Ultrasound accelerates transport of recombinant tissue plasminogen activator into clots

Ultrasound

in Med.

Pergamon

& Biol., Vol. 21. No. 3. DD. 419-424. 1995 Copyright 0 1995 Eiievier Science Ltd Printed in the USA. All tights reserved 0301.5629/91$9.50 + .OO

0301-5629(94)00119-7

@Original Contribution ULTRASOUND TISSUE

ACCELERATES PLASMINOGEN

TRANSPORT ACTIVATOR

OF RECOMBINANT INTO CLOTS

W. FRANCIS,+ ALES BLINC,+ SIMONE LEE+ and CHRISTOPHER +Hematology Unit, Department of Medicine and ‘Department of Biostatistics, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA

CHARLES

(Received

24 May

1994; in final form

22 July

Cox*

1994)

Abstract-Fibrinolysis is accelerated in vitro in an ultrasound field, and externally applied high frequency ultrasound also accelerates thrombolysis in animal models. Although the mechanism of this effect is not known, ultrasound does not cause mechanical disruption of clots but rather accelerates enzymatic fibrlnolysis. To determine if accelerated fibrinolysls could be related to increased transport of enzyme into clot, we have examined the effect of insontication on the distribution of plasminogen activator between clot and surrounding fluid in vi&u. Plasma clots were overlayed with plasma containing ‘251-radlolabeled, activesite-blocked recombinant tissue plasminogen activator @t-PA) and incubated in the presence of l-MHz ultrasound at 4 W/cm* or in the absence of ultrasound. The rate of uptake of rt-PA was signlftcantly faster in the presence of ultrasound, reaching 15.5 ? 1.4% at 4 h compared to 8.2 2 1.0% in the absence of ultrasound @ < 0.0001). Similarly, ultrasound increased transport of enzyme from the clot into the surrounding fluld. To determine the effect of ultrasound on the spatial distribution of enzyme, plasma clots were overlayed with plasma containing radiolabeled r&PA and incubated in the presence or absence of ultrasound. The clots were then snap-frozen, and the radioactivity in serial cryotome sections was determined. Exposure to ultrasound altered the r&PA distribution, resulting in significantly deeper penetration of rt-PA into the clots. We conclude that exposure to ultrasound increases uptake of r&PA into clots and also results in deeper penetration. These effects of ultrasound on enzyme transport may contribute to the accelerated fibrinolysis observed in an ultrasound field. Key Words: Ultrasound, Fibrinolysis, Tissue plasminogenactivator, Thrombosis, Thrombolysis.

INTRODUCTION

senschein et al. 1990; Ariani et al. 1988), but the acceleration of fibrinolysis observed at lower intensities and at higher frequencies of over 200 kHz is not associated with mechanical disruption and requires enzymatic activity (Francis et al. 1992; Tachibana 1992; Lauer et al. 1992; Blinc et al. 1993; Luo et al. 1993; Harpaz et al. 1993). Most nonthermal bioeffects of ultrasound are mediated by acoustic cavitation which refers to the growth, oscillation and collapse of gas bubbles in a fluid medium (Flynn 1964). Oscillating bubbles can generate local fluid motion termed microstreaming (Nyborg 1982), and bubble collapse may also cause fluid motion (Flynn and Church 1988). The acceleration of fibrinolysis by ultrasound in vitro is greater at higher intensities and duty cycles, and maximum effects are seen at frequencies between 1 and 2.2 MHz with a reduced effect at higher frequencies, properties that are consistent with a cavitation-dependent mechanism (Blinc et al. 1993). We have hypothesized that fluid motion resulting from ultrasound-induced cavita-

Fibrinolysis in vitro is accelerated in an ultrasound field (Francis et al. 1992; Tachibana 1992; Lauer et al. 1992; Blinc et al. 1993; Luo et al. 1993; Harpaz et al. 1993), and experiments in rabbits (Komowski et al. 1994; Kashyap et al. in press) and dogs (Kudo 1989) have demonstrated that externally applied, high frequency ultrasound accelerates clot lysis in vivo after administration of fibrinolytic agents. However, the mechanism by which ultrasound potentiates fibrinolysis has not been determined. The minor degree of heating observed in most experiments is insufficient to account for the accelerated fibrinolysis (Francis et al. 1992; Blinc et al. 1993), although Higazi et al. (1993) concluded that thermal effects play a major role. Exposure to ultrasound at lower frequencies of 20 to 25 kHz and higher intensities of up to 20 W/cm2 can mechanically disrupt clots (Hong et al. 1990; RoAddress correspondence to: Charles W. Francis, M. D., Hematology Unit, P.O. Box 610, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY 14642, USA. 419

