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(Circulation. 1997;95:1886-1891.)
© 1997 American Heart Association, Inc.

Articles

₂-Antiplasmin Causes Thrombi to Resist Fibrinolysis Induced by Tissue Plasminogen Activator in Experimental Pulmonary Embolism

Anjum N. Butte, MB, BS; Aiilyan K. Houng, BS; Ik-Kyung Jang, MD, PhD; Guy L. Reed, MD

From the Cardiovascular Biology Laboratory, Harvard School of Public Health (A.K.H., G.L.R); Harvard Medical School (A.N.B., I.-K.J., G.L.R.); and Massachusetts General Hospital (A.N.B., I.-K.J., G.L.R.), Boston.

Correspondence to Guy L. Reed, MD, Harvard School of Public Health, II-127, 677 Huntington Ave, Boston, MA 02115. E-mail reed{at}cvlab.harvard.edu

	Abstract

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Background In patients with pulmonary embolism, thrombi resist fibrinolysis induced by plasminogen activators. Because the molecular basis of this thrombus resistance is poorly understood, we used a potent inhibitor to examine the potential role of ₂-antiplasmin (2AP) in experimental pulmonary embolism.

Methods and Results Lysis of experimental pulmonary emboli was measured 4 hours after embolization in anesthetized ferrets. All animals received heparin (100 U/kg). Five experimental groups were studied: (1) no recombinant tissue plasminogen activator (rTPA); (2) rTPA at 1 mg/kg; (3) rTPA at 2 mg/kg; (4) rTPA at 1 mg/kg plus a control monoclonal antibody (MAb); and (5) rTPA at 1 mg/kg plus an 2AP inhibitor (MAb 77A3). In comparison with ferrets receiving no rTPA (15.6±10.5% lysis, mean±SD), rTPA–treated groups showed significantly greater lysis (P<.01). Animals treated with rTPA and 2AP inhibitor (56.2±4.7% lysis) showed significantly greater lysis than all other treatment groups, including ferrets treated with the same dose of rTPA alone (38.5±6.3%, P<.01), with twice the rTPA dose alone (45.0±6.5%, P<.05), or with a control MAb (35.2±4.6%, P<.01). The combination of rTPA treatment and 2AP inhibition caused no consumption of fibrinogen.

Conclusions Inhibition of 2AP significantly amplified the lysis of experimental pulmonary emboli by rTPA without increasing fibrinogen consumption. These results suggest that 2AP may play an important role in thrombus resistance in patients with venous thromboembolism.

Key Words: 2-antiplasmin • fibrinolysis • embolism • plasminogen activators

	Introduction

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Venous thrombosis and pulmonary embolism are major causes of morbidity and mortality in the United States, accounting for about 270 000 hospitalizations a year.¹ In addition, it is estimated that about 50 000 to 200 000 patients a year die of a pulmonary embolism.² In surprising contrast to the mortality rate for myocardial infarction, the mortality rate for pulmonary embolism (estimated at 9.2% in treated patients) has not improved in the past 30 years.^{2 3} Moreover, survivors of venous thromboembolism are known to be at risk for recurrent thrombosis, postphlebitic syndrome, and pulmonary hypertension.^{4 5}

Standard therapy for venous thromboembolism is heparin, which potentiates thrombin and factor Xa inhibition by antithrombin III.⁶ Although heparin decreases new thrombus formation, clinical studies suggest that there is little early endogenous lysis of the large thrombi that often exist at the time of diagnosis in patients with venous thromboembolism.^{7 8 9 10} Because large thrombi are associated with an increase in morbidity and mortality, several studies have examined the effects of plasminogen activators in patients with venous thromboembolism.^{7 8 9 10} Compared with heparin alone, plasminogen activators cause significant increases in the lysis of venous thromboemboli, but patients are frequently left with large amounts of residual thrombi in the lungs or deep veins immediately after therapy.^{7 8 9 10} Moreover, none of the randomized, controlled trials of patients with pulmonary embolism have demonstrated a mortality benefit from plasminogen activators, although this may well be due to the small numbers of patients enrolled in these studies.

