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Rebound increase of plasminogen activator inhibitor type I after cessation of thrombolytic treatment for acute myocardial infarction is independent of type of plasminogen activator used.

Impaired fibrinolysis may contribute to the development of coronary artery disease and myocardial infarction [1]. The fibrinolytic activity of blood depends on the balance between the circulating concentrations of tissue-type plasminogen activator (t-PA) (4) and plasminogen activator inhibitor type 1 (PAI-1), which are both secreted by endothelial cells [2]. Patients with coronary artery disease showed increased plasma PAI-1 activity, which diminishes fibrinolytic capacity and thus predisposes to thrombotic disorders including myocardial infarction [3-5]. Coronary thrombosis is the major cause of myocardial infarction [6]. Thrombolytic therapy has been shown to reduce mortality and to improve left ventricular function of acute myocardial infarction (AMI) patients [7,81. Spontaneous thrombolysis also occurs in some patients and may confer similar benefits [9]. However, not all patients treated with a thrombolytic agent successfully reperfuse the thrombosed infarct-related coronary artery, and treatment is further limited by recurrent ischemia and rethrombosis in 10-25% of patients [10]. One factor that could potentially influence the response to thrombolytic treatment and the occurrence of spontaneous thrombolysis is PAI-1 [9]. Higher PAI-1 concentrations might interfere with the spontaneous thrombolytic process and might make pharmacologic thrombolytic intervention less effective [11,12]. Recently, a marked increase in PAI-1 activity and antigen was demonstrated after Alteplase infusions for AMI [13,14].

The purpose of the present study was to compare the time courses of PAI-1, t-PA, and D-dimer in AMI patients treated with different thrombolytic agents (i.e., rt-PA, streptokinase, and urokinase) to elucidate whether concentration changes are dependent on the administered fibrinolytic drug.

Materials and Methods


We studied 55 patients (44 men and 11 women, ages 42-89 years; mean, 61 years) presenting with nitroglycerol-resistant chest pain of <6 h duration (mean, 145 min; SD, 70 min), typical infarct-related electrocardiogram changes and an increase in myocardial enzymes and proteins [i.e., creatine kinase and its MB isoenzyme (CKMB) and myoglobin].

Two patients died from cardiogenic shock on days 3 and 4, respectively, and another one died from cardiac rupture on day 4. The latter three and two additional patients with incomplete data were excluded from analysis. The remaining 50 patients received intravenous thrombolytic therapy either with streptokinase (n = 23, 1.5 X [10.sup.6] U for 60 min), urokinase (n = 17, 2 X [10.sup.6] U for 15 min), or rt-PA (n = 10, given in a bolus of 10 mg over 2 min, then 50 mg during the first hour and 20 mg each during the second and third hours after admission). In all patients, heparin treatment was initiated with an intravenous bolus of 5000 units immediately before starting thrombolytic therapy. After the thrombolytic infusion was stopped, heparin therapy was continued with an intravenous infusion of 1000 units/h for the next 3 days titrated to maintain the partial thromboplastin time approximately twice that of the control.

The institutions responsible ethical committee approved this investigation, and all patients gave their informed consent. All procedures followed were in accordance with the Helsinki declaration of 1975, as revised in 1983.


Peripheral venous blood was collected through an indwelling forearm catheter (Venflon [R]) before thrombolytic therapy was started in the coronary care unit (time 0), then serially on day 1 (e.g., 1, 2, 4, 6, 8, 12, and 24 h after initiating therapy), and then daily until day 7 after admission. After discarding the first 5 mL of blood, 4.5 mL of venous blood was collected in tubes containing 0.5 mL of sodium citrate (0.1 mol/L). All samples were immediately centrifuged at 1500g for 20 min, and the plasma was frozen within 30 min after blood sampling and stored in aliquots at -70 [degrees]C until analysis was performed.

