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Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:1277-1284

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:1277-1284.)
© 1996 American Heart Association, Inc.

Articles

High Concentrations of Active Plasminogen Activator Inhibitor-1 in Porcine Coronary Artery Thrombi

William P. Fay; Joseph G. Murphy; Whyte G. Owen

the Department of Internal Medicine (Cardiology), University of Michigan Medical School, Ann Arbor, and the Ann Arbor Veterans Affairs Hospital (W.F.), Division of Cardiovascular Diseases and Internal Medicine (J.M.) and the Section of Hematology Research and Department of Biochemistry and Molecular Biology (W.O.), Mayo Clinic, Rochester, Minn.


*    Abstract
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 *Abstract
down arrowIntroduction
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 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Addition of exogenous plasminogen activator inhibitor-1 (PAI-1) to fibrin clots inhibits fibrinolysis in vivo. However, it is unknown whether the localized concentrations of active PAI-1 necessary to produce this antifibrinolytic effect can be recruited to acute arterial thrombi by endogenous mechanisms. We measured PAI-1 activity and antigen in porcine coronary artery thrombi that formed in response to acute vascular injury. Mean PAI-1 activity in thrombi (n=5) was 36±5.1 µg/mL, which is >2000 times its concentration in normal porcine plasma. The presence of markedly elevated concentrations of active PAI-1 in thrombi was confirmed by an immunoactivity assay and by demonstrating formation of sodium dodecyl sulfate–stable complexes after addition of 125I-urokinase to thrombus extracts. Comparative analysis of PAI-1 antigen by Western blotting and urokinase inhibition assay suggested that approximately one third of thrombus-associated PAI-1 was active. Histological examination of coronary thrombi revealed that they consisted predominantly of dense aggregates of platelets with interspersed islands of fibrin, which closely resemble the histological appearance of thrombi in patients with myocardial infarction and unstable angina pectoris. Washed porcine platelets prepared from peripheral blood contained sufficient PAI-1 antigen and activity to account for the concentrations observed in coronary artery thrombi. However, the specific activity of human platelet PAI-1 was lower than that of porcine platelet PAI-1 (2% versus 50% active, respectively), and human platelets inhibited in vitro fibrinolysis to a lesser extent than did porcine platelets. These results indicate that active PAI-1 accumulates in porcine coronary artery thrombi in concentrations markedly higher than those present in plasma and that PAI-1 may be an important determinant of the known resistance of platelet-rich thrombi to lysis by tissue-type plasminogen activator. These studies also underscore the importance of considering possible species differences in protein function when comparing animal models of thrombosis to acute coronary thrombosis in humans.


Key Words: myocardial infarction • thrombosis • thrombolysis • platelets


*    Introduction
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 up arrowAbstract
 *Introduction
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 down arrowResults
 down arrowDiscussion
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PAI-1, the primary inhibitor of TPA and UK, is a major regulator of fibrinolysis.1 Abnormal expression of PAI-1 in plasma appears to be an important determinant of human disease. PAI-1 deficiency results in abnormal bleeding.2 3 Conversely, an elevated plasma PAI-1 level is associated with myocardial infarction,4 5 and plasma PAI-1 levels peak in the early morning hours, coincident with the peak time of onset of myocardial infarction.6 7 These observations suggest that elevated plasma PAI-1 may alter the normal balance between blood coagulation and fibrinolysis, thereby predisposing to thrombus formation.

In addition to its role in plasma, a localized function of PAI-1 is to inhibit plasmin formation at sites of fibrin deposition, thereby producing a clot-stabilizing effect that is important in maintaining normal hemostasis. This antifibrinolytic effect may be particularly potent in the case of platelet-rich clots, since platelets contain PAI-18 and factors released from activated platelets stimulate vascular endothelial and smooth muscle cells to secrete PAI-1.9 10 However, in the pathological setting of acute coronary artery thrombosis, inhibition of fibrinolysis by clot-associated PAI-1 would be disadvantageous, since rapid restoration of arterial blood flow is strongly associated with improved clinical outcome.11 12 Several studies suggest that accumulation of PAI-1 within clots may contribute to the resistance of platelet-rich thrombi to lysis by TPA.13 Anti–PAI-1 monoclonal antibodies accelerate fibrinolysis within platelet-rich clots.14 15 In addition, clots formed in the presence of PAI-1–deficient platelets lyse more rapidly than do those that contain normal platelets.16 However, the relevance of these observations to in vivo settings is poorly defined, since the PAI-1 content of clots generated in vitro may differ substantially from that of thrombi that form within arteries, where shear forces and thrombin formation can lead to intense platelet deposition and PAI-1 release from the blood vessel wall may become incorporated in the forming thrombus.17 Marsh et al18 have demonstrated that incorporation of exogenous PAI-1 into experimental thrombi inhibits fibrinolysis in vivo. However, it is unknown whether the local concentrations of active PAI-1 necessary to produce this effect can be recruited to thrombi by endogenous mechanisms. Although plasma PAI-1 levels have been measured in a variety of clinical settings,1 the concentration of this inhibitor in coronary thrombi is not known. The purpose of this study was to measure the concentration of PAI-1 in thrombi formed in vivo in a porcine model of platelet-dependent coronary artery occlusion.


