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Circulation. 1995;92:1320-1325

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(Circulation. 1995;92:1320-1325.)
© 1995 American Heart Association, Inc.

Articles

Visualization of Thrombi in Pulmonary Arteries With Radiolabeled, Enzymatically Inactivated Tissue-Type Plasminogen Activator

Presented in part at the 67th Scientific Sessions of the American Heart Association, Dallas, Tex, November 14-17, 1994, and published in abstract form in Circulation. 1994;90(pt 2):I-369.

Van H. De Bruyn, MD; Steven R. Bergmann, MD, PhD; Bruce A. Keyt, PhD; Burton E. Sobel, MD

From the Cardiovascular Division, Washington University School of Medicine, St Louis, Mo (V.H.DeB., S.R.B.); Cardiovascular Research, Genentech, Inc, South San Francisco, Calif (B.A.K.); and the Department of Medicine, the University of Vermont, Burlington (B.E.S.).

Correspondence to Van H. De Bruyn, MD, Cardiovascular Division, Box 8086, Washington University School of Medicine, 660 S Euclid Ave, St Louis, MO 63110.


*    Abstract
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 *Abstract
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Background Despite the high frequency of pulmonary thromboembolism and its significant morbidity and mortality, diagnosis remains suboptimal. We have been developing a method for prompt detection with the use of radiolabeled, inactivated tissue-type plasminogen activator (TPA) and performed the present study to determine whether its use permits rapid scintigraphic visualization of pulmonary thrombi in vivo.

Methods and Results The thrombolytic, but not fibrin-binding, property of TPA was inactivated with a tripeptide chloromethyl ketone (YPACK) that had already been iodinated with 123I to radiolabel the TPA. Pulmonary arterial thrombosis was induced in nine dogs with the use of guide wires modified to provide thrombogenic tips. 123I-YPACK-TPA (1.1 to 7.8 mCi, 0.5 to 7.8 mg) was infused for 5 minutes into either the systemic or the pulmonary circulation. Clearance of radioactivity from the blood was rapid and indistinguishable from that of unlabeled, thrombolytically active TPA, with only 6.7±1.0% (mean±SEM) of peak radioactivity remaining after 60 minutes and minimal release of labeled fragments from the liver during this interval. Thrombi were visualized with single photon emission computed tomography and/or planar imaging 40 to 120 minutes after infusion of tracer in all seven animals given at least 3.7 mCi of 123I-YPACK-TPA. Ratios of radioactivity in thrombus (wet mass, 610±64 mg) to blood were high (14±3:1).

Conclusions The use of radiolabeled TPA in which thrombolytic activity is inactivated permits prompt scintigraphic detection of thrombi in pulmonary arteries in vivo.


Key Words: plasminogen activators • embolism • thrombosis • lung


*    Introduction
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 *Introduction
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Pulmonary embolism afflicts roughly 600 000 patients in the United States annually and is associated with a mortality of 30%.1 2 Treatment with fibrinolytic agents or anticoagulants alone cannot be undertaken lightly because of the risk of hemorrhage, stroke, or death.3 4 Although pulmonary angiography is the gold standard for diagnosis, it is also costly, not always immediately feasible, and invasive. Ventilation-perfusion scans exhibit low diagnostic specificity.5 6 7 The potential value of scintigraphic detection of thromboemboli is evident from the diverse approaches being explored, including those involving fibrinogen, antifibrin antibodies, and synthetic peptides.8 9 10 11 12 13 14 15 16 17 18 19 20 21