Why venous thromboemboli resist fibrinolysis is unknown. Physical characteristics such as size, retraction, exposure to blood flow, and age may affect the lysis of these large, fibrin-rich thrombi.¹¹ However, it is also likely that the fibrinolytic resistance of these thrombi is regulated by specific molecular factors such as factor XIII, PAI-1, and 2AP.^{12 13 14 15 16 17 18 19 20 21 22 23} Because 2AP is an ultrafast covalent inhibitor of plasmin, the enzyme that degrades thrombi, 2AP is a particularly likely cause of thrombus resistance.^{12 13 14} Moreover, 2AP is the only fibrinolytic inhibitor that is covalently cross-linked to the fibrin surface.¹⁵ This cross-linking (by activated factor XIII) concentrates 2AP on the fibrin surface, where it inhibits the initiation of fibrinolysis.¹⁵ Previous in vitro studies have shown that clots from 2AP-deficient patients lyse spontaneously, suggesting that 2AP plays a critical role in thrombus resistance to endogenous plasminogen activators.^{24 25} These observations led to the hypothesis that 2AP is a molecular mediator of the thrombus resistance seen in patients with pulmonary embolism. To test this hypothesis, we generated a specific inhibitor of 2AP and used it to determine the role played by 2AP in the regulation of lysis of experimental pulmonary emboli.

	Methods

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Materials were obtained from the following suppliers: rTPA with a specific activity of 580 000 IU/mg, Genentech; ketamine (100 mg/mL), Fort Dodge Laboratories; acepromazine maleate, Fermenta Animal Health Co; heparin (1000 U/mL), Elkins-Sinn Inc; sodium iodide, Aldrich Chemical Co; calcium chloride, Mallinckrodt; normal saline for intravenous use, Travenol Laboratories; 2AP assay kit, Stachrom; purified 2AP and fibrinogen, American Diagnostica; goat anti-mouse antibody, Cappel Organon Technika; human plasma pooled from random donors, Massachusetts General Hospital; bovine thrombin, Parke-Davis; [¹²⁵I]NaI, Dupont-NEN; Bard Parker surgical blade, Becton Dickinson; 4-0 silk sutures, American Cyanamid Co; Surflo IV catheter and 20-gauge 1.25-in Venoject tubes with K₃EDTA, Terumo Medical Corp; sterile three-way stopcock, Mallinckrodt Critical Care; auto syringe infusion pump, Baxter Health Care Corp; infusion pump tubing and microbore 60-in extension set, McGaw of Puerto Rico; surgical instruments, VWR; tubing, Namic; ferrets (0.8 to 1 kg), Marshall Farms; aprotinin, Sigma; and microcentrifuge tubes, National Scientific Supply Co.

MAb Production, Purification, and Characterization
Two BALB/c mice were immunized subcutaneously with 25 µg of purified human 2AP fragments derived from the trypsin digest of a human plasma clot. The 2AP fragments were affinity purified with a Sepharose-coupled MAb, RWR,²⁶ against human 2AP. Mice were initially immunized with complete Freund's adjuvant and boosted 90 days later with 50 µg of 2AP fragment in incomplete Freund's adjuvant. The antisera titer was tested in a solid-phase radioimmunoassay²⁷ with 2AP immobilized in the wells of a microtiter plate. Four days before fusion, the mouse with the highest titer of 2AP antibody was hyperimmunized with 100 µg 2AP intraperitoneally. Somatic cell fusion was performed as described.²⁸ Hybridomas were tested for the production of antibodies to the 2AP fragment and for their ability to inhibit 2AP as we have described.²⁷ The binding of MAbs to ¹²⁵I-2AP was tested in a solid-phase radioimmunoassay. Wells of a microtiter plate were coated with goat anti-mouse antibody (25 µL, 5 µg/mL) for 2 hours. The wells were rinsed, and nonspecific protein binding sites were blocked with 1% BSA in TBS, pH 7.4, for 1 hour. After a wash, 25 µL of hybridoma supernatant was added to the wells and incubated for 1 hour. The wells were rinsed, and ¹²⁵I-2AP was added (25 µL, 60 000 cpm) for 1 hour. The ¹²⁵I-2AP was then removed, and the wells were rinsed and gamma-counted.

Cloned hybridomas were expanded into ascites in pristane-primed BALB/c mice. Antibodies were purified from filtered ascites by precipitation with 40% ammonium sulfate, dialysis into 10 mmol/L KH₂PO₄, pH 7.2, and ion-exchange chromatography on DEAE–Affigel Blue Sepharose with a linear gradient from 0 to 100 mmol/L NaCl.