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tion accelerates fibrinolysis by promoting the transport of activators and plasminogen to their target sites on fibrin within the clot. In the present report we have examined the effect of ultrasound on the transport of plasminogen activator into clots in vitro, measuring both rate of transport and the effect of ultrasound on the spatial distribution of activator within clots. METHODS Inactivation and labeling of rt-PA Recombinant tissue plasminogen activator (Activase’) was kindly provided by Genentech (South San Francisco, CA) and was inactivated by incubation with a loo-fold molar excess of D-Phe-Pro-Arg-chloromethylketone (PPACK) (Bachem, Torrance, CA). Excess PPACK was removed by gel-filtration chromatography on Sephadex G-25 (Bio-Rad Laboratories, Hercules, CA), and the inactivated rt-PA showed no enzymatic activity when incubated with the chromogenic substrate H-D-Ile-L-Pro-L-Arg-P-nitroanalide (S-2288) (Kabi Vitrum, Stockholm, Sweden). Radioiodination of inactivated rt-PA was performed using the iodogen method (Fraker and Speck 1978), and unbound “‘1 was removed by Sephadex G-25 chromatography. The radiolabeled, inactivated rt-PA was affinity purified using a fibrin celite column (Husain et al. 1989). The fibrin-binding form that eluted with 0.5 M arginine hydrochloride comprised approximately 90% of the total inactivated, labeled i-t-PA, and this was used for transport experiments. Its mobility on sodium dodecyl sulfate polyacrylamide gel electrophoresis was identical to active, unlabeled rt-PA. Clot preparation Normal pooled human plasma was prepared from five single donor units obtained through the American Red Cross (Rochester Region, New York), anticoagulated with acid citrate dextrose, pooled and stored at -70°C in aliquots until use. The titrated plasma was recalcified to a final concentration of 50 mmol/Liter by addition of calcium chloride prior to clotting. Clots were prepared in thin-walled, 8-mm-diameter nitrocellulose tubes (Beckman, Palo Alto, CA) with an attenuation of less than 0.8 dB at 1 MHz as determined by insertion loss measurements when the water-filled tube was placed between the ultrasound source and a needle hydrophone (Type 80-0.5-4.0, Imotek GmbH, Wurselen, Germany). To prepare clots, aliquots of 160 /JL of recalcified plasma were transferred to the tubes, and 20 PL of bovine thrombin (Calbiochem, LaJolla, CA) was added to a final concentration of 1 U/r& Clots were incubated for 1 h at 37°C prior to use. Plasma clots were overlaid with 820 /JL of plasma containing 5 U/mL heparin (Riker Labs, Inc., Northridge, CA).