*    Methods
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 up arrowIntroduction
 *Methods
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Materials
Recombinant, human, single-chain TPA (580 000 IU/mg) was obtained from Genentech, Inc. LMW human UK (albumin free) was from Abbot Laboratories. Human glu-plasminogen, HMW UK, and spectrozyme UK were from American Diagnostica, Inc. Human thrombin was from Calbiochem-Novabiochem. Purified porcine PAI-1 and fluorescein-labeled human fibrinogen were prepared as previously described.16 19 Recombinant hirudin was a gift of Dr Robert B. Wallis, CIBA-GEIGY Pharmaceuticals. Affinity-purified, rabbit anti-human PAI-1 polyclonal antibody and purified, recombinant, human PAI-1 were gifts from Dr David Ginsburg, University of Michigan.20

Induction of Coronary Artery Thrombi
All animal care and experimental procedures complied with the Principles of Laboratory Animal Care established by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH publication No. 86-23, revised 1985). Platelet-rich thrombi were induced in juvenile domestic crossbred pigs (weight, 25 to 35 kg) by percutaneous implantation of a tantalum wire coil within a proximal coronary artery, as previously described.21 Induction of coronary thrombosis was not intentional. However, high-pressure inflation of an oversized angioplasty balloon during coil deployment caused deep injury to the arterial media in some animals, thereby inducing acute coronary artery thrombosis. The thrombi analyzed in this study completely occluded coronary blood flow and resulted in sudden death, presumably by precipitating ventricular arrhythmias. Thrombi were retrieved by dissection <=20 minutes after animal death and were frozen at -70°C until analyzed for PAI-1 content. Prior to freezing, portions of some thrombi were fixed and subsequently analyzed by light and electron microscopy.

Preparation of Thrombus Extracts
Thrombus volumes were determined after centrifugation of each thrombus in a graduated polypropylene microcentrifuge tube calibrated in 5-µL increments. Nine volumes of 0.02 mol/L Tris-HCl (pH 8.0) containing 0.01% Tween 80, 50 KIU/mL aprotinin, 0.05% (vol/vol) 2-mercaptoethanol, and 1.0 µg/mL recombinant hirudin were added to each thrombus, which was then subjected to 15 strokes of a close-fitting miniature pestle, gently rocked overnight at 4°C, and centrifuged at 14 000g for 2 minutes. The supernatant (thrombus extract) was removed and assayed for PAI-1 content. The concentration of PAI-1 in thrombi was calculated by multiplying the concentration of PAI-1 in the corresponding thrombus extract by 10.

Preparation of Washed Platelets and Platelet-Rich Clots
Washed platelets were prepared from peripheral porcine and human blood.16 Platelet extracts were prepared from washed platelet pellets by the same methods used to prepare thrombus extracts. Extracts of human platelet-rich clots were prepared by mixing washed platelets (1.5x1010·mL-1), human fibrinogen (1 mg/mL), and thrombin (1 U/mL) in Tyrode's buffer. After 30 minutes at 37°C (during which time clot retraction occurred) the clot supernatant was removed. The platelet-fibrin clot was retrieved, gently blotted, and then extracted as described above. Clot lysis in vitro was studied by mixing washed platelets (5x108·mL-1), fluorescein-labeled human fibrinogen (1 mg/mL), human glu-plasminogen (180 µg/mL), human TPA (0 to 10 ng/mL), and thrombin (1 U/mL) in Tyrode's buffer.16 After 60 minutes at 37°C, clots were centrifuged and percent clot lysis was determined by measuring the fluorescence of the clot supernatant, as described previously.16

Assays of PAI-1 Activity
Thrombus extracts were diluted 2-fold to 16-fold in 0.05 mol/L sodium phosphate (pH 7.5) containing 0.1 mol/L NaCl and 0.1% BSA (buffer A). To microtiter wells containing 25 µL buffer A were added 10 µL of LMW UK (2.0 µg/mL) and 10 µL thrombus extract, either undiluted or diluted as indicated above. After a 5-minute incubation at room temperature, 160 µL of 0.02 mol/L Tris-HCl (pH 8.4) containing 0.02 mol/L imidazole, 0.3 mol/L NaCl, and 125 µmol/L Spectrozyme UK were added to each well to yield final reaction mixtures of 205 µL. The change in absorbance of each well at 405 nm was measured in a Vmax kinetic microplate reader (Molecular Devices). PAI-1 activity in extracts was determined by assessment of residual UK activity and quantified by comparison with a standard curve constructed from samples containing 0 to 2.0 µg/mL UK. Separate control experiments verified that thrombus extracts did not hydrolyze Spectrozyme UK and that the thrombus extraction buffer did not inhibit UK. The UK used for measurements of PAI-1 activity was >95% pure by SDS-PAGE analysis, and enzyme concentrations (in micrograms per milliliter) were determined with the BCA reagent (Pierce) with BSA as the standard. Active-site titrations of UK preparations were performed by incubating UK with PAI-1 (molar ratio of PAI-1 to UK=2:1) and resolving the reaction products by SDS-PAGE. Complete incorporation of UK into SDS-stable UK–PAI-1 complexes was observed, confirming that the UK used in these experiments was fully active. Molecular weights of 34 000 for LMW UK and 47 000 for PAI-1 were used for calculating PAI-1 concentration (in micrograms per milliliter) from residual plasminogen activator activity.