Clot Lysis Assays In Vitro
Pooled, fresh-frozen, citrated ferret plasma (1100 µL) was mixed with 15 µL of ¹²⁵I-labeled human fibrinogen (40 000 cpm/clot). Ferret plasma (35 µL) was mixed with 35 µL of TBS containing 10 mmol/L CaCl₂ and thrombin (1 U/mL) in 12x65-mm plastic tubes and clotted for 1 hour at 37°C. The clots were washed in TBS, the supernatant was removed, and then 100 µL of TBS or 25 µg of purified MAb (RWR or 77A3 [the antibody described below]) was added to tubes in duplicate. Clot lysis was initiated by the addition of 0.1 U rTPA per tube. The clots were incubated at 37°C for 5 hours, and the amount of lysis was determined by sampling for the release of radiolabeled fibrin degradation products into the supernatant, as described in Reference 27²⁷ .

Pulmonary Embolism Experiments
Male ferrets were anesthetized by injection (0.4 mL IM) of a mixture of ketamine and acepromazine (two parts acepromazine [10 mg/mL] to one part ketamine [100 mg/mL]). Intraperitoneal injections were repeated as necessary to keep the animals anesthetized. After an anterior midline incision had been made in the neck, the jugular vein and the carotid artery were exposed by blunt dissection and cannulated with 20-gauge catheters that were secured at the proximal and distal ends with 4-0 silk sutures. The catheters were capped with three-way stopcocks.

Pooled, citrated human plasma was mixed with ¹²⁵I-fibrinogen to achieve 1 000 000 cpm/mL. Individual clots were formed by mixing ¹²⁵I-fibrinogen–labeled plasma (45 µL) with 2.5 µL bovine thrombin (100 U/mL) and 2.5 µL calcium chloride (0.4 mol/L). These clots were incubated at 37°C for 90 minutes, compressed, and washed thoroughly with saline three times to remove unbound proteins. The radioactive content of the clots was measured in a gamma counter immediately before clot injection. Blood samples were drawn at baseline and at the end of the experiment. Sodium iodide (10 mg) was injected to block thyroid uptake. Clots were embolized into the lungs by injection through the internal jugular vein. Ferrets weighing <1 kg received three clots; those weighing 1 kg received four clots. Successful embolization was evinced by the accumulation of radioactivity in the thorax. After the clots had been injected, the ferrets were turned on their sides to ease breathing.

All animals received weight-adjusted heparin at 100 U/kg (bolus), a dose sufficient to keep the aPTT >150 seconds throughout the procedure. The 2AP inhibitor (sterile-filtered, 14 mg/mL in saline) or a purified control MAb (antidigoxin) was given as a single dose (22.5 mg/kg IV). The rTPA was given as a continuous infusion over a period of 2 hours (1 or 2 mg/kg in 5 mL normal saline). Animals were observed for a total of 4 hours after pulmonary embolization and then killed by lethal injection of anesthesia or by CO₂ inhalation. The thorax was dissected, and all intrathoracic structures were removed for gamma counting to detect residual thrombi. The percentage of clot lysis was determined for each ferret by dividing the total residual radioactivity in the thorax (cpm) by that in the initial thrombi.

This experimental protocol was approved by the Harvard Medical Area Standing Committee on Animals. The Harvard Medical School animal management program is accredited by the American Association of Laboratory Animal Care, and the procedures were conducted in accordance with National Institutes of Health standards, as set forth in the Guide for the Care and Use of Laboratory Animals (DHHS publication NIH 85-23, revised 1985), the Public Health Service Policy on the Humane Care and Use of Laboratory Animals by Awardee Institutions, and the NIH Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training.

Statistical Tests
The data were analyzed by a one-way ANOVA followed by a Bonferroni-Dunn procedure for multiple-comparison testing.