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Radiolabeled, inactivated rt-PA was added to the overlying solution in a final concentration of 100 rig/ml,. In some experiments, radiolabeled rt-PA was incorporated throughout the clot by mixing with plasma before clotting. Ultrasound apparatus The clots were exposed to ultrasound using an apparatus described previously (Francis et al. 1992). Briefly, up to six tubes were suspended in a circular test-tube rack with a diameter of 2.5 cm and immersed in a tank containing water maintained at 37°C. The rack was placed in the near-field of an ultrasound transducer and was rotated at a frequency of 6 to 8 rpm to give an equal average exposure to all tubes. The active element of the transducer was a piezoelectric crystal with a diameter of 2.5 cm operating at a frequency of 1 MHz. The total acoustic intensity from the source was 4 W/cm2 as measured with a radiation force meter (Carstensen et al. 1983) at a distance corresponding to the closest approach of the sample tube to the transducer during rotation. At intervals of 1, 2 and 4 h, tubes were removed from the apparatus, the overlying solution decanted and radioactivity in the clot and overlay was measured in a gamma counter (Minigama 1275, LKB Wallac, Finland). Clot sectioning To determine the spatial distribution of radiolabeled rt-PA, the clots were serially sectioned. After exposure to radiolabeled, inactivated r&PA in the overlay for 2 h, the overlay was decanted and the clot snap-frozen in an ethanol dry-ice bath. Clots were then carefully removed from the nitrocellulose tubes and embedded (Tissue-Tek, O.C.T. Compound, Miles, Inc., Pittsburgh, PA), mounted and serially sliced every 480 pm using a cryotome (Cryotome 1500, Shandon Lipshaw, Pittsburgh, PA). Individual slices were counted in a gamma counter. Statistical analysis Comparison of total uptake of radiolabeled, active-site-blocked rt-PA in the presence or absence of ultrasound used Student’s two-tailed t test for unpaired data. The spatial distribution of r&PA in clots with and without ultrasound exposure was compared after calculating the mean and median of each distribution. These values were then compared using a two-way analysis of variance (ANOVA) considering both the treatment and the individual experiments in the analysis. Each ANOVA included an examination of residuals as a check on the required assumptions of normally distributed errors with constant variance.

421

Acceleration of r&PA transport l C. W. FRANCIS et al.

2o r

I

2 3 T I ME (hours)

4

Fig. 1. Uptake of rt-PA into clots in the presenceor absence of ultrasound.Plasmaclots wereoverlayed with heparinized plasmacontaining1251-radiolabeled inactivatedrt-PA andincubatedin the ultrasoundapparatusin the presenceor absenceof l-MHz ultrasoundat 4 W/cm*. Clot uptake is expressedasa percentageof the total radioactivity in the tube at the beginning of the experiment. Each point represents the mean + SD for 12 clots. Statistical significanceof the difference between the meansof the clots exposedor not exposedto ultrasoundwasp < 0.005, 0.0001, and 0.0001 at 1, 2 and 4 h, respectively.

To determine whether ultrasound also increased transport of enzyme from the clot into the surrounding fluid the radiolabeled and inactivated enzyme was incorporated into the clot during formation. The enzymecontaining clot was then overlaid with plasma, and the movement of enzyme into the surrounding fluid was measured over time (Fig. 2). At time 0, representing sampling after overlaying with plasma and placement in the apparatus, a total of 1.1% of enzyme was in the overlay in all tubes. In the absence of ultrasound, the amount of radioactivity increased to 8.3 -+ 0.6% at 1 h and was 12.9 + 1.3% at 4 h. Transport out of the clot was more rapid with ultrasound, reaching 12.2 t 2.0% at 1 h, 18.0 + 3.0% at 2 h and 24.3 t 1.8% at 4 h. These were all significantly higher than the counts in the overlay in the absence of ultrasound @ < 0.0001 for each). To characterize the spatial distribution of &PA, overlying fluid was removed, the clot was snap-frozen and then sectioned serially using a cryostat. The distribution of counts throughout one of two clots exposed to radiolabeled, active-site-blocked rt-PA in the presence and absence of ultrasound is shown in Fig. 3. The

RESULTS Nonretracted plasma clots were prepared in plastic tubes with low ultrasound attenuation and overlaid with plasma containing ‘?-radiolabeled, active-siteblocked rt-PA. The uptake of radiolabeled enzyme into the clot was measured over time either in the absence of ultrasound or after rotating through a l-MHz ultrasound field at 4 W/cm’. In the absence of ultrasound there was a progressive increase in the amount of enzyme in the clot from 4.2 + 1.2% at 1 h to 8.2 + 1 .O% at 4 h (Fig. 1). The increase was more rapid in the presence of ultrasound reaching 6.2 + 1.7%, 9.2 + 1.6% and 15.5 + 1.4% at 1, 2 and 4 h, respectively. At all times there was significantly greater uptake of enzyme into the clot in the presence of ultrasound @ < 0.005, 0.0001 and 0.0001 at 1, 2 and 4 h, respectively). Since exposure to 1 MHz at 4 W/cm2 increases clot temperature approximately 4°C (Blinc et al. 1993), we compared uptake at 37°C and 41°C. The 4-h uptake of enzyme at 41°C was 10.0 t 0.6% which was significantly greater than the uptake at 37°C without ultrasound (8.2 -t 1.0%) (p < O&OS), but also significantly less than the 15.5 + 1.4% uptake at 37°C in the presence of ultrasound @ < 0.0001).

