Laser-induced stimulation of thromboxane B2 synthesis in human blood platelets: Role of superoxide radicals

Laser-induced stimulation of thromboxane B2 synthesis in human blood platelets: Role of superoxide radicals Exposure of platelet-rich plasma to laser radiation at 3.5 W for 30 seconds reduced the threshold concentrations of adenosine diphosphate and L-epinephrine needed from complete platelet aggregation by 20% to 60% and by 30% to 50%, respectively. The irradiation of platelet-rich plasma with laser also increased the basal level of thromboxane A2 from <0.5 pmol/lO* platelets for each second of exposure. In contrast, the exposure of gel-filtered platelets to laser produced no effect on the prostanoid formation. However, the addition of laser-exposed platelet-free plasma to gel-filtered platelets stimulated the synthesis of thromboxane A2 in these cells. The effect of laser was completely blocked by adding superoxide dismutase or catalase to the platelet-rich plasma, indicating that the radiation-induced stimulation of thromboxane AZ production was mediated through the generation of superoxide radicals. Electron microscopic studies indicated that the laser-induced stimulation of thromboxane A2 production in platelet can occur without any noticeable damage in the cellular structure. (AM HEART J 1993;125:357.)

Rohit R. Arora, MD,a Hiltrud S. Mueller, MD,b and Asru K. Sinha, PhDb New York and Bronx, N.Y.

Percutaneous balloon angioplasty is a well-established method of treating selected patients with coronary arterial and peripheral vascular disease.le5Because of the resistance to passage of the balloon catheter, technical failure may occur in severe stenosis. Further, in complete occlusions use of the catheter is limited only because of the difficulty in crossing them. Although the use of laser radiation could be a potentially valuable technique in luminal improvement, progress is hampered by a high incidence of vessel wall perforation and inadequate recanalization with a potential for reocclusion.1-6 Studies on the effects of laser energy on arterial luminal surface have so far been limited largely to the morphologic studies of atherosclerotic lesions in animal& 6 and in human postmortem vasculature.7 In animal models, it has been reported that the exposure of endothelial cells to laser radiation reduced the formation of prostacyclin.8 However, alteration of clotting mechanism,

From the ‘Division of Cardiology, Department of Medicine, Columbia University College of Physicians and Surgeons, New York, and the bDivision of Cardiology, Department of Medicine, Montefiore Medical Center, Albert Einstein College of Medicine, Bronx. Received

for publication

May

11, 1992;

accepted

Aug.

17, 1992.

Reprint requests: Rohit R. Arora, MD, Columbia-Presbyterian Center, Cardiovascular Laboratory, Milstein Hospital Building, Washington Ave., New York, NY 10032.

Copyright 0002.S’iO:i/93/$1.00

1993 by Mosby-Year + .IU

Book,

10/4/l/42643

Medical 177 Ft.

thrombogenicity, and platelet function induced by laser energy remains largely unknown.gy lo METHODS Materials.

Superoxide dismutase(from bovine kidney) and catalase (from bovine liver) were purchased from Sigma Chemical Co., St. Louis, MO. Thromboxane Bz radioimmunoassaykit was obtained from New England Nuclear, Boston, Mass.All other chemicalswere of analytic grade. Exposure

of platelet-rich

plasma

to laser radiation.

Laser energy was delivered with an Innova laser system (model920;Coherent, Palo Alto, Calif.) usinga continuouswave argon laser (wavelength 488 nm) and a red krypton laser (wavelength 647 nm). The delivery system consisted of 400 pm silica-clad quartz fiber. Power was delivered through a 220V, 100amp, three-phaseelectrical line. The lasersystemwascooledwith a noncirculating water pump at a rate of 2.5 gallons/min at a temperature of 30” C. The angle of the beam had a divergence from 8 to 15 degrees. The power of fluctuations waslessthan 1%. The width of the slit was 61 cm. Platelet-rich plasma (PRP) was exposed in siliconized glass tubes (10 X 1.6 cm) placed at a distance of 10 cm from the power source. The spot size was kept constant at 100pm for all experiments, and the pulseduration was 1 second. The power output was kept constant at 3.5 W, but the number of exposures (pulses) was varied from 5 to 300 for different experiments. These parameters resulted in total energy delivered in the range of 30 to 1050 joules. The percentage of energy loss through reflection was

Inc.