Fibrinogen Assays
Blood samples were collected on K₃EDTA (0.15% solution final) with aprotinin (50 KIU/mL). Platelet-poor plasma was obtained by centrifugation of whole blood²⁹ and assayed for fibrinogen by the sodium sulfite method.³⁰

2AP Assays
To measure 2AP levels, we collected ferret blood on sodium citrate (1/10 volume) and centrifuged it to obtain plasma.²⁹ The plasma was tested for functional 2AP with a chromogenic substrate assay for plasmin inhibition (Stachrom kit) as described.²⁷

	Results

Top Abstract Introduction Methods Results Discussion References

 
From a panel of hybridomas, we selected 77A3, an MAb that bound tightly to human 2AP. Fig 1A shows that compared with a control, anti-digoxin MAb in a radioimmunoassay, 77A3 bound specifically to ¹²⁵I-2AP. MAb 77A3 was purified from mouse ascites by ion-exchange chromatography, and its purity was confirmed by SDS-PAGE (Fig 1B). To study the role of 2AP in experimental pulmonary embolism in vivo, we tested purified 77A3 in several different animal plasma clot lysis assays to determine whether it could bind to and inhibit a nonhuman 2AP. Of various small animal plasmas tested (eg, hamster, gerbil, guinea pig, rat), 77A3 appeared to cross-react only with ferret plasma. Fig 2 compares the lytic effects of 77A3 with those of another MAb inhibitor of human 2AP, RWR,²⁶ and with buffer alone. Fig 2 shows that in comparison with the control (buffer alone), 77A3 accelerated the lysis of ferret plasma clots induced by a low dose of rTPA (0.1 U). In contrast, RWR, which inhibits human 2AP²⁶ but does not cross-react with nonhuman 2AP, had no detectable effect. This experiment indicated that 77A3 inhibited ferret 2AP and amplified ferret clot lysis in vitro.

View larger version (39K): [in this window] [in a new window]   Figure 1. Left, Binding of 125I-2AP to 77A3 and a control MAb. Wells of a microtiter plate were coated with goat anti-mouse antibody. Wells were incubated in duplicate with 77A3 or a control (antidigoxin) MAb.37 After a wash, 125I-2AP (60 000 cpm) was added for 1 hour. Wells were rinsed, and the amount of bound 125I-2AP was measured in a gamma counter. Right, Reduced SDS-PAGE of 77A3 purification. Ascites containing 77A3 was harvested and purified. Lane 1, protein standards (Std) with molecular mass in kilodaltons (left); lane 2, supernatant after precipitation with 40% ammonium sulfate; lane 3, purified 77A3. Reduced 77A3 immunoglobulin consists of bands of 50 kD, corresponding to heavy chain, and 25 kD, corresponding to light chain.

View larger version (18K): [in this window] [in a new window]   Figure 2. Effect of 77A3 on rate of lysis of ferret plasma clots in vitro. Ferret plasma clots formed with trace amounts of 125I-labeled human fibrinogen were incubated with 100 µL TBS (control) or purified MAb (25 µg, 77A3 or RWR). Clot lysis was initiated by 0.1 U rTPA added per tube. Clots were incubated at 37°C, and amount of lysis was determined by sampling for release of radiolabeled fibrin degradation products into supernatant as described.27

The cross-reactivity of 77A3 allowed us to investigate the role of 2AP in a ferret model of pulmonary embolism. In humans, pulmonary embolism is usually treated with heparin.⁶ Consequently, ferrets were treated with a weight-adjusted bolus dose of heparin at 100 U/kg. This dose was sufficient to keep the aPTT >150 seconds throughout the experiment (n=3). To investigate the effects of intravenous MAb 77A3 on the activity of 2AP in the blood, we selected a dose, 22.5 mg/kg, that was in molar excess of the level of ferret 2AP. Our ex vivo measurements of ferret 2AP activity, 1 and 4 hours after intravenous dosing, showed that 75% of ferret 2AP activity was inhibited at this dose (Fig 3, n=2).

View larger version (19K): [in this window] [in a new window]   Figure 3. Effect of in vivo administration of MAb 77A3 on functional 2AP levels in ferrets. In dose-finding experiments, two anesthetized ferrets (A and B) were given 77A3 (22.5 mg/kg IV), and amount of functional 2AP was measured in citrated plasma samples drawn before (time 0) and 1 and 4 hours after infusion. Data represent mean±SD inhibition of 2AP in plasma samples.