of-y-0

2 T I M E (hours)

3

4

Fig. 2. Lossof rt-PA from clots in the presenceor absence of ultrasound.Plasmaclots containing ‘Z51-radiolabeled, inactivated r&PA were overlayed with heparinizedplasmain the ultrasoundapparatus.They were either exposedto lMHz ultrasoundat 4 W/cm* or not exposedto ultrasound for timesup to 4 h. The lossof radiolabelfrom the clot is expressedas a percentageof the total radioactivity in the clot at thebeginningof the experiment.Eachpoint represents the mean+- SD of four to eight clots. At eachtime increment the loss of radioactivity from the clot was greater in the ultrasound-exposed group @ < 0.0001).

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section from the top of the clot was in contact with the overlying fluid. However, the counts in the first sections are low because of the presence of a meniscus at the top of the clot. The meniscus had a total depth of 1 mm, and it was the same in clots exposed or not exposed to ultrasound. Because of the presence of the meniscus, the counts increased in the first two or three sections until the sections were below the level of the meniscus and included the full thickness of the clot. The distribution of counts in clots exposed or not exposed to ultrasound differed, with deeper penetration of enzyme into the ultrasound-exposed clot. This is suggested by the visual appearance of the distributions and also by statistical analysis of the distribution of counts. For control clots, the mean of the distribution was at 1489 2 81 (SEM) pm and the median was 1200 2 10 1 pm. In contrast, for the ultrasound-exposed clots the mean was at 2275 + 203 and the median at 2053 + 192 pm. The analysis of variance showed a significant difference between ultrasound and control for both the mean (p = 0.001) and median (p = 0.0004).

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DISCUSSION The results presented in this report demonstrate that exposure of a clot to ultrasound increased transport of rt-PA from the surrounding fluid into the clot. The amount of uptake was 48% greater at 1 h in the presence of ultrasound, and the rate of uptake remained faster so that the difference increased to 84% at 2 h and 89% at 4 h. Ultrasound also increased transport from the clot into the surrounding fluid with the net effect representing results of both processes and governed by the concentration gradient. In addition to increasing total uptake by the clot, ultrasound also altered the distribution, with deeper penetration of t-t-PA in the presence of ultrasound. For these experiments we used t-t-PA with the active site blocked to avoid any fibrin degradation which would complicate interpretation. Also, t-t-PA binds to fibrin, and this could affect its uptake and distribution. Since iodination can affect the fibrin binding properties of &PA (Husain et al. 1989; Tate et al. 1987), we used fibrin-binding r&PA that had been affinity purified on fibrin celite (Husain et al. 1989). In the absence of ultrasound, transport of molecules from the surrounding fluid into a clot can occur by thermally mediated diffusion or by permeation. Diffusion results from random movement of individual molecules due to the thermal energy in the system, and it results in exponentially declining concentration profiles (Crank and Park 1968). It is a very inefficient mechanism of transport beyond a few tens of micrometers. Ultrasound causes heating of the clot (Blinc et al. 1993) and heating caused some increase in enzyme

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E Q 0

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Fig. 3. Distribution of u-PA in clots in the presenceor absence of ultrasound. Plasma clots were overlayed with heparinized plasma containing ‘ZI-radiolabeled, inactivated n-PA and incuhated with rotation in the ultrasound appmtus. They wem either exposedto l-MHz ultrasound at 4 W/c& or not exposedfor 2 h. They were then embed&d and serially sszctionedevery 480 pm using a ciyotorne, and individual sectionswerecounted. The figure shows the distribution of counts in the clot in (a) the absmce or (b) presenceof ultrasound The data includes four separateclots exposed to ultrasound and three that were not exposed.Values are plotted at the middle of each se&on (e.g., thesectionfromOto48o~misplottedat24o~).Boththe ineansandmediansofthedislributionsweresiguificantly~erent @ = 0.001, p = 0.0004, respectively).