Fibrinolytic variables. PAI-1, t-PA, and D-dimer were measured in citrated plasma by commercially available enzyme immunoassays (COALIZA-ELISA systems from Chromogenix, Molndal, Sweden). The detection limit of the PAI-1 assay is 2.5 [micro]g/L, and the assay is specific for total PAI-1. In our laboratory, its intra- and interassay CVs were 3.5% and 8.4%, respectively. The t-PA assay is highly specific for t-PA antigen (one-chain and two-chain). The presence of PAI-1 does not interfere with the t-PA quantification. The detection limit of the t-PA antigen assay is 0.5 [micro]g/L, and its intra- and interassay CVs were 4.5% and 8.3%, respectively. The D-dimer antigen assay is specific for cross-linked fibrin derivatives. No binding was found in fibrinogen concentrations up to 5 g/L. Fibrinogen at physiological concentrations and fibrinogen degradation products (>3.0 g/L) do not markedly interfere with the assay. This assay is characterized by strong reactivity toward D-dimer, its detection limit is 25 [micro]g/L, and its intra- and interassay CVs were 5.6% and 10.3%, respectively.

Thirty healthy volunteers (18 men and 12 women, ages 18-54 years; mean, 27 years) served as controls to verify the reference limits given by assay manufacturers. Because some investigators showed a diurnal variation in fibrinolytic variables [15-17], blood was obtained from these controls in the morning (from 0800 to 1200; mean, 0920). In these controls, citrate plasma concentrations of PAI-1 ranged from 3 to 51 [micro]g/L (mean, 20 [micro]g/L), t-PA concentrations from 2 to 15 [micro]g/L (mean, 6 [micro]g/L), and D-dimer concentrations from 100 to 680 [micro]g/L (mean, 357 [micro]g/L). These values were in good agreement with the reference limits given by the manufacturers.

Cardiac markers. Total creatine kinase activity was measured by means of an N-acetylcysteine-activated, optimized ultraviolet test from Merck. The plasma concentration of CKMB mass was determined by a commercially available enzyme immunoassay on an IMX-analyzer from Abbott. The upper limits of the reference interval for creatine kinase activity (25 [degrees]C) were 70 U/L in women and 80 U/L in men, and for CKMB mass concentration 5 [micro]g/L. Myoglobin was measured by immunoturbidimetry (Turbiquant [R] Myoglobin, Behringwerke AG), and its upper limit of the reference interval was 70 [micro]g/L.


Repeated-measures ANOVA was performed for continuous data in different subgroups. A Student's t-test for independent samples was performed to assess differences between two groups and one-way ANOVA for more than two groups. A Student's t-test for dependent samples was used to determine significant changes between two observations within groups. [chi square] tests were used for nominal data. All descriptive statistics and tests were computed by means of the statistical software package SPSS (Superior Performing Software System, Inc.) on an IBM-compatible personal computer. Significance was defined as P <0.05.



Clinical characteristics and cardiac markers did not differ significantly (P >0.19) between the three subgroups of AMI patients that were treated with different thrombolytic agents (Table 1).


PAI-1. On admission, PAI-1 plasma concentrations did not differ significantly (P = 0.24) among groups (Figs. 1 and 2A). During the subsequent hours after thrombolytic therapy, PAI-1 increased significantly in each group (P <0.01 compared with admission concentrations) and peaked at 4 h in both the streptokinase and urokinase group and at 6 h after start of thrombolytic therapy in the rt-PA-treated group. Time to peak values and time courses differed significantly (P <0.01) between groups. In each group, PAI-1 peaked ~3 h after stop of thrombolytic infusion, corresponding to 4 h after start of streptokinase and urokinase infusion and to 6 h after start of rt-PA infusion. However, PAI-1 peak concentrations did not differ significantly (P >0.82) among groups (Fig. 2A). In each group, PAI-1 plasma concentrations returned nearly to admission concentrations 24 h after admission, and PAI-1 remained stable until day 7 after admission.