Using heparin at 100 U/kg and 77A3 at 22.5 mg/kg, we then investigated the effects of these agents and rTPA on the lysis of pulmonary emboli (Fig 4). All animals received heparin. Control animals (n=8), which received no rTPA, showed 15.6±10.5% (mean±SD) lysis of their pulmonary emboli. Animals receiving rTPA at 1 mg/kg (n=4) over 2 hours showed 38.5±6.3% lysis, which was significantly greater than lysis obtained in those receiving heparin alone (P<.01). Similarly, animals receiving rTPA at 1 mg/kg and a control (antidigoxin) MAb (n=3) showed 35.2±4.6% lysis. Ferrets treated with rTPA at 2 mg/kg (n=4) showed a minimal increase in lysis over those treated at 1 mg/kg (45.0±6.5% versus 38.5±6.3%, P<.05). However, animals receiving rTPA at 1 mg/kg together with the 2AP inhibitor (n=4) showed greater lysis (56.2±4.7%) than those receiving an equivalent dose of rTPA alone (P<.01), with or without the control (antidigoxin) MAb (P<.01), or those receiving twice the dose of rTPA alone (P<.05).

View larger version (24K): [in this window] [in a new window]   Figure 4. Effect of rTPA and 2AP inhibition on lysis of pulmonary emboli in vivo. Anesthetized ferrets were given a heparin bolus (100 U/kg), and 125I-labeled fibrin clots were embolized into lungs. After embolization, three groups of ferrets were given rTPA (0, 1, or 2 mg/kg IV) over 2 hours (open bars). Two other groups of ferrets also received rTPA (1 mg/kg) and a control MAb (antidigoxin, solid bar, 22.5 mg/kg) or a MAb that inhibits 2AP (77A3, hatched bar, same dose). Graph shows amount of lysis (mean±SD) for each treatment group. Number of ferrets in each treatment group is shown, and P values for differences between groups are indicated.

In addition to inhibiting plasmin on the thrombus surface, 2AP and other inhibitors inactivate plasmin in the blood.^{12 13 14} We measured fibrinogen levels in the blood to determine whether inhibition of 2AP led to nonspecific plasminolysis of a circulating clotting factor. Fig 5 shows residual fibrinogen levels expressed as a function of their initial values in four treatment groups. In animals that received no rTPA, fibrinogen levels varied moderately but did not diminish during the experiment. Ferrets receiving 1 and 2 mg/kg of rTPA alone showed no significant change in fibrinogen level. Similarly, animals receiving the combination of rTPA and the 2AP inhibitor showed no detectable change in circulating fibrinogen levels.

View larger version (22K): [in this window] [in a new window]   Figure 5. Residual fibrinogen levels in animals treated with heparin, rTPA, and an 2AP inhibitor. Blood samples were collected (on EDTA with aprotinin) from ferrets before pulmonary embolization and at end of experiment. Residual fibrinogen levels were measured as described.30 Graph shows mean±SD percentage residual fibrinogen level for animals receiving rTPA alone (0, 1, or 2 mg/kg; open bars) and those receiving rTPA and 2AP inhibitor (hatched bar).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Clinical and experimental studies suggest that pulmonary emboli and venous thrombi resist endogenous fibrinolysis and lysis induced by plasminogen activators.^{6 7 8 9 10} This resistance to lysis is due in part to specific molecular factors in the thrombus that act to oppose fibrinolysis. During thrombus formation, 2AP is covalently cross-linked to fibrin by activated factor XIII.¹⁵ Studies in vitro indicate that when 2AP in the clot is absent or inhibited by MAbs, clots undergo spontaneous lysis.^{24 25 26 27} Conversely, when levels of 2AP in clots are increased by supplementation in vitro, fibrinolysis is inhibited.¹⁵ In the present study, we investigated the hypothesis that 2AP plays a major regulatory role in fibrinolysis and that it contributes to the thrombus resistance obtained in pulmonary embolism.

We measured the effect of rTPA, with and without 2AP inhibition, on the net lysis of pulmonary emboli in ferrets. Because heparin is the established therapy for humans with pulmonary embolism, we considered animals treated with heparin alone to be the control group. The weight-adjusted bolus dose of heparin given to the ferrets was sufficient to maintain a high level of anticoagulation throughout the experiment. In animals treated with rTPA at a dose comparable to that used in humans (1 mg/kg), lysis of pulmonary emboli was enhanced significantly in comparison with lysis in animals treated with heparin alone. Increasing the dose of rTPA to 2 mg/kg, a dose higher than is safe in humans, led to a minimal increase in lysis. A similar plateau in the dose response for TPA-induced lysis has been noted in experimental studies of pulmonary embolism in dogs.³¹ However, specific inhibition of 2AP markedly potentiated the lysis of experimental pulmonary emboli by rTPA (1 mg/kg), causing significantly more lysis than was seen in ferrets treated with the same dose of rTPA, alone or with a control MAb. The lysis achieved with 2AP inhibition was also greater than that achieved in ferrets treated with high-dose rTPA (2 mg/kg). At the same time, despite the higher total lysis obtained in animals treated with the 2AP inhibitor, there was no significant consumption of circulating fibrinogen. In these studies of experimental pulmonary embolism, 2AP played an important role in thrombus resistance to lysis induced by rTPA. Further studies will be necessary to establish the relative quantitative roles of circulating and thrombus-bound 2AP in this process.