The concentrations of active PAI-1 in plasma and in thrombus or platelet extracts were also determined by modification of a previously reported immunoactivity assay.2 22 Ninety-six–well Immulon 2 microtitration plates (Dynatech Laboratories) were coated with TPA (150 ng per well), washed with phosphate-buffered saline, and blocked with 3% BSA as described.2 Samples (100 µL) of thrombus or platelet extract (prepared by 10-fold to 160-fold dilution of each extract into 0.01 mol/L Tris-HCl, 0.14 mol/L NaCl, 1 mg/mL BSA, and 0.01% Tween 80, pH 7.5) or pooled citrated plasma (prepared from 30 normal pigs) were added to the wells and incubated for 2 hours at room temperature. Wells were extensively washed with 0.9% NaCl and 0.05% Triton X-100, and binding of PAI-1 to immobilized TPA was detected by sequential incubation with biotinylated rabbit anti-human PAI-1 antibody, streptavidin-conjugated alkaline phosphatase (GIBCO-BRL), and phosphatase substrate (Sigma Chemical Co) as previously described.20 Concentrations of PAI-1 in samples were determined by comparison with standard curves generated from purified porcine PAI-1 (specific activity, 425 000 U/mg) or recombinant, human PAI-1 purified under conditions that allowed isolation of the active conformer (gift of Dr Joseph Shore, Henry Ford Hospital).23

Assays of PAI-1 Antigen
Western blotting was used to demonstrate the presence of PAI-1 antigen in extracts prepared from porcine coronary thrombi and platelets. Extracts and samples of purified porcine PAI-1 were subjected to SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore Co) by using the PhastTransfer semidry system (Pharmacia). After the membranes were blocked, they were incubated with rabbit anti–PAI-1 polyclonal antibody (1:2500 dilution) for 2 hours at room temperature and then developed with the BM Chemiluminescence Western Blotting Kit (Boehringer Mannheim). PAI-1 concentrations were determined by densitometric analysis (NIH Image software, version 1.59) of developed x-ray films. Concentrations of PAI-1 antigen in human platelet lysates and platelet-rich clots were determined by ELISA.2

Radioautography of PAI-1–UK Complexes
125I-UK (LMW) was prepared by using the Iodo-Gen reagent (Pierce). Buffer A (10 µL) containing 10 ng 125I-UK (determined by titration with purified PAI-1) was incubated with 10 µL of thrombus extract or a dilution of thrombus extract at room temperature for 30 seconds. The reaction mixtures were adjusted to 1% in SDS, heated to 90°C for 5 minutes, and subjected to SDS-PAGE. Gels were dried and developed as radioautographs.

Analyses of Binding of PAI-1 to Other Thrombus Components
Measurement of PAI-1 binding to coronary thrombi was determined by a modification of the method of Wagner et al.24 The insoluble fraction remaining after removal of the thrombus extract was resuspended in 0.5 mL buffer A and centrifuged for 1 minute at 16 000g. The supernatant was discarded and the washing process was repeated four times. The washed pellet was resuspended in buffer A (9x the volume of the original thrombus) containing 1.0 µg/mL 125I-labeled UK and rocked overnight at room temperature. The resulting suspension was adjusted to 1% in SDS, rocked for 1 hour at room temperature, and centrifuged. A sample of the supernatant was subjected to SDS-PAGE and radioautography as described above. Binding of PAI-1 to the soluble factors in extracts prepared from thrombi or platelets was analyzed by native (ie, nondenaturing) PAGE and Western blotting with anti–PAI-1 polyclonal antibodies as described above.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
Coronary thrombi were retrieved from five consecutive animals that suffered occlusive, lethal coronary thrombosis in response to balloon angioplasty. In each case the stimulus for thrombus formation was deep arterial injury, ie, disruption of the internal elastic lamina (Fig 1ADown). Thrombus volumes ranged from 30 to 70 µL (mean, 40 µL). The platelet-rich nature of each thrombus was evident on gross inspection and confirmed by electron microscopy, which revealed that thrombi consisted essentially of dense masses of aggregated platelets with interspersed islands of fibrin (Fig 1BDown).



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  		Figure 1. Light and electron microscopy of coronary thrombus. A, Cross section of porcine coronary artery showing occlusive, platelet-rich thrombus (T) and underlying myocardium (M). Smaller arrows denote indentations in arterial wall caused by wire coil, which has been removed. Larger, open arrow denotes the internal elastic lamina. Note balloon-induced disruption of the internal elastic lamina at the 2 o'clock position (original magnification x12). B, Electron photomicrograph of a portion of thrombus, demonstrating densely packed platelet composition and interspersed islands of fibrin (arrow). Note retention of granular contents within a high proportion of platelets. Erythrocytes (E) and leukocytes (L) are present (original magnification x250).