t-PA. On admission, t-PA concentrations did not differ significantly (P = 0.27) between groups (Fig. 2B). During thrombolytic infusion, t-PA increased only slightly in the streptokinase- and urokinase-treated patients, whereas, as expected, t-PA increased significantly (P = 0.0001) in the rt-PA group. In the latter group, t-PA antigen concentrations were significantly higher (P <0.01) than in the streptokinase- and urokinase-treated patients from start of thrombolytic infusion onward until 8 h thereafter. This difference in t-PA concentrations between the rt-PA group vs the other two groups is obviously because of additional detection of the therapeutically administered exogenous rt-PA by the t-PA assay (Fig. 2B). In both the streptokinase and the urokinase groups, t-PA increased only slightly from 12 to 24 h after admission. t-PA did not differ significantly between the streptokinase and urokinase treated group, neither in time course (P = 0.40) nor in maximum concentrations (P = 0.30; Fig. 2B). In each group, t-PA concentrations decreased to below admission concentrations on day 3 after admission and remained stable until day 7 after admission (no significant difference between groups; P >0.43).

D-dimer. D-dimer concentrations did not differ significantly between groups on admission (P = 0.45; Fig. 2C). In each group, D-dimer concentrations increased significantly (P <0.01) after start of thrombolytic therapy and peaked 4 h after admission. Afterward, D-dimer concentrations decreased from 6 to 12 h after admission to nearly the admission concentrations and remained stable from day 2 until day 7 after admission (P >0.10 compared with admission concentrations). D-dimer concentrations did not differ significantly between groups treated with different thrombolytic agents, neither in time course (P = 0.78) nor in maximum concentrations (P = 0.54; Fig. 2C).


We investigated PAI-1, t-PA, and D-dimer plasma concentrations before, during, and after thrombolytic therapy in AMI patients treated with three different thrombolytic agents, i.e., streptokinase, urokinase, and rt-PA. In each group, PAI-1 plasma concentrations increased significantly during the hours after thrombolytic therapy was stopped.

Recently, a PAI-1 increase was shown after Alteplase infusion for myocardial infarction [13,141. Our findings of an increase in PAI-1 concentrations during and after rt-PA infusion confirm these results. However, the PAI-1 increase is not restricted to only rt-PA-treated patients; it can be also observed after thrombolytic therapy with streptokinase and urokinase. In each group, PAI-1 peaked ~3 h after stop of thrombolytic infusion, i.e., 4 h after start of streptokinase and urokinase infusion and 6 h after start of rt-PA infusion. This difference in time-to-peak values between patients treated with different thrombolytic drugs is most probably due to the different administration regimen. Streptokinase and urokinase were infused in 1 h and 15 min, respectively, and rt-PA was given over 3 h. Thus, in the rt-PA group, the PAI-1 peak occurs 2 h later than in both other groups, obviously due to longer infusion time of rt-PA. However, the amount of the PAI-1 increase did not depend on the thrombolytic agent used, because PAI-1 maximum concentrations did not differ between groups. Thus, this PAI-1 increase may reflect a common, drug-independent reaction to thrombolytic infusion, which supports the hypothesis of an antifibrinolytic rebound phenomenon of the organism after thrombolytic therapy [18].



This rebound PAI-1 increase may interfere with thrombolytic therapy and may thus diminish the success of such interventions or may be responsible for spontaneous reocclusion after successful reopening of the coronary vessel after thrombolysis. In several diseases, including AMI, increased PAI-1 concentrations were accompanied by a decreased fibrinolytic activity, which predisposes to thrombotic disorders [2-5].