Besides 2AP, other molecular factors may regulate the thrombus resistance of pulmonary emboli. A leading candidate is PAI-1, a serine protease inhibitor of TPA and urinary-type plasminogen activator (UPA or urokinase).^{20 21 22 23} Unlike 2AP, PAI-1 is not specifically cross-linked to fibrin in the thrombus, although it has been shown to bind to fibrin in vitro.²⁰ By adding recombinant PAI-1 to developing thrombi, Marsh et al²³ have shown that PAI-1–enriched clots can suppress the spontaneous lysis of pulmonary emboli in a canine model; however, the role of PAI-1 in the lysis of autologous thrombi was not investigated. Pathological studies of pulmonary emboli extracted by thrombectomy have suggested that PAI-1 expression increases in the endothelial cells at the margins of fresh thrombi but is not detectable in the thrombi themselves.²² Since PAI-1–deficient mice (by gene deletion) are less likely than regular mice to develop venous thrombosis induced by endotoxin,²¹ the expression of PAI-1 in endothelial cells at the margin of the developing thrombus may be functionally important. Nonetheless, the role of PAI-1 in thrombus resistance to pharmacological plasminogen activators is less clear: in patients given TPA, the inhibitory capacity of PAI-1 is overwhelmed completely,³² and thrombus resistance is also observed in patients given streptokinase, against which PAI-1 has no effect.

Another potential cause of thrombus resistance in pulmonary embolism is activated factor XIII. Several studies in vitro suggest that this coagulation enzyme renders the fibrin in clots more resistant to degradation by plasmin by cross-linking fibrin chains together and by cross-linking 2AP to fibrin.^{15 16 17 18 19} However, little is known about activated factor XIII and thrombus resistance in vivo. This is probably because a potent inhibitor of factor XIII function has only recently become available.³³ One study has suggested that when factor XIII is partially inhibited, coronary thrombi lyse at accelerated rates in response to TPA.³⁴ This observation argues that factor XIII, through its effects on fibrin-fibrin and 2AP-fibrin cross-linking, also contributes to thrombus resistance.

Improving the lysis of thrombi in patients with pulmonary embolism and deep venous thrombosis remains a challenge. Unfortunately, increasing the dose of plasminogen activators is not a promising approach. High-dose TPA has been associated with an unacceptable increase in the risk of cerebral bleeding.³⁵ In addition, in the present study and others,³¹ high-dose TPA (2 mg/kg) produced only minimal increases in net lysis. The current FDA-approved doses of urokinase and streptokinase cause plasminogen "depletion"; thus, increasing the doses of these agents is also not likely to have an effect on net lysis.³⁶ Several potent inhibitors of thrombin generation and activity are under development. Although these agents may further reduce the formation of new thrombi, they will not directly improve lysis of the large thrombi that typically exist in patients at the time they are diagnosed. These considerations suggest that fundamental insights into the molecular factors that oppose physiological or pharmacological lysis in thrombi will be necessary to spark improved treatments for venous thromboembolism. The results of the present study indicate that 2AP is a major contributor to thrombus resistance in experimental pulmonary embolism, and they suggest that inhibiting 2AP might improve lysis in patients with thrombotic disease.

	Selected Abbreviations and Acronyms

 

2AP = 2-antiplasmin aPTT = activated partial thromboplastin time MAb = monoclonal antibody PAI-1 = plasminogen activator inhibitor 1 rTPA = recombinant tissue plasminogen activator TBS = Tris-buffered saline

	Acknowledgments

 
This work was supported in part by an NIH Clinical Investigator Award to Dr Reed (HL-02348) and through a sponsored research agreement with Bristol-Myers Squibb. The authors gratefully acknowledge the previous contributions of Edgar Haber and Gary R. Matsueda and the editorial assistance of Thomas J. McVarish.

Received September 16, 1996; accepted November 18, 1996.

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