We have been developing a potential diagnostic approach entailing the use of radiolabeled tissue-type plasminogen activator (TPA) in which the thrombolytic property had been enzymatically inactivated; such TPA retained its high binding affinity to fibrin and its rapid clearance from plasma.22 23 24 TPA was inactivated with a peptidyl chloromethyl ketone (PPACK); dilactitol tyramine (DLT) was used as a residualizing agent to increase hepatic retention of extracted, radiolabeled, inactive TPA fragments and thus obviate release of radiolabeled fragments into the blood, with the subsequent degradation of image quality.23 These maneuvers permitted visualization of femoral arterial thrombi in experimental animals and pulmonary thrombi ex vivo. However, uptake in the bone marrow and other tissues precluded imaging pulmonary thrombi in vivo.23 24 Accordingly, we modified this initial approach further and performed the present study with TPA inactivated and radiolabeled with 123I-tyrosylprolylarginyl chloromethyl ketone (YPACK), which also served as a hepatic residualizing agent. This 123I-YPACK-TPA retained the highly specific clot-binding properties of TPA and allowed us to scintigraphically visualize thrombi in pulmonary arteries in vivo.


*    Methods
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 *Methods
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This protocol was approved by the Animal Studies Committee of Washington University.

Experimental Preparation
Adult, fasted, conditioned dogs weighing 22 to 28 kg were anesthetized with thiopental (12.5 mg/kg IV) and {alpha}-chloralose (72 mg/kg IV). They were intubated and ventilated with room air and supplemental oxygen. A femoral artery and vein were catheterized. A thyroid-blocking dose of sodium iodide (67 mg/kg IV) was administered.

Thrombosis in pulmonary arteries was induced with distally placed guide wires to which thrombogenic cotton thread had been attached. The wires were prepared with 100% cotton mercerized thread tied to heparin-coated guide wires (diameter, 0.038 in) (Cook, Inc) with 60 to 90 knots on the distal 4 to 5 cm of the guide wire (Fig 1ADown). The most distal 1 cm of the guide wire was left bare to facilitate insertion through catheters. The threads were aligned so that they were parallel and were trimmed to extend roughly 0.8 cm from either side of the wire, yielding a total diameter of roughly 1.6 cm. Dimensions were recorded so that the net mass of thrombus formed in each case could be determined after completion of each study. Thrombi formed with cotton threads are typically composed of platelet aggregates surrounded by red thrombus.25



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  		Figure 1. Top, Photograph of tip of guide wire with thrombogenic cotton thread attached. Placement of tip into pulmonary artery led to formation of thrombi. Knots were placed on 4 to 5 cm of the catheter; the distal 1 cm was left uncovered to facilitate guide wire placement. Bottom, Anteroposterior (AP) and right lateral chest radiographs demonstrating placement of a guide wire with thrombogenic thread into the left lower lobe.

The guide wires were placed under fluoroscopy after a Swan-Ganz catheter was positioned and wedged in a distal pulmonary artery, and a cardiac biopsy catheter was advanced over the Swan-Ganz catheter. The Swan-Ganz catheter was then removed, and the guide wire with thrombogenic cotton thread was advanced through the biopsy catheter. The tip of the guide wire with thread attached was placed in a low segment of the left lung in seven of the nine studies (Fig 1BUp). In one dog, the tip was placed in a low segment of the right lung, and in another, the tip was placed in the central left lung. The biopsy catheter was then removed. Lidocaine (1 to 1.5 mg/kg IV bolus) was administered as needed to suppress catheter-induced ventricular arrhythmias.

For pulmonary arterial infusions of radiolabeled, inactive TPA, a catheter was advanced over the guide wire to an appropriate location. For systemic infusions, catheters were placed in the brachiocephalic or femoral veins. All infusion and biopsy catheters were flushed with saline. Saline was infused at a minimal rate to prevent formation of thrombi within the catheters.

123I-YPACK-TPA (1.1 to 7.8 mCi in 5 to 10 mL) was administered by continuous infusion over a period of 5 minutes beginning 10 to 30 minutes after placement of the guide wire. Systemic infusions were via the femoral vein (n=2), into the right ventricular cavity (n=1), the right atrium (n=3), or a brachiocephalic vein (n=1). Two dogs were given infusions via the proximal pulmonary artery. Aliquots of blood were obtained for assay of radioactivity at selected intervals after initiation of each infusion, placed in tubes with sodium citrate as the anticoagulant, mixed, placed on ice, and centrifuged to yield plasma that was assayed intact and after precipitation with trichloroacetic acid (20% wt/vol) for determination of protein-bound radioactivity.