Activity of Thrombus-Associated PAI-1
The mean PAI-1 concentration within thrombi as determined by UK inhibition was 36±5.1 µg/mL (39, 41, 35, 38, and 28 µg/mL for thrombi 1 through 5, respectively). This concentration of inhibitor at sites of arterial injury contrasts with plasma levels of PAI-1, which are usually <20 ng/mL.1 To confirm the presence of active PAI-1 antigen, thrombus extracts were analyzed by an immunoactivity assay as described in "Methods." The concentrations of PAI-1 in two thrombi assayed by this method (thrombi 4 and 5) were 26.2±3.5 and 26.8±1.2 µg/mL, respectively (mean±SD of duplicate experiments). Incubation of thrombus extract (3.3 µL) with TPA (42 ng) prior to addition to the immunoactivity assay completely inhibited binding of PAI-1 to immobilized TPA. In addition, binding of active PAI-1 to control wells coated with BSA only was not detectable. These results strongly suggested that the immunoactivity assay detected only active PAI-1 (as opposed to latent PAI-1 or TPA–PAI-1 complexes) and that nonspecific binding of PAI-1 to TPA-coated wells was insignificant.

Citrated porcine plasma was analyzed for PAI-1 content by using the immunoactivity assay. Purified porcine PAI-1 was used as the standard, and negative and positive controls (human PAI-1–deficient plasma and PAI-1–deficient plasma "spiked" with porcine PAI-1 [5 to 20 ng/mL], respectively) were included.3 Mean PAI-1 concentration in pooled plasma (n=30 pigs) was 9.0±3.6 ng/mL (mean±SD of triplicate measurements), a value similar to those reported for humans. Hence, the concentration of active PAI-1 in coronary thrombi exceeded that in normal plasma by >2000:1.

Thrombus-Associated PAI-1 Antigen
Samples of thrombus extract were subjected to SDS-PAGE and Western blot analysis. As shown in Fig 2Down, anti–PAI-1 polyclonal antibodies identified a band of {approx}45 000 MW in thrombus extracts, confirming the presence of PAI-1 antigen. Videodensitometry was used to quantify PAI-1 antigen in extracts prepared from two thrombi (4 and 5), yielding concentrations of 7.0 and 12.8 µg/mL (mean, 9.9 µg/mL), which corresponds to a calculated concentration of PAI-1 antigen within thrombi of 99 µg/mL. These results suggest that although high concentrations of active inhibitor are present in coronary thrombi (mean, 36 µg/mL), most of the thrombus-associated PAI-1 is inactive. By Western blot analysis we were unable to detect SDS-stable PAI-1–PA complexes in thrombus extracts to which either TPA or HMW UK had been added (data not shown). The anti-human PAI-1 antibody used in these experiments exhibited significantly reduced reactivity to porcine PAI-1 in complex with TPA or UK than to free PAI-1. Therefore, our inability to detect this complex by Western blotting was likely an issue of sensitivity. Consistent with the aforementioned results, SDS-stable TPA–PAI-1 complexes were weakly detected by Western blotting when TPA was reacted with purified porcine PAI-1 at a concentration of 20 µg/mL but were not detected when TPA was reacted with lower concentrations (<=3 µg/mL) that reflected those of the active inhibitor detected in thrombus extracts. Therefore, we used radioautography to confirm that addition of a PA to thrombus extracts resulted in complex formation. Thrombus extract was incubated with 125I-labeled UK and subjected to SDS-PAGE. An increase in the apparent MW of labeled UK (from 34 to 78 kDa) was observed after incubating UK with thrombus extracts for 30 seconds, consistent with rapid complex formation between UK and an inhibitor of {approx}45 kDa (Fig 3Down). The electrophoretic mobility of complexes generated in thrombus extracts by addition of UK was indistinguishable from that of complexes formed by incubation of labeled UK with purified PAI-1 (Fig 3Down, lane 2).



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  		Figure 2. Western blot analysis of coronary thrombus extract. Equal volumes of purified porcine PAI-1 or thrombus extract were subjected to SDS-PAGE and Western blot analysis with anti–PAI-1 antibodies. Lanes 1-3, purified PAI-1, 0.5, 1.0, and 2.0 µg/mL, respectively; lane 4, coronary thrombus extract diluted 5-fold. MW standards (in kDa) are indicated.



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  		Figure 3. Demonstration of PAI-1–UK complex formation by addition of UK to coronary thrombus extract. Samples of thrombus extract were incubated with an equal volume of 125I-UK (LMW) for 30 seconds and subjected to SDS-PAGE and radioautography. Lane 1, 125I-UK (incubated with equal volume of buffer); lane 2, 125I-UK+equal volume of purified PAI-1 (80 µg/mL); lanes 3-6, 125I-UK+16-, 8-, 4-, and 2-fold dilutions of thrombus extract, respectively; lane 7, 125I-UK+undiluted extract. MWs of UK and UK–PAI-1 complex determined from standards are indicated. Note incomplete incorporation of labeled UK into enzyme-inhibitor complex upon incubation with excess PAI-1 (lane 2), indicating that a minor fraction of UK was inactivated during radioiodination.

