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;ACC Vol. 19 . No. 2 February 1992:44 1-3

Fibrinolytic Activity After Vessel Wall Injury YI SRI . MD, PrD. DONALD NARDONE, MD . ANTONIO HERNANDEZ-MARTINEZ, MD, PAUL WALINSKY, MD, FACC, THORIR D . BJORNSSON, MD, PHD, ANDREW ZALEWSKI, MD Philadelphia, Pennsylvania

The goal of this study was to assess fibrinolytic activity after vessel

wall injury and to correlate changes in fibrinolytic activity with anglographic and histologic findings . Accordingly, in 18 athcrosclerotic rabbits, vessel wall injury was produced by means of iliac artery balloon angioplasty (the Injury group), whereas 8 atherosclerotic rabbits served as a control group . In all rabbits from the injury group, deep vessel wall injury was documented by either angtography or histologic study. Plasminogen activator inhibitor-I activity in plasma :^rr.zsed significantly, from 21 .79 z 1 .29 arbitrary unitslml tAUlmll at baseline study to 32 .05 € 1 .47 AUJmt at 6 h after vessel wall in jury (p < 0 .011, whereas adis y remained unchanged thronghoat the 24-h period in the control

Acute coronary syndromes are associated with several procoagulant mechanisms that lead to intmcoronary thrombosis and impaired coronary blood low . Plasma levels of plasminogen activator inhibitor-I have been found to be elevated both in acute myocardial infa-ction and . more recently, in unstable angina at rest (1). These clinical observations raise the possibility that vascular wall injury such as plaque fissuring may be responsible for impaired fibrinolytic activity . Accordingly, the goals of this study were to 1) assess fbrinolytic activity after vascular wall damage in an experimental model of arterial injury, and 2) correlate changes in iibrinolytic activity with angiographic and histologic findings.

Methods Experimental protocol . Twenty-six New Zealand White rabbits were fed an atherogenic diet consisting of 1% cholesterol and 3% peanut oil . After 6 weeks, the animals werandomized to either the injury group (n = 18) or the could .I

From the Divisions ofCardialogy and Clinical Phaoracology, Deaanmeni of Medicine and the Department of Pathology and Cell Biology, Thomas Ieterson University . Philadelphia, Pennsylvania This study was sopponed to part by Groin HL45593 and by Biomedical Research Support Gram

2SO7RR0541429fmm!he National Institutes of Health . Bethesda, Maryland and was presented in pan at the 40th Annual Scientific Session of the American College of Cardiology. Aam,ea, Georgia, March, 1991 . Manuscript received June 3 . 1991 : revised manuscript received July 29, 1991, accepted Augost 23. 1991 . Address 'or eee,int . : Andrew Zatewski, MD, D,vision of Cardiology . Thomas Jefferson University Suite 5360 Gihbov Onildier . Ill South I Ith

St- Philadelpiie . Pennsyl+ania 19(07 .

01992 by the American College of Cardiology

group . Plasma levels of tissue plasminogen activator activity were similar in both groups . Intravascular thrombus was found in five of six a&4lonal rabbits 6 h after vessel wall injury, that Ev, at the time of impaired fibrinolytic activity, whereas no thrombus was found in the control group tp < 0 .05) . B Is concluded that deep vessel wait injury is associated with reduced fibrinolytic activity . In addition to other precoagulant factors, elevated plasminogen activator inhibitor-I activity may lead to intravaseular thrombosis and Impaired resolution of thrombus. (J Am Coil Cardiol 1992;19:441-3)