Imaging
Single photon emission computed tomography (SPECT) was performed 40 to 70 minutes after infusions except for two studies, in which imaging was delayed until 120 minutes after infusion because of technical difficulties (overall mean, 61 minutes). SPECT images were obtained with a Siemens Orbiter 750 with a medium-energy collimator connected to a devoted MacIntosh 2CI with MACLEAR nuclear medicine software. Sixty-four 60-second windows were obtained over 360°. The same collimator was used to obtain subsequent planar images (1 million counts per view) in multiple views. Image reconstructions were performed on a devoted Digital Electronics Corp workstation. SPECT and planar images were reviewed by two independent observers. Thrombi were identified as unilateral areas of increased activity present in all views of each SPECT (frontal, lateral, and cross-sectional) or planar (anteroposterior and left and right lateral) set of images.

After imaging, cardiac arrest was induced with thiopental (12.5 mg/kg IV) and potassium chloride (60 mL of saturated solution IV). Tissue radioactivity was assayed 3 to 4 hours after infusion of tracer with a Beckman 4000 gamma counter. Clot-to-blood ratios of radioactivity were expressed as the quotient of counts per minute (cpm)/g thrombus divided by cpm/mL whole blood 60 minutes after the onset of infusion. Clot-to-tissue ratios of radioactivity were expressed as cpm/g thrombus divided by cpm/g tissue. Radiolabeled and endogenous plasma TPA concentration was determined with an ELISA (American Diagnostica, Inc) after it had been demonstrated that the sensitivity of the ELISA to 123I-YPACK-TPA was indistinguishable from that to wild-type TPA.

Radiolabeling of TPA
D-Tyrosyl-L-prolyl-L-arginyl chloromethyl ketone (YPACK) and recombinant human wild-type TPA were provided by Genentech, Inc. Anhydrous sodium iodide, sodium metabisulfite, chloramine T, Tween 20 (polyethylene-20-sorbitan monolaurate), and all other chemicals of reagent grade were obtained from Sigma Chemical Co. YPACK was radiolabeled with a modification of the chloramine T–catalyzed iodination method.26 27 Although multiple variations of the iodination were evaluated, the following protocol produced the highest and most consistent yield. The ratio of iodine (unlabeled NaI+[123I]NaI):YPACK:TPA used was 1:3:10. Unlabeled NaI (14.5 nmol in 0.1 mol/L NaOH) was added to a polypropylene reaction vessel, followed by [123I]NaI (20 mCi, 0.145 nmol in 0.1 mol/L NaOH acquired from Nordion International). Given the high specific activity of 123I, the unlabeled NaI was found to be necessary to quench side reactions. A molar ratio of at least 100:1 for unlabeled NaI:[123I]NaI was needed for yields >=35% of 123I incorporated into the TPA. Tris-HCl/Trizma buffer (1 mL of 1 mol/L, 75%/25% by volume, pH 6.4) was added to the reaction vessel and the pH titrated to 7.5 with pH indicator strips (pH 4.5 to 10, P1119-6A, Baxter Scientific Products), a titration generally requiring 15 to 20 µL of 0.1 mol/L HCl. YPACK (44 nmol, 100 µg/mL in 12 mmol/L HCl) was then added to the reaction vessel. Iodination was initiated by adding chloramine T (10 mg/mL in pH 7.5 PBS with 0.01% Tween 20) to yield a concentration of 100 µg/mL. After the mixture had been incubated from 45 to 60 seconds, iodination was stopped by addition of excess sodium metabisulfite (10 mg/mL in pH 7.5 PBS with 0.01% Tween 20) to yield a concentration of 200 µg/mL. After incubation for another 60 seconds, recombinant, wild-type human TPA (50 mg/mL, 145 nmol in 0.2 mol/L arginine phosphate; see below) was added to the reaction vessel and incubated for 60 minutes at room temperature for inactivation and radiolabeling of the TPA.