Additional Characterization of Thrombus-Associated PAI-1
Because PAI-1 binds to fibrin,24 25 measurement of PAI-1 in soluble thrombus extracts could underestimate the total thrombus PAI-1 content if significant amounts of inhibitor remained bound to fibrin or other insoluble components. To examine this possibility, the pelleted fraction remaining after removal of thrombus extract was washed and resuspended in buffer containing 125I-labeled UK. Formation of UK–PAI-1 complexes was detected by adding SDS to the suspension and subjecting a sample of the supernatant to SDS-PAGE and radioautography. Only trace amounts of radiolabeled complex were detected (data not shown), suggesting that either very little of the PAI-1 activity within platelet-rich coronary thrombi was bound to insoluble thrombus components or that the binding affinity was low. We also studied binding of PAI-1 to human platelet-rich clots by forming fibrin clots in the presence of washed platelets (1.5x1010·mL-1) and measuring the amount of PAI-1 antigen in (1) the clot supernatant after clot retraction (ie, PAI-1 released from the clot), (2) aqueous extracts of the clot (ie, PAI-1 retained within the clot but not bound to it), and (3) 8 mol/L urea extracts of the insoluble pellet remaining after generation of the clot extract (ie, PAI-1 retained within the clot and bound to it). These studies revealed that 82% (886±136 ng) of total platelet PAI-1 (1080 ng) was released into the supernatant during clot formation and retraction, whereas only 18% (191 ng) remained associated with the clot. Of the total clot-associated PAI-1, 95.8% (183±61 ng) was recovered in clot extracts, while only 4.1% (7.8±2.4 ng) remained bound to the insoluble clot pellet (ie, recovered in the 8 mol/L urea extract). Therefore, PAI-1 does not bind to platelet-fibrin clots with high affinity, and calculation of thrombus PAI-1 concentrations from analysis of aqueous thrombus extracts appears valid.

Although PAI-1 does not appear to bind to insoluble thrombus components, it is possible that it may bind to soluble factors within the environment of the clot, such as vitronectin.26 To explore this possibility, extracts prepared from porcine coronary thrombi and from human platelet-rich clots formed in vitro were subjected to native PAGE and then analyzed by Western blotting with anti–PAI-1 polyclonal antibodies. In contrast to experiments performed under denaturing conditions (Fig 2Up), the electrophoretic mobility of clot-associated PAI-1 under nondenaturing conditions was significantly reduced compared with that of purified PAI-1 (Fig 4Down), suggesting that PAI-1 in platelet-rich clots was complexed with another factor or factors.



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  		Figure 4. Nondenaturing Western blot analysis of thrombus-associated PAI-1. Samples of purified PAI-1 or PAI-1 extracted from clots or platelets were subjected to native PAGE and Western blot analysis. Left, Porcine samples: lane 1, purified platelet PAI-1 (5 µg/mL); lane 2, extract of washed platelets; lane 3, extract of coronary artery thrombus. Right, Human samples: lane 1, purified recombinant PAI-1 (2 µg/mL); lane 2, extract of platelet-rich clot formed in vitro by addition of thrombin to washed platelets and fibrinogen; lane 3, extract of washed platelets. For both species, note the retarded electrophoretic mobility of PAI-1 from platelets or clots compared with purified PAI-1, consistent with complex formation between PAI-1 and another factor in extracts. Additional studies (not shown) demonstrated that purified human PAI-1 analyzed in this experiment (right, lane 1) consisted of approximately equal amounts of active and inactive PAI-1. Therefore, the altered electrophoretic mobilities of platelet and clot-associated PAI-1 compared with those of purified PAI-1 are unlikely secondary to conformational changes.