group (n = 8) . All rabbits were anesthetized with a intramuscular injection of 35 mg/kg body weight ketamine . The injury group was subjected to v, :,sel wall injury by means of bilateral common iliac artery angioplasty using a 3-mm balloon catheter . The control group underwent the same handling, anesthetic procedures and blood sampling as the injury group . Because our preliminary observations (unpublished data) indicated that vascular trauma during arterial cannulation and surgical exposure of the arterial vessels are associated with a transient increase in plasminogen activator inhibitor-I, those procedures (except for veniponcture) were pronibited in the control group . Peripheral angiography was performed immediately after balloon injury in nine rabbits from the injury group. The surgical and angiographic procedures were carried out under sterile conditions. The angiographic variables analyzed include the presence and lengsh of vessel wail dissection and the presence o`an intravascular thrombus. The rabbits were allowed to recover and were killed 4 weeks later. The animal experiments conform to the "Position of the American Heart Association on Research Animal Use" adopted November II, 1984 . Biochemical assays. Blood samples were obtained from the ear vein at baseline and at 1 . 6 and 24 h after the vessel injury and at the same time intervals in the control group . To account for possible circadian variations in measured variables, the time of blood sampling was matched in the two groups (€2 h). . Venous blood (0.9 ml) and sodium citrate (0 .1 ml, 0.13 M; pH 7 .5, were mixed, placed an ice and centrifuged (2,(00 g for 5 min at 4 °C) . For measurements of rs5ue plasminogen activator (t-PA) activity, the citrated pI sma samples were immediately acidified with sodium 0735-1U)7N7iao.e



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SHIET AL 'LASMINOOEN ACTIVATOR INHIBITORI

acetate buffer (I M ; pH 3 .9) . The acidified and citrated plasma samples were stored at -70'C until analyzed . Plasminogen activator inhibitor-1 and t-PA activities were determined by chromogenic assay as described before (1,2) . Plasminogen activator inhibitor-I activity was expressed in arbitrary units (AU)/ral. One arbitrary unit of plasminogen activator inhibitor-I is defined as the amount that inhibits I JU oft-PA activity for 15 min . Histology . With use of in situ perfusion fixation with a4% formaldehyde solution, iliac arteries in nine rabbits of the injury group were preserved for histologic study . Arteries were sectioned (every 4 mm), dehydrated in graded ethanol and embedded in paraffin. Sections from each block were stained with hematoxyiin-eosin, trichrome (for connective tissue) and Verhoeff-van Gieson stains (for elastic fibers) . In six additional atherosclerotic rabbits, histologic features of the iliac artery were assessed at 6 h after vessel wall injury (that is, at the time of maximal changes in the fibrinolytic activity) and compared eith those of three control rabbits not subjected to the balloon injury . Variables analyzed by light microscopy included the presence of deep vessel wall injury (defined as a disruption of the internal elastic lamellae) and intravascular thrombus. Microscopic evaluation was performed by an observer who had no knowledge of the randomization data . Statistical analysis . All results are expressed as mean values A SEM . The biochemical data in the control and injury groups were analyzed with use of two-way analysis of variance . Subsequent comparisons between the groups or different sampling times were carried out with the unpaired and paired t tests and Bonferroni correction for multiple group comparisons ; the Fisher exact test was used to compare the incidence of intravascular thrombus .

30-

= E

at.

20-

In-

0 0

6

24

Time (hr.) Figure 1 . Levels of tissue plasminogen activator ft-PA) in plasma in the control group In = 8) (open drdes) and in the injury group In = 181 )dosed circles) are comparable .

iliac artery dissection . There was no correlation between the length of dissection and the level of maximal plasminogen activator inhibitor-1 activity (r = 0 .35, p = NS) . In addition, only three of the nine rabbits showed intravascular thrombus immediately after vessel wall injury . Histology. Of the nine rabbits from the injury group that were studied by light microscopy 4 weeks after the procedure, all showed signs of deep vessel wall injury (disruption of the internal elastic lamellae, smooth muscle cell proliferation and foci of fibrosis). Intravascular thrombus was found in five of six additional rabbits evaluated 6 h after vessel wall injury (that is, at the time of impaired fibrinolytic activity) (Fig. 2), whereas three control rabbits showed intact inten,al elastic lamellae and no thrombus formation (p < 0 .05).