The TPA was concentrated with a Centricon-10 microconcentrator (Amicon) by first dissolving 50 mg (TPA plus arginine phosphate, obtained from Genentech, Inc) in 10 mL sterile H2O, yielding a TPA solution of 5 mg/mL in 1 mol/L arginine phosphate. This was concentrated by ultrafiltration until 50 mg TPA/mL in 1 mol/L arginine phosphate was obtained. The solution was diluted fivefold with deionized H2O and reconcentrated to yield a final solution of 50 mg TPA/mL in 0.2 mol/L arginine phosphate.

One hour after addition of TPA to the reaction medium, an excess of PPACK (730 nmol in 12 mmol/L HCl, Calbiochem) was added to inhibit any remaining, unreacted TPA, and the labeling mixture was incubated for an additional hour. Previous studies had shown that a 1.1 molar excess of YPACK completely inactivated the thrombolytic capability of TPA.23 In these experiments, a fivefold molar excess of PPACK inhibitor was used to ensure complete inactivation. 123I-YPACK-TPA was separated from unconjugated material by placing up to 1000 µL of the reaction mixture onto a prepacked, disposable Sephadex G-25M PD-10 column (bead dimensions, 1.5x5 cm, 9 mL volume) (Pharmacia LKB Biotechnology) and eluting with 1-mL aliquots of 0.2 mol/L arginine phosphate containing Tween 20 (0.01% by volume). The reaction mixture was typically divided in half and placed on two columns to improve separation. If <1 mL of reaction mixture was placed on a column (eg, 0.8 mL), a supplemental aliquot of column buffer was added onto the column (eg, 0.2 mL) before elution so that uniform 1-mL fractions were produced. Trichloroacetic acid (TCA) (20% wt/vol) precipitates were obtained from each fraction for determination of precipitable counts. The labeled and unlabeled TPA was generally in the second, third, and fourth fractions, and radioactivity in these fractions was consistently >95% precipitable. Fractions with >90% precipitable counts were combined and stored at room temperature for <2 hours before administration by infusion.

For the Chandler tube studies needed to define binding to aged thrombi in vitro (see below), 125I (0.204 nmol, Amersham) was used instead. All other reactants were adjusted such that the same molar ratios as those delineated above were maintained. Only 50 µL of Tris/Trizma buffer was used, and the chloramine T and sodium metabisulfite stock solutions were constituted at 1 mg/mL. For the Chandler tube studies, the concentration of TPA was determined by a Bradford assay before the TPA was added to the tubes (BioRad Protein Assay, BioRad Laboratories).

Binding of 125I-YPACK-TPA to Thrombi of Diverse Ages In Vitro
Since clinical pulmonary emboli typically develop from deep vein thromboses, in vitro studies were performed to determine whether the age of the thrombus affected the binding of labeled TPA. In vitro thrombi were formed from human blood as described by Chandler28 and Fry et al.22 29 Briefly, venous whole blood was collected without anticoagulants from a single volunteer via a 19-gauge needle and transferred immediately to a 27-cm length of Tygon tubing (ID, 1/8 in; OD, 3/16 in) (Fisher Scientific Products). The ends of the tube were brought together to form a loop and joined with a 1-cm-long collar (ID, 3/16 in.; OD, 5/16 in.) of Tygon tubing. Tubes were rotated at 37°C for 30 minutes at an angle of 60° and at 30 rpm on a tube rotator so that thrombi formed in a moving column of blood. To age thrombi longer, tubes were rotated for 24 or 48 hours at 4°C so that the morphology of the thrombi remained unchanged (unpublished data) and resembled thrombi formed in vivo.28

Tubes were opened after 0.5, 24, or 48 hours of rotation, and 166.7 µL of either diluted or undiluted radiolabeled TPA was added to triplicate samples to yield TPA concentrations of 0.5, 1.0, and 10 µg/mL, spanning the peak plasma concentrations measured in vivo. The tubes were closed and rotated again for 60 minutes at 37°C. The contents of each were then poured onto preweighed polyethylene mesh filters (Spectrum Medical Industries, Inc) and washed with 0.9% NaCl containing 0.01% (vol/vol) Tween 80. Radioactivity in washed thrombi was measured with a gamma counter (Isoflex Automatic Gamma Counter, Micromedic Systems, Inc). The filters and thrombi were dried to constant weight. Thrombus mass was determined by subtracting the known filter mass. Bound TPA (nanograms) per milligram thrombus was determined for purposes of comparison.