Comparison of Human and Porcine Platelet PAI-1
Thrombus-associated PAI-1 may originate from platelets, the surrounding blood vessel wall, or plasma. To examine this issue, washed porcine platelets were prepared from peripheral blood and centrifuged to generate platelet pellets. Extracts of platelet pellets were prepared and analyzed for PAI-1 antigen and activity by the same methods used for the coronary thrombi. Porcine platelet extracts contained 10.3±3.1 µg/mL PAI-1 antigen (Fig 5Down). PAI-1 activity in porcine platelet extracts was 5.1±1.2 µg/mL by the immunoactivity assay. These values correspond to calculated PAI-1 concentrations of 103 µg/mL (antigen) and 51 µg/mL (activity) in platelet pellets. Incubation of porcine platelet extracts with TPA prior to SDS-PAGE and Western blotting resulted in significantly reduced intensity of the band corresponding to free PAI-1 (compare lanes 4 and 5 in Fig 5Down), consistent with incorporation of active PAI-1 into TPA–PAI-1 complex. However, similar to the results obtained with thrombus extracts, we were unable to visualize TPA–PAI-1 complexes by Western blotting. These results indicate that porcine platelets contain sufficient PAI-1 antigen and activity to account for the concentrations detected in coronary thrombi and that porcine platelet PAI-1 is {approx}50% active. They also support the hypothesis that thrombus PAI-1 is predominantly of platelet origin, consistent with the histological analyses of thrombi, which revealed that their major components were dense masses of aggregated platelets. For comparison, washed human platelets were extracted and assayed for PAI-1 activity simultaneously with porcine platelets. Human platelet extracts contained 1.6±0.1 µg/mL PAI-1 antigen (determined by quantitative ELISA), which corresponds to a PAI-1 antigen concentration of 16 µg/mL in densely packed platelets. In contrast, human platelet extracts contained only 30±7.6 ng/mL active PAI-1, indicating that the specific activity of human platelet PAI-1 (ie, 0.03/1.6=1.9% active) is significantly lower than that of porcine PAI-1. We also studied the activity of PAI-1 in human platelet-rich clots formed in vitro. Extracts prepared from these clots (n=2, formed in the presence of 1.5x1010·mL-1) contained 731±242 ng/mL PAI-1 antigen (determined by ELISA) and 66±5.5 ng/mL active PAI-1 antigen (determined by the immunoactivity assay), yielding a specific activity of 9.0%, which is less than that of PAI-1 from porcine coronary thrombi (3.6 µg/mL /9.9 µg/mL=36% active). Consistent with these species differences in platelet PAI-1 content and specific activity, washed porcine platelets (5x108·mL-1) inhibited in vitro clot lysis to a significantly greater extent than did human platelets (Fig 6Down). However, since platelets contain other antifibrinolytic factors (eg, {alpha}2-antiplasmin, factor XIII) that may vary in concentration between species, the greater antifibrinolytic effect of porcine platelets cannot necessarily be attributed to differences in PAI-1 activity.



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  		Figure 5. Western blot analysis of porcine platelet PAI-1. Extracts of washed platelets were prepared and subjected to SDS-PAGE and Western blot analysis with anti–PAI-1 polyclonal antibodies. Lanes 1-3, purified PAI-1, 0.5, 1.0, and 2.0 µg/mL, respectively; lane 4, 10-fold dilution of porcine platelet extract; lane 5, porcine platelet extract diluted 10-fold in Tris-buffered saline containing TPA (13 µg/mL); lane 6, purified PAI-1 (2.0 µg/mL) preincubated with TPA (33 µg/mL); lane 7, purified PAI-1 (20 µg/mL) preincubated with TPA (250 µg/mL). Note the significant reduction in intensity of the band corresponding to free PAI-1 upon reaction of platelet extract with TPA (compare lanes 4 and 5), consistent with incorporation of active PAI-1 into TPA–PAI-1 complex. Reduced reactivity of anti–PAI-1 antibody with porcine PAI-1 in complex with TPA is demonstrated by lanes 3 and 6, which contain equal amounts of purified PAI-1 (the former free, the latter in complex with TPA). PAI-1–TPA complex is faintly detected in lane 7, which contains 10 times as much PAI-1 as lane 6. Markers to right of lane 7 denote electrophoretic mobilities of PAI-1–TPA complex, free PAI-1, and cleaved PAI-1, respectively. SDS-PAGE analysis (not shown) of the purified porcine PAI-1 in these experiments revealed that it was essentially completely consumed upon incubation with a molar excess of TPA, reacting predominantly to form TPA–PAI-1 complex and minor amounts of cleaved PAI-1.



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  		Figure 6. Inhibition of fibrinolysis by human and porcine platelets. Platelet-rich fibrin clots were formed by addition of thrombin (1 U/mL) to fluorescein-labeled fibrinogen (1 mg/mL), glu-plasminogen (180 µg/mL), TPA (0-10 ng/mL), and human ({blacksquare}) or porcine ({blacktriangleup}) platelets (5x108·mL-1). After 1 hour at 37°C, percent clot lysis was calculated. Data points represent mean±SD of duplicate experiments (lack of error bars for some data points indicates SD was less than the size of data symbol). Parallel experiments (not shown) revealed that fibrin clots lacking platelets were completely dissolved by TPA (2.5 ng/mL) in <1 hour. Note greater inhibition of fibrinolysis by porcine platelets.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
This study examined the concentration and activity of thrombus-associated PAI-1 under experimental conditions that are highly relevant to acute coronary artery thrombosis in humans. Coronary thrombi that form in response to atherosclerotic plaque rupture usually consist of dense platelet aggregates with interspersed islands of fibrin.27 Therefore, the experimental thrombi in this series appear to be a good model for human coronary thrombi. In contrast to the 2-fold to 3-fold elevation in plasma PAI-1 that has been associated with thromboembolic disease,1 coronary thrombi contained >2000 times the normal plasma PAI-1 concentration (ie, calculated concentration of active PAI-1 in coronary thrombi was 36 µg/mL versus 9.0 ng/mL in plasma). This high concentration of PAI-1 within platelet-rich thrombi suggests that PAI-1 may contribute significantly to the inherent resistance of arterial thrombi to lysis.13 Addition of exogenous PAI-1 (10 µg/mL) to experimental clots inhibits thrombolysis in vivo.18 However, it has been unknown whether PAI-1 concentrations of this magnitude can be recruited to acute arterial thrombi by endogenous mechanisms. Our studies indicate that PAI-1 concentrations sufficient to produce an antifibrinolytic effect in vivo are present in acute platelet-rich thrombi.