Results Plasma t-PA and plasminogen activator inhibitor-I activity. Base'iae t-PA activity in plasma was 13 .52 € 2.07 Wind in the control group and 11 .89 € 1 .02 IUlml in the injury group (p = NS) (Fig. 1) . Tissue plasminogen activator activity did not change significantly throughout the experiment within the groups, nor did it differ significantly between the control and injury groups . Baseline plasminogen activator inhibitor-I activity in plasma was 21 .8 `- 1 .34 AU/ml in the control group and 21 .79 * 1 .29 AU/ml in the treated group (p = NS) (Fig. 2) . In the control group, plasminogen activator inhibitor-I activity remained unchanged at I, 6 and 24 h. In contrast, in the injury group, plasminogen activator inhibitor-I activity increased to 32 .05 ._ 1 .47 AIUml at 6 h (p < 0.01 vs . the control group at baseline and at 1, 6 and 24 h) and retotned to baseline at 24 h (23 .99 € 1 .16 AU/ml), Phtsminogen activator inhibitor-I activity at 6 h after vessel wall injury was significantly higher than at any other time in the injury group (p < 0.01) (Fig . 2) . Angiography. Seven of the nine rabbits from the injury group subjected to peripheral angiography demonstrated

Figure 2 . Plasminogen activator inhibitor-t (PAW) activity in plasma is significantly increased at 6 h after vessel wall injury . •p < 0 .01 compared with activity levels at baseline and at 1 .6 and 24 h in the control group (open elrdes) and at baseline and at I and 24 h in the injury group (closed dreks) . AU - arbitrary units.

6

24

Time (hr .)



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Pebrvary 1992.441-3

Discussion The major finding of this study was that deep vessel wail injury resulted in transient elevation of plasminogen actisator inhibitor-I activity . Furthermore, a temporal relation between impaired fibrinolytic activity and increased incidence of intravascular thrombus was observed . Mechanism of plasminogen activator Inhibitor elevation, An increase in plasminogen activator inhibitor-I activity after vessel wall injury could be related either to direct release of plasminogen activator inhibitor-! from the activated platelets or to its enhanced de novo synthesis stimulated by released growth factors . The latter was recently demonstrated both in vitro (3) and in vivo (4) . Platelet lysates increase^O plasminogen activator inhibitor-1 activity, whereas antibodies to transforming growth factor beta markedly attenuated this response (4) . The alternative mechanism may involve conformational change in the plasminogen activator inhibitor-I molecule after vessel wall injury . The exposure of extracellular matrix to a variety of binding proteins could lead to conversion of the latent plasminogcn activator inhibitor-1 to its active form (5). Because plasminogen activator inhibitor-1 activity increased in the injury group, but not in the control rabbits, the possibility of in vitro release of the inhibitor is collected anticoagulated blood samples was excluded. To avoid bacterial contamination, all surgical procedures were carried out under sterile conditions ; therefore, an endotoxin-induced increase in plasminogen activator inhibitor-] activity seems unlikely (6). Pathophysiologic effects of plasminogen activator inhibitor-I elevation. The exact cause and effect relation between increased plasminogen activator inhibitor-1 activity and intravascular thrombosis has not been eairely determine"., although high plasma levels of the inhibitor have been accompanied by prolonged clot lysis (7). Elevated plasminogen activator inhibitor-1 activity has been associated with failure of thrombolytic therapy to achieve sustained reperfusion in acute myocardial infarction (8) and with increased risk of reinfarction over a long follow-up period (9) . Furthe,more, our previous study (I) in patients with unstable angina demonstrated that impaired fibrinolytic activity was associated with a 50% incidence rate of intracoronary thrombus at the site of vessel wall injury by coronary angiography . The present study suggests a procoagulant role of elevated plasma levels of plasminogen activator inhibitor-t, as indicated by microscopic evidence of intravascular thrombosis at the time of impaired fibrinolytic activity . Experimental model and limitations. There are several limitations of this study . To achieve medial injury, the iliac artery was intentionaily injured with an oversized balloon . Therefore, the extent of impairment of fibrinolytic activity in lesser forms of vessel wall injury remains to be determined . Furthermore, this study did not address the exact location of plasminogen activator inhibitor-I formation (for example, at the site of iliac artery dissection or arteriotomy) . However, our results indicate that vessel wall injury in conjunction