Recently, possible mechanisms were reported that may be responsible for the observed marked PAI-1 release. In humans, high amounts of PAI-1 concentrations are localized in thrombocytes, endothelial cells, and hepatocytes [3]. Activation of thrombocytes [14] did not contribute to the marked PAI-1 release observed after Alteplase infusion. Furthermore, thrombolytic therapy with rt-PA was suggested to affect the vascular intima to produce or release an increased amount of endogenous t-PA and PAI-1 from endothelial cells [13,19]. In the present study, a concomitant marked increase in PAI-1 and t-PA was detected in the rt-PA group only in the subsequent hours after start of thrombolytic infusion. However, as mentioned above, the marked t-PA increase in this group is obviously because of additional detection of the therapeutically administered exogenous rt-PA by the t-PA assay. By contrast, in both the streptokinase- and urokinase-treated group, PAI-1 plasma concentrations increased markedly in the subsequent hours after stop of thrombolytic infusion, whereas t-PA did not significantly increase after thrombolytic therapy. Thus, our data suggest that PAI-1 is selectively released from endothelial cells.

A possible stimulator of PAI-1 release from endothelial cells is the production of fibrin degradation products (e.g., D-dimer) [20], which appear in human blood after plasmin digestion of cross-linked fibrin [20,21]. D-dimer is a useful marker of thrombin activation with subsequent fibrinolysis and was found to be increased after thrombolytic therapy for AMI [21]. In each group of the present study, D-dimer increased significantly after thrombolytic therapy compared with admission concentrations. However, there was no close correlation between plasma concentrations of D-dimer and PAI-1. Furthermore, D-dimer time courses did not differ significantly between groups with different thrombolytic agents.

The acute-phase response after myocardial infarction was not deemed to contribute markedly to the observed PAI-1 increase [14, 19, 22]. Our results also do not support a major role of acute-phase response for the observed increase in PAI-1, because acute-phase proteins appear in the human plasma ~1 or 2 days after the onset of myocardial infarction [23], whereas in the present study, PAI-1 peaked 4 and 6 h after admission, respectively.

Circadian fluctuation might be another possible cause for increased PAI-1 plasma concentrations, because increased PAI-1 was found in the early morning hours in healthy volunteers [15,16] and in patients with coronary artery disease [15,17]. Thus, increased PAI-1 was found to contribute to the reported higher morning incidence of thrombotic cardiovascular events [16]. However, in the present study, only 18 patients (36%) were admitted between 0100 to 1200, and groups did not differ in time of day, neither when patients were admitted nor when PAI-1 peaks occurred. Thus, our data support earlier reports that the early peaking of PAI-1 after thrombolytic therapy is apparently independent of physiological diurnal fluctuations [14].

In conclusion, our results demonstrate a marked PAI-1 increase 3 h after stop of thrombolytic therapy, which occurs independently of the thrombolytic agent used. Thus, this PAI-1 increase seems to be a common, drug-independent reaction of the organism in response to enhanced therapy-induced plasminogen activation, suggesting an antifibrinolytic rebound phenomenon after thrombolytic therapy. These increasing PAI-1 concentrations may impair the success of thrombolytic therapy.

Received April 21, 1997; revision accepted October 9, 1997.


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[16.] Andreotti F, Davies GJ, Hackett DR, Khan MI, De Bart ACW, Aber VR, et al. Major circadian fluctuations in fibrinolytic factors and possible relevance to time of onset of myocardial infarction, sudden cardiac death and stroke. Am J Cardiol 1988;62:635-7.

[17.] Angleton P, Chandler WL, Schmer G. Diurnal variation of tissue-type plasminogen activator and its rapid inhibitor (PAI-1). Circulation 1989;79:101-6.

[18.] Lucore CL, Sobel BE. Interaction of tissue-type plasminogen activator with plasma inhibitors and their pharmacologic implications. Circulation 1988;77:660-9.

[19.] Juhan-Vague I, Alessi MC, Joly P, Thirion X, Vague P, Declerck PJ, et al. Plasma plasminogen activator inhibitor-1 in angina pectoris. Arteriosclerosis 1989;9:362-7.

[20.] Sprengers ED, Kluft C. Plasminogen activator inhibitors. Blood 1987;69:381-7.