An important aspect of this study is that PAI-1 activity within thrombi was measured. PAI-1 is secreted from endothelial cells in an active form that spontaneously converts to an inactive, or latent, conformation with a half-life of 1 to 2 hours under physiological conditions.1 Latent PAI-1, although incapable of inhibiting PAs, can be reactivated in vitro by exposure to denaturants like SDS and guanidine HCl.28 The activity of thrombus-associated PAI-1 was confirmed in our studies by inhibition of UK, binding to TPA, and formation of SDS-stable complexes with labeled UK. Since the thrombus extracts assayed in these experiments were prepared under nondenaturing conditions, these results suggest that the PAI-1 activity detected within them reflects high concentrations of active PAI-1 that were present within porcine coronary thrombi in situ. This is significant in regard to the potential antifibrinolytic impact of thrombus-associated PAI-1, since animal studies indicate that only active PAI-1 inhibits fibrinolysis in vivo.29

Binding of PAI-1 to Other Factors in Thrombi
Although PAI-1 binds to fibrin,24 25 we detected very little binding of PAI-1 to the insoluble fraction of coronary thrombi, which would include fibrin. Similarly, analysis of human platelet-rich clots formed in vitro revealed that only a minor fraction (<5%) of clot-associated PAI-1 was bound to the insoluble clot fraction. These results are consistent with the relatively low affinity of PAI-1 for fibrin (Kd=3.8 µmol/L)25 and the relatively low fibrin content of coronary thrombi, which consist predominantly of dense masses of aggregated platelets. However, analysis of coronary thrombus extracts by nondenaturing gel electrophoresis and Western blotting suggested that PAI-1 within thrombi was bound noncovalently to a soluble factor (or factors), as evidenced by the reduced electrophoretic mobility of thrombus-associated PAI-1 compared with purified PAI-1 (Fig 4Up). Although we did not identify the PAI-1–binding protein(s) in thrombus or platelet extracts, it is most likely vitronectin, since Preissner et al26 have demonstrated that PAI-1 released from platelets is associated with vitronectin.

Source and Distribution of Thrombus-Associated PAI-1
It was not possible from our studies to determine the source of thrombus-associated PAI-1. Given the platelet-rich nature of these coronary thrombi, it is likely that most of their PAI-1 content was platelet derived. Our experiments with washed, porcine platelets confirm that they contain sufficient PAI-1 antigen and activity to account for the concentrations thereof detected in coronary thrombi. However, it is also possible that vascular cells at the site of arterial injury secrete significant amounts of PAI-1 into the forming thrombus, as has been demonstrated in vitro.17 This hypothesis is supported by reports that factors released by activated platelets, including transforming growth factor-ß and platelet-derived growth factor, stimulate vascular endothelial and smooth muscle cells to secrete PAI-1.9 10 The methods used to extract PAI-1 from coronary thrombi disrupted platelet membranes. Therefore, we were unable to determine whether PAI-1 within the in situ thrombus was predominantly intracellular or extracellular. However, electron microscopy of coronary thrombi (Fig 1BUp) revealed that most platelets had retained their granular contents. These results are consistent with studies by Owen et al30 of porcine carotid artery thrombi. The histological appearance of these carotid thrombi, which were induced by mechanical injury, was essentially identical to that of porcine coronary artery thrombi. Isolated platelets, recovered by disaggregating carotid artery thrombi in vitro, released ADP (a dense-granule component) with kinetics and yield similar to those of basal platelets obtained from peripheral blood, indicating that although having undergone selective deposition in response to vascular injury, platelets within arterial thrombi had retained a majority of their dense granules. Of note, release of platelet dense granules precedes release of {alpha}-granules.31 Together, these observations suggest that a high percentage of PAI-1 (an {alpha}-granule protein) within acute coronary thrombi was intracellular, although immunohistochemical studies would be necessary to directly confirm this. It is also important to note that the presence of PAI-1 within platelets in freshly formed thrombi does not imply that this pool of PAI-1 is not eventually secreted. PAs as well as factors generated during thrombolysis, including thrombin and plasmin, stimulate platelet secretion.32 33 34 In addition, platelets within porcine arterial thrombi undergo spontaneous cytolysis 7 to 24 hours after initial clot formation, which would result in release of PAI-1.35

Comparison With Other Studies
Potter van Loon et al36 measured PAI-1 concentration in a series of human thrombi retrieved from the peripheral arterial circulation by embolectomy or from the venous system at autopsy. These investigators detected PAI-1 concentrations in arterial thrombi (2031 ng/g thrombus) that were 150 times greater than those in plasma. No coronary thrombi were analyzed in their series, and PAI-1 activity was not directly measured. Robbie et al37 found high concentrations of PAI-1 antigen (1.6 µg/g thrombus) in aortic and femoral artery thrombi from patients undergoing nonemergent surgery for peripheral vascular occlusion. PAI-1 activity was not measured. In addition to the species differences, our experiments differ from those studies in that coronary thrombi were retrieved <20 minutes after formation, whereas thrombi retrieved at surgery were likely present hours to days before removal. During this time conversion of active PAI-1 to latent and/or release of PAI-1 from the thrombus into the circulation may occur. Biemond et al38 showed that anti–PAI-1 monoclonal antibodies accelerate thrombolysis in a canine model of acute coronary artery occlusion. PAI-1 concentration in those thrombi was not measured. The thrombi in this study were formed by clotting whole blood within an occluded segment of coronary artery. Therefore, thrombus composition probably did not reflect that of platelet-rich thrombi that form in response to arterial injury.