44 3

with elevated plasminogen activator inhibitor-I activity is associated with increased intravascular thrombosis at the site of medial injury . Although transient plasminogen activator inhih^or-I elevation is not uncommon, its adverse effect appears to be expressed only in the presence of deep vessel wall injury . The procoagulant role of increased plasminogen activator inhibitor-I activity was established only indirectly, because vessel wall injury is associated wills other prothrombotic mechanisms, such as platelet activation (10) and thrombin generation (1l) . Additional studies using antiplatelet agents (for exam ;ue ; aspirin and monoclonal antibodies against Ilbdlla receptors) or autithromhotic agents (for example, hirudin) will be needed to elucidate further the source of elevated plasminogen activator inhibitor-I activity and its response to therapy . Moreover, it remains to be determined if a similar phenomenon exists not only in patients with spontaneous vessel wall injury (1) but also in patients undergoing an intracoronary intervention such as angioplasty or atherectomy . Conclusions. Experimental deep vessel wall injury is associated with impaired fibrinolytic activity . In addition to other procoagulant factors, increased plasminogen activator inhibitor-I activity in plasma can contribute to increased risk of intravascular thrombosis and impaired resolution of thrombus

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activity in unstable argina at rest: clinical, biochemical, and angiog aphic orrelaies . Circulation s991 :93 :1685-91 . 2 . Chmielewska J. Booby M. Wiman B. Evidence for a rapid inhibitor to tissue plasminogen activator in plasma . Tbmmb Res 1483:31 :427-36. 3 . Fujii S . Lucon CL, Hopkins WE, Billadeao 1J, Sobel BE. Potential attenuation of fibriuotysis by growth factors released from patelets and their pharnaacatogic implications, Am 1 Cardiol 198903 :1505-t 1 . 4. Fuji S . Sobel BE. Induction of plasminogcn activator inhibitor by products released from platelets . Circulation 1990;82:1405 13 . 5. tan uen 1W, Cammenoa M, came BW, Menens K, Pannekuek H, Van Mounk JR . Activation or human endothelial cell-type plasminogen Rat ,A ., inhibitor (PAI-11 by negatively charged phosphdipds . I Blot Chic

1987 ::'0- :17492-6 . 6. Engebr~~tsen L, Kieiulf P, Brandtraee P. Extreme plasminogen activator inhibit, and endotoxin values in patients with meningococcaldisease . 7brona Res 1986:42:713-6. 7. Brommer El, Boles AL, Koopman 1, Haverkate F Protraction of whole blood and plasma clot lysis in patients with high Ievets of an inhibitor of

tissue-type pasminogcn activator . Thromb Res 1905;39:271-g0. S. Barhash GI. Hod H, Roth A, et al. Conelalion of baseline plasm boacn

activator inhibitor activity with pa ency of the infant artery after thimnbolyr:c therapy in acute myecard :al infarction . Am 1 Cardiol 1989 ;64 : 1271-5 . 9 . HamnlrrA,deFaireU,WalldiusG,etal .Plasminogenactivator inhibitor in plasma : risk factor for recurrent myocardial infantion . Lancet 1987;2: 19. 10 . Wilma 1R, Sanbom TA, Ha-drnsebild CC, Valeri CR, Ryan TJ, Faxon DP . Platelet accumulation in expenaa :nul angioplasty: time course nod

on to vascutar ;ojury. Circulation 1907:7fi636-42. 11. Hems M, Cbesebro 1H, Webvlne MWI, et a1 . Himdin, heparin, and placebo during deep arterial i fury to the pig : the in vivo role of thrombia

in platelet-mediated thrombosis . Circulation 1996:82 :1476-84.

Fibrinolytic activity after vessel wall injury.

The goal of this study was to assess fibrinolytic activity after vessel wall injury and to correlate changes in fibrinolytic activity with angiographi...
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