Statistics
Results are presented as mean±SEM.


*    Results
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Clearance of 123I-YPACK-TPA
Nine studies were performed in dogs subjected to insertion of a guide wire with attached thrombogenic cotton threads. In two studies, the 123I-YPACK-TPA was given as a bolus. In seven subsequent studies, it was given by constant infusion over 5 minutes; peak plasma TPA concentration after infusion was 1.8±0.2 µg/mL. Clearance of labeled TPA from blood was biexponential. After 60 minutes, only 6.7±1.0% of the peak concentration remained in circulating blood (Fig 2Down).



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  		Figure 2. Graph showing percentage of peak radioactivity remaining in the circulating blood in dogs given 123I-YPACK-TPA intravenously. Values are mean±SEM from four to seven dogs at each interval.

Imaging of Thrombi
Pulmonary thrombi were visualized with SPECT in all seven dogs given >=3.7 mCi of 123I-YPACK-TPA, regardless of the site of infusion (Fig 3Down and TableDown). With local (pulmonary artery) infusions, clots were visible with planar imaging (Fig 3CDown). The only thrombi that could not be visualized were in two dogs given <=1.6 mCi of tracer (TableDown).



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  		Figure 3. Examples of SPECT and planar images. Top, SPECT images after a local pulmonary arterial infusion of 123I-YPACK-TPA with the thrombus clearly visualized in all views (arrows); middle, SPECT images after systemic infusion of 123I-YPACK-TPA with the thrombus visible in all three views (arrow) and the sternum (S), vertebrae (V), and thyroid (TD) also visualized but not interfering with detection of the thrombus; and bottom, planar images after local infusion of 123I-YPACK-TPA with uptake in the clot (arrows). Uptake is also seen in the shoulder joints (J) and liver (L). R indicates right; A, anterior; and AP, anteroposterior.


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  		Table 1. Tissue Distribution and Clot Binding of Radiolabeled, Enzymatically Inactivated TPA

Images after local infusion were superior, consistent with a higher thrombus-to-blood and thrombus-to-tissue ratio (see below). Images after right atrial infusion were comparable to those after peripheral venous infusion. Thrombus size and net thrombus activity did not markedly influence visualization. Embolization was evident in some SPECT images, which showed regions of increased uptake in the periphery of the lung fields.

Thrombus, Blood, and Tissue Radioactivity
Thrombus-to-blood ratios of radioactivity were consistently high, although thrombus mass and activity were highly variable (TableUp). No clear correlation was present between the clot-to-blood or clot-to-tissue ratios and clot mass, the quantity of radioactivity injected, or clot-bound radioactivity (Figs 4Down and 5Down). Liver radioactivity was far higher than that in other tissues, consistent with the known high hepatic extraction of TPA. Dosimetry calculations indicate a peak organ (liver) dose of 1.5 rad/mCi injected.



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  		Figure 4. Bar graph showing clot-to-tissue radioactivity ratios. Trough blood levels were assayed in samples obtained 60 minutes after initiation of systemic infusions. Values are mean±SEM.



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  		Figure 5. Bar graph showing thrombus and tissue radioactivity normalized to the amount of radioactivity infused systemically. Values are mean±SEM.

Binding of 125I-YPACK-TPA to Thrombi In Vitro
Studies in vitro demonstrated that tracer bound to relatively older thrombi at tracer concentrations comparable to those observed in vivo (Fig 6Down). As expected, higher concentrations of the labeled TPA led to increased binding. Thrombi 48 hours old bound roughly 50% less tracer than young (30 minutes) thrombi at the lower concentrations of labeled TPA.