[21.] Lew AS, Berberian L, Cercek B, Lee S, Shah PK, Ganz W. Elevated serum D-dimer: a degradation product of cross-linked fibrin (XDP) after intravenous streptokinase during acute myocardial infarction. J Am Coll Cardiol 1986;7:1320-4.

[22.] Munkvad S, Jespersen J, Gram J, Kluft C. Interrelationship between coagulant activity and tissue-type plasminogen activator (t-PA) system in acute ischaemic heart disease. Possible role of the endothelium. J Intern Med 1990;228:361-5.

[23.] Andreotti F, Roncaglioni MC, Hackett DR, Khan MT, Regan T, Haider AW, et al. Early coronary reperfusion thrombolysis blunts the procoagulant response of plasminogen activator inhibitor-1 and von Willebrand factor in acute myocardial infarction. J Am Coll Cardiol 1990;16:1553-7.


Departments of [1] Internal Medicine and [2] Medical Chemistry and Biochemistry, University of Innsbruck, A-6020 Innsbruck, Austria.

(4) Nonstandard abbreviations: t-PA, tissue-type plasminogen activator; PAI-1, plasminogen activator inhibitor type 1; rt-PA, recombinant t-PA; AMI, acute myocardial infarction; and CKMB, MB isoenzyme of creatine kinase.

* Address correspondence to this author at: Institut fur Medizinische Chemie & Biochemie, Fritz-Pregl Str. 3, A-6020 Innsbruck, Austria. Fax 43 512 507 2876; e-mail
Table 1. Clinical characteristics and myocardial markers of AMI
patients treated with different thrombolytic agents.

Clinical characteristics Streptokinase

n 23
Male sex, n (%) 18 (78)
Age, yr 63.0 [+ or -] 9.5
Delay, min 130 [+ or -] 45
Time of admission (a), n (%)
 0001-1200 8 (35)
 1201-2400 15 (65)
Infarct location (b)
 Inferior 16 (70)
 Anterior 7 (30)
Myocardial markers (c)
 Myoglobin ([micro]g/L) 1290 [+ or -] 975
 CK (U/L) 1065 [+ or -] 930
 CKMB mass ([micro]g/L) 265 [+ or -] 205

Clinical characteristics Urokinase

n 17
Male sex, n (%) 13 (76)
Age, yr 57.6 [+ or -] 12.8
Delay, min 150 [+ or -] 75
Time of admission (a), n (%)
 0001-1200 8 (47)
 1201-2400 9 (53)
Infarct location (b)
 Inferior 12 (71)
 Anterior 5 (29)
Myocardial markers (c)
 Myoglobin ([micro]g/L) 1005 [+ or -] 780
 CK (U/L) 835 [+ or -] 610
 CKMB mass ([micro]g/L) 235 [+ or -] 120

Clinical characteristics rt-PA

n 10
Male sex, n (%) 8 (80)
Age, yr 58.5 [+ or -] 15.2
Delay, min 175 [+ or -] 95
Time of admission (a), n (%)
 0001-1200 2 (20)
 1201-2400 8 (80)
Infarct location (b)
 Inferior 7 (70)
 Anterior 3 (20)
Myocardial markers (c)
 Myoglobin ([micro]g/L) 1390 [+ or -] 1095
 CK (U/L) 910 [+ or -] 865
 CKMB mass ([micro]g/L) 220 [+ or -] 130

The groups did not differ significantly in clinical characteristics
or myocardial markers. Data are presented as means

(a) Time of day was divided because of observed circadian fluctuation
of fibrinolytic variables [16, 17].

(b) The location of the infarcted area was determined by

(c) Peak concentrations.
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Title Annotation:Enzymes and Protein Markers
Author:Genser, Norbert; Lechleitner, Peter; Maier, Josef; Dienstl, Franz; Artner-Dworzak, Erika; Puschendor
Publication:Clinical Chemistry
Date:Feb 1, 1998
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