输注,总量15mg;重组组织型纤溶酶原激活剂（rt-PA）组106例,8mg静注,42mg在90min内输注,总量50mg。全部患者给予阿司匹林和静脉输注肝素,于用药90min行冠状动脉造影,对TIMI血流0～2级者行补救性PCI。结果主要终点：用药90min冠状动脉通畅率（TIMI血流2级或3级）,r-Sak组明显高于rt-PA组（77.8％比63.6％,P=0．0277）,TIMI 3级者两组间差异无统计学意义（57．6％比48．5％,P=0．1929）;1个月内死亡（8,7％比5,7％,P=0．3997）、非致死性再梗死（2,9％比3．8％,P=1．0000）、心肌缺血复发（8,7％比16．0％,P=0.1043）和复合临床终点（18．3％比21．7％,P=0。5345）两组间差异均无统计学意义。次要终点：r-Sak组出血发生率（28．8％）与rt-PA组（27．4％）比较,差异无统计学意义（P=0,8105）,其中严重或威胁生命的出血,两组间差异亦无统计学意义（1．9％比3．8％）,r-Sak组脑出血1例（0．96％）,rt-PA组脑出血4例（3．85％）。无其他药物相关的严重不良反应及过敏反应发生。结论r-Sak是一种安全、有效的治疗AMI的溶栓药物,其疗效及安全性至少与rt-PA 50mg相似。     

S-nitrosylation of tissue-type plasminogen activator confers vasodilatory and antiplatelet properties on the enzyme.

Tissue-type plasminogen activator (t-PA) reacts upon exposure to endothelium-derived relaxing factor (EDRF) by way of the enzyme's single free sulfhydryl (Cys-83) to form a stable S-nitrosothiol protein adduct. S-nitrosylation endows t-PA with potent vasodilatory and antiplatelet properties that are accompanied by elevations in intracellular cyclic GMP analogous to those induced by low molecular weight (e.g., S-nitroso amino acid) S-nitrosothiols. Moreover, this chemical modification does not adversely affect the catalytic efficiency of t-PA, the fibrin stimulation of this activity, the binding of t-PA to fibrinogen, or the interaction of the enzyme with its physiologic serine protease inhibitor, plasminogen-activator inhibitor type I. The coupling of vasodilatory, antiplatelet, and fibrinolytic properties in one molecule makes the S-nitrosylated t-PA a unique molecular species and may provide insight into the mechanisms by which the endothelium maintains vessel patency. These data also suggest a pharmacologic approach to treatment of thromboocclusive disorders.     

A randomized, blinded, placebo-controlled trial of recombinant human tissue-type plasminogen activator in patients with unstable angina pectoris

Twenty-four patients with unstable angina pectoris, defined as chest pain at rest with transient ST segment deviation of at least 1 mm, were randomly assigned to blinded treatment with either placebo or intravenous recombinant human tissue-type plasminogen activator (rt-PA). Before randomization, all patients were treated with oral beta-blockers, calcium antagonists, nitrates, and continuous intravenous heparin for a monitoring period of 12 to 28 hr. After this monitoring period the 24 patients were randomly assigned to receive either placebo or 1.75 mg/kg intravenous rt-PA given over 12 hr at a rate of 0.75 mg/kg over 1 hr, 0.5 mg/kg over 4 hr, and 0.5 mg/kg over 7 hr. One patient, assigned to receive placebo, developed acute myocardial infarction after randomization but before receiving the study drug. Ischemic events were recorded during a hospital follow-up period of at least 4 days unless a further intervention was indicated or the coronary angiogram was normal. The follow-up period was 7 +/- 5 days (mean +/- SD) after the placebo infusion and 8 +/- 4 days after the infusion of rt-PA. Unstable angina pectoris persisted after the completion of the infusion in six of 11 patients receiving placebo and only one of 12 patients receiving rt-PA (p less than .03). Coronary angiography, performed 38 +/- 19 hr after the infusion, demonstrated subocclusive thrombus in eight of 11 patients receiving placebo but in none of 11 patients treated with rt-PA (p less than .002). One patient on rt-PA refused coronary angiography.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vivo thrombin inhibition enhances and sustains arterial recanalization with recombinant tissue-type plasminogen activator

The effects of heparin and the synthetic competitive thrombin inhibitor (2R,4R)-4-methyl-1-[N2-(3-methyl-1,2,3,4-tetrahydro-8-quinolinesulfon yl)-L-arginyl]-2-piperidinecarboxylic acid monohydrate (Argatroban) on thrombolysis with recombinant tissue-type plasminogen activator (rt-PA) was studied in groups of six or seven rabbits with arterial thrombosis. The model consisted of a whole-blood clot produced in a 1-cm isolated femoral arterial segment with superimposed endothelial damage and distal high-grade stenosis. rt-PA was injected as an intravenous bolus of 0.45 mg/kg body wt at 15-minute intervals until recanalization, or up to a maximum of four boluses. In seven rabbits given an intravenous injection of 17 mg/kg aspirin, rt-PA induced transient reflow in only one animal. In seven rabbits that received intravenous heparin (200 units/kg over 60 minutes), rt-PA administration produced reflow in five animals, which was persistent in two rabbits. Combined administration of aspirin and heparin in seven rabbits was associated with similar rt-PA-induced recanalization. rt-PA administration in six rabbits given intravenous Argatroban (100 micrograms/kg/min for 60 minutes) caused recanalization in five, with persistent patency in three. In six rabbits given aspirin and Argatroban, rt-PA caused recanalization in all, with persistent patency in five animals. Reflow occurred significantly more rapidly with Argatroban (14 +/- 7 minutes) than with heparin (35 +/- 11 minutes), reflow was obtained with fewer boluses of rt-PA in combination with Argatroban (median value of one bolus) than with heparin (median value, three boluses), and reocclusion after reflow was less frequent with Argatroban (0 of 11 versus 5 of 10 rabbits). Furthermore, the degree of thrombolysis determined by pathological analysis was significantly more extensive with Argatroban than with heparin, and patency persisted during a 3-hour observation period, despite elimination of Argatroban from the circulation. Thus, Argatroban, relative to heparin, enhances and sustains thrombolysis with rt-PA. It may offer promise as an adjunctive agent for thrombolytic therapy of arterial thrombosis.