Acceleration

of &PA

transport

transport, probably by accelerating diffusion. Most of the ultrasound effect, however, was not thermally mediated. Permeation, or bulk flow, is a collective, directional movement driven by a pressure gradient, and it can result in much more rapid transport. However, in our system there was no pressure gradient, so permeation could not have contributed to the uptake. Ultrasound could increase transport by other mechanisms including phonophoresis and cavitation. Transfer of ultrasonic momentum to the medium via absorption results in macroscopic streaming in the direction of sound propagation which is typically seen in fluids. Such radiative forces may also act on the fluids in gels, a process termed phonophoresis. It has been suggested that this effect may increase uptake of topical medications into skin exposed to ultrasound (Williams 1990; Williams et al. 1990). In our system, it is unlikely that phonophoresis contributed to increased transport, since ultrasound was applied at right angles to the direction of the concentration gradient. Other bioeffects of ultrasound are related to cavitation and result from the effects of gas bubbles generated by the pressure changes in the liquid associated with the propagation of sound (Flynn 1964). Transient cavitation results in violent collapse of bubbles and can generate high local pressure and shock waves that could result in localized fluid motion (Flynn and Church 1988). If this occurs at a fluid-solid interface, the motion may be asymmetric and create fluid jets which could penetrate the gel. Ultrasound may also generate stable gas bodies that resonate at the ultrasound frequency, creating streaming of fluid in its proximity (Nyborg 1982). The frequency dependence of ultrasonic acceleration of fibrinolysis is consistent with such a cavitation-dependent mechanism (Blinc et al. 1993). Bubbles could form either at the interface between the clot and surrounding fluid or within the clot which contains pores with diameters up to 5 pm (Carr and Hardin 1987; Blomblck and Okada 1982) and, therefore, are large enough to accommodate the expected gas bodies. Gas bubbles have been observed to form and grow in agar gels exposed to ultrasound (Daniels et al. 1987). The accelerated transport that we have observed could contribute to the accelerated fibrinolysis observed with ultrasound, since transport of fibrinolytic enzymes into clots is an important determinant of the rate of fibrinolysis. Mathematical models of fibrinolysis predict that clot lysis occurs along a front where the concentrations of plasminogen and activator generate a sufficient plasmin concentration (Zidansek and Blinc 1991; Zidansek et al. 1993; Diamond and Anand 1993). These models predict that enzyme transport into the clot is the major determinant of the lysis rate which will be very slow if limited by diffusion. Clot lysis is

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faster if plasminogen activator is incorporated throughout the clot than if it diffuses from the surrounding fluid (Matsuo et al. 1982), and fibrinolysis can be accelerated in vitro (Bookstein and Saldinger 1985) and in animal models (Valji and Bookstein 1987; Kandarpa et al. 1988) by directly injecting activator into a clot. Blinc et al. (1991) compared the rate of lysis using a simple in vitro model and found that lysis was increased 59-fold at a flow rate of approximately 50 pL/ min driven by a pressure gradient of 37 cm of water through an occlusive clot as compared to that with diffusion alone. Clinical experience also suggests that enzyme transport into a clot is an important determinant of therapeutic success. In acute myocardial infarction, higher rates of reperfusion have been observed with intracoronary administration of plasminogen activator that result in a higher concentration at the site of thrombosis (Marder and Sherry 1988). Treatment of peripheral arterial occlusion is more successful if a catheter can be advanced into the clot for direct intrathrombic infusion rather than administration into the blood proximally (Hess et al. 1982; Gardiner et al. 1986). Also, lysis of upper extremity venous clots was found to be more successful with direct intraclot infusion of activator than with local administration into the blood (Fraschini et al. 1987). Such mechanical approaches to increasing activator concentration within the clot are effective, but they are not feasible in many clinical situations in which small vessels are involved, when technical problems prevent passage of the catheter into a clot or in treatment of extensive venous thrombi. In such cases, the use of ultrasound to increase transport of activator into a thrombus could be a useful therapeutic adjunct to accelerate fibrinolysis. Acknowledgements-The authors gratefully acknowledge useful discussions with Prof. Edwin Carstensen. The assistance of Carol Weed in preparation of the manuscript is appreciated. This work was supported in part by Grant HL-30616 from the National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD.

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