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  		Figure 6. Graph showing binding of 125I-YPACK-TPA (t-PA) to thrombi in vitro. Thrombi were formed in Chandler tubes for 0.5, 24, or 48 hours and incubated with tracer for 1 hour.


*    Discussion
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*Discussion
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Several radiopharmaceuticals have been evaluated as potential imaging agents for detection of thromboemboli, including 125I-fibrinogen,8 99mTc-plasmin,14 131I-plasminogen,15 131I-urokinase,16 99mTc- and 131I-streptokinase,16 18 99mTc-antifibrin antibody,9 10 99mTc-antifibrin antibody fragments,11 and 99mTc–synthetic peptides.12 Most have half-lives in the circulation of several hours and cannot be used for imaging for prolonged intervals after injection. Other approaches such as computed tomography30 31 and magnetic resonance imaging32 33 exhibit better specificity than that of ventilation-perfusion scans, but specificity remains suboptimal. The approach developed in the present study using radiolabeled TPA that has had its thrombolytic capability inactivated appears to merit further investigation. Thrombus-to-blood ratios were high. Dosimetry calculations indicate a radiation burden to the liver (1.5 rads/mCi injected) that is well within limits acceptable for human diagnostic studies. This burden could be reduced if isotopes with a shorter half-life, such as 99mTc, were used or if the method developed were adapted to positron emission tomography with an 18F-containing residualizing label.

Our results suggest that planar scintigraphy after systemic infusions will be practical with only modest modifications. Planar scintigraphy was limited in the present study by the relatively low but diffuse background radioactivity seen particularly after systemic infusions, consistent with binding of the tracer to bone marrow. SPECT overcame the difficulties attributable to such background activity. To successfully image thrombi by planar scintigraphy after systemic infusions of tracer, background activity may be decreased with the use of novel mutants of TPA such as T103N,N117Q,KHRR(296-299)AAAA-TPA (TNK-TPA),34 which is devoid of the high mannose carbohydrate responsible for binding of TPA to extrahepatic receptors in tissues such as bone marrow.24

The variation in thrombus-associated and tissue radioactivity between individual dogs reflected differences in the amount of tracer injected in each case, probable variation in harvesting thrombi postmortem, and variable effects attributable to endogenous lysis and embolization. Movement of the thrombogenic catheter tip affecting blood flow past the thrombus probably contributed as well. The 5-minute infusion interval was designed to maximize the interval of exposure to TPA while minimizing persistence of tracer in the blood pool.

Although the thrombi in this in vivo study were relatively young at the time of infusion of tracer, it is clear from in vitro work that the tracer binds to older thrombi as well. The in vitro studies reported here demonstrate that 123I-YPACK-TPA binds reasonably well to thrombi as old as 48 hours at concentrations similar to those induced in vivo in dogs, although binding is somewhat decreased compared with that with younger thrombi. Thus, the label may be suitable for imaging deep vein thrombi as well as pulmonary emboli. It is anticipated that use of radiolabels such as 99mTc with SPECT and 18F with positron emission tomography may enhance clot imaging even further with the approach reported here.


*    Acknowledgments
 
This work was supported in part by NIH grant HL-17646, SCOR in Coronary and Vascular Diseases, and NIH training grant 5-T32-HL-07081-19 (Dr De Bruyn). The authors appreciate technical assistance by Pamela K. Lundius, John J. Botz, Lea T. Berleau, Denise Nachowiak, Roland Zheng, Certified Nuclear Medicine Technologist, and Dana S. De Bruyn. They are grateful to Dr Alan Daugherty, who graciously shared laboratory facilities; Dr John Eichling for dosimetry calculations; and Kelly Hall and Barbara Donnelly for secretarial assistance.


*    Footnotes
 
Dr Keyt is employed by Genentech, Inc; Dr Sobel has served as a consultant to Genentech.

Received December 14, 1994; revision received February 21, 1995; accepted February 27, 1995.


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*References
 

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