Species Variation in Platelet and Clot-Associated PAI-1
Controversy exists regarding the specific activity of platelet PAI-1. In earlier studies we purified active PAI-1 from porcine platelets under nondenaturing conditions but did not measure PAI-1 antigen in platelets, thereby precluding precise determination of specific activity.19 Studies by Schleef et al22 and Booth et al39 indicate that most ({approx}95%) PAI-1 in washed human platelets is inactive, and studies by Lang et al40 suggest that the specific activities of porcine and human platelet PAI-1 are similar. However, Deguchi et al41 concluded that human platelet PAI-1 is predominantly active. Of note, these investigators observed significantly lower PAI-1 activity in washed platelets than in platelets activated in plasma. In this report we measured PAI-1 antigen and activity in extracts prepared from porcine coronary artery thrombi, human platelet-rich clots formed in vitro, and washed platelets from peripheral human and porcine blood. We found that porcine platelet PAI-1 was {approx}50% active (5.1 µg/mL activity per 10.3 µg/mL antigen), whereas the specific activity of PAI-1 in extracts of porcine coronary thrombi was slightly lower (3.6 µg/mL activity per 9.9 µg/mL antigen=36% active). In contrast <2% PAI-1 from washed human platelets was active (0.03 µg/mL activity per 1.6 µg/mL antigen), whereas the specific activity of PAI-1 in extracts of human platelet-rich clots generated in vitro was 9% (66 ng/mL activity per 731 ng/mL antigen). Consistent with these results, porcine platelets inhibited clot lysis in vitro to a greater extent than did human platelets. These data suggest that significant differences exist between pigs and humans in the intracellular concentration and specific activity of platelet PAI-1, at least in samples prepared from washed platelets. Of note, species differences in the concentration and function of other platelet proteins, such as von Willebrand factor, have been observed.42

Functional Significance of Thrombus-Associated PAI-1
Generation of a fibrinolytic state depends on the local balance of PAs and PAIs. The concentrations of endogenous TPA attained at sites of vascular injury are probably in the nanogram per milliliter range.37 Therefore, thrombus-associated PAI-1 very likely inhibits endogenously mediated fibrinolysis, which is relevant to the pathophysiology of unstable angina pectoris, in which mural platelet-rich thrombi are frequently present at sites of atherosclerotic plaque rupture.43 In addition it is possible that high local concentrations of PAI-1 may inhibit pharmacological lysis of platelet-rich thrombi,13 particularly when local arterial blood flow is absent, since the systemic plasma TPA concentration attained in this setting is {approx}1 µg/mL.44 However, additional studies are necessary to address the potential role of PAI-1 in thrombolysis resistance, since measurement of PAI-1 concentration in thrombi does not define its function in this environment and the concentrations of PAI-1 in thrombi that form in response to plaque rupture may vary more than observed in experimental systems.45 Mutant forms of TPA that are highly resistant to inhibition by PAI-1 should prove useful in testing the functional significance of thrombus-associated PAI-1 in animal models of arterial thrombosis and in ongoing clinical trials.46 47 48 Given the inherent instability of PAI-1, analyses of its activity within thrombi of different ages also should provide important information regarding its role in thrombolysis resistance. Finally, analyses of acute human arterial thrombi (eg, <4 hours old), although difficult to obtain, are necessary to better define the relevance of animal studies to acute coronary artery thrombosis in humans.


*    Selected Abbreviations and Acronyms
 
BSA = bovine serum albumin
ELISA = enzyme-linked immunosorbent assay
(H/L)MW = (high/low) molecular weight
PA(s) = plasminogen activator(s)
PAGE = polyacrylamide gel electrophoresis
PAI-1 = plasminogen activator inhibitor-1
TPA = tissue-type plasminogen activator
UK = urokinase


*    Acknowledgments
 
This work was supported in part by a Department of Veterans Affairs Medical Research Service Advisory Group Grant (to Dr Fay) and by National Institutes of Health grants HL-02728 (to Dr Fay) and HL-47469 (to Dr Owen). The Mayo Clinic Electron Microscopy Core Facility prepared the electron micrographs analyzed in this study.


*    Footnotes
 
Reprint requests to William P. Fay, MD, University of Michigan Medical Center, 7301 MSRB III, 1150 W Medical Center Dr, Ann Arbor, MI 48109-0644. E-mail wfay@umich.edu.

Received February 10, 1995; revision received June 11, 1996;
*    References
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 *References
 

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