357

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Arora, Mueller,

and Sinha

estimated to be 4 I:, with approximately 960; of energy delivered to PRP. Preparation of PRP. All blood donors abstained from medicationsfor at least 2 weeksbefore blood donationsand had normal serum lipoprotein values. Venous blood was collectedthrough a siliconizedneedleinto a plastic syringe and anticoagulated by mixing 9 vol of blood with 1 vol of trisodium citrate (final concentration, 0.013 mol/L). PRP was prepared by centrifuging the blood for 15 minutes at 2OOg.l’Platelet-free plasmawasprepared by centrifuging PRP at 10,OOOg for 10 minutes at 23” C. This preparation contained 1 to 3 x 10s cells/ml. Platelet counts were performed in a Coulter Counter (Model ZB, Coulter Electronics, Hialeak, Fla.) equipped with a 50-pm-aperture tube. Gel-filtered platelets (GFP) wereprepared from PRP asdescribedbefore with the useof Sepharosemedium (2B column).‘” Platelet aggregation studies. PRP (0.5 ml) wasplaced in a cylindrical cuvette (8 mm diameter) containing a Teflon-coated stirring bar. Aggregation wasstudied by adding a threshold concentration (minimum concentration necessary for completeaggregation)of adenosinediphosphateor L-epinephrinein an aggregometer(Chrono-Log Corp., Havertown, Pa.) with a stirring rate of 1200rpm at 37’ C as describedbefore.ll The apparatus was calibrated so that the difference in light transmittance betweenplatelet-rich and platelet-poor plasmawas defined as 100%. Effects of exposureof platelets to laser energy on the aggregationof thesecells by various aggregatingagentswassimilarly determined and compared with control platelets. Radioimmunoassayof thromboxane AZ.Thromboxane As (TXA2) is a labile compound that is rapidly converted to the more stable derivative known as thromboxane Bs. We assayedTXA2 by determining thromboxane Bs with the radioimmunoassaykit. Statistical analysis. Parametersfrom the laser-exposed platelets and control platelets were compared by using Student’s paired t test; p value <0.05 wasconsideredstatistically significant. Electron microscopy. Immediately after laserexposure, the 1 ml sampleof PRP wasfixed by adding 1 ml of 5%) glutaraldehyde at room temperature in 0.06 mol/L phosphate buffer. The sample was centrifuged between each successivestep of the preparation. After a rinse in phosphate buffer, the platelets were postfixed in 1% osmicacid in chrome buffer, dehydrated in alcohol, and embeddedin epoxy after two changesin propylene oxide. Thin sections were prepared, stained with uranyl and lead salts, and viewed in an electron microscope (Model 101, Siemens Medical Instrumentation, Inc., Iselin, N.J.). RESULTS Effects of laser radiation of PRP on platelet aggregation. To determine the effect of laser energy on platelet aggregation, PRP was exposed to radiation for various times. It was found that PRP had to be

exposed to laser for at least 25 to 30-second pulses before the hyperactivity of these platelets to aggre-

American

February 1993 Heart Journal

gating agents became apparent. At 30 pulses of exposure, the threshold concentration of adenosine diphosphate and L-epinephrine was significantly (p < 0.05, n = 6) decreased in comparison with the control values (Fig. 1). The minimal quantities of agonists required to induce complete platelet aggregation (lOO’rl) in the case of laser-exposed PRP decreased between 2070 and 60”;) in the case of L-epinephrine and between 30 (‘; and 50 ‘:; in the case of adenosine diphosphate in comparison with unexposed PRP. Effects of laser energy on TXA2 production in PRP.

The exposure of PRP (0.5 ml) to laser radiation at 3.5 W, l-second pulse duration, increased the production of TXAs from a basal level of <0.50 pmol/lOs cells to 1.37 + 0.05 pmol/108 cells (p < 0.01, n = 15) for each pulse in the absence of any added agonists. The production of TXA2 on exposure to radiation linearly increased, and at 70 seconds of exposure it was 92 i 3 pmol/lOs cells (Fig. 2). Incubation of PRP with 1.0 mmol/L of aspirin for 30 minutes before laser exposure totally decreased the increased formation of TXAs in platelets (10.5 pmol/lO’ cells). This indicated the involvement of cyclooxygenase in the production of laser-induced TXA2. Addition

of’

ethyl-

enediaminetetraacetic acid, 1 mmol/L, to PRP also completely inhibited the effect of laser radiation on TXA2 production. The effect of laser radiation on TXAz production was not related to the particular type of radiation used. Whereas the argon laser produced 1.676 t 0.5 pmol TXAs/lO’ platelets per second, the krypton laser increased the prostanoid formation to 1.74 + 0.5 pmol/lO’ platelets per second (n = 3) compared with <0.005 pmol/108 platelets in

control experiments

(p, not significant;

n = 6).

Effects of laser radiation on GFP. In contrast to PRP, the exposure of GFP to laser radiation produced no effect on the production of TXA2 compared with control platelets (Table I). The amounts of TXA2

produced remained less than 0.5 pmol/108 cells when the GFP were exposed to radiation for 28 seconds. In contrast, when the PRP was exposed to radiation for 28 seconds, the TXA2 level increased from 10.5 to 35.5 pmol/lOs platelets. These results indicated that the effect of laser radiation on platelet TXA:! production might be mediated through plasma. To determine the role of plasma in the effect of the laser radiation, we exposed platelet-free plasma to radiation as described above. The laser-exposed plasma was then immediately (<5 seconds) added t.0 a GFP preparation that had not been exposed to radiation. After incubation at 37* C for 2 minutes, the TXA2 production was compared with control experiments by using platelet-free plasma unexposed to the radi-

Volume Number

125 2, PaIt

Effects of laser on platelet

1

thromboxane

359

3.5

L-EPINEPHRINE

3.0

2.5

2.0

1.5

1.0

0.5

0’

I

0

CONTROL

LASER

I

CONTROL

LASER

Fig. 1. Reduction of threshold concentrations of L-epinephrine and adenosinediphosphate (ADP) to induce complete aggregation of laser-exposedPRP. PRP (0.5 ml) was irradiated with argon laser at 3.5 W for 30 seconds,asdescribedin the Methods section.The aggregationof platelets were studied by using either L-epinephrine or ADP. Each point indicates the average of six experiments.

of the laser-exposed plasma to GFP stimulated the production of TXA2 to 5.2 pmol/108 cells. In contrast, the addition of unexposed plasma to the laser-treated GFP had no effect on TXAa production. When the stimulation of TXA2 production by the laser-treated plasma in GFP was corrected for the dilution caused by the addition of plasma to GFP, the laser-exposed plasma was approximately 28% efficient compared with the effect of laser itself on PRP under the conditions described above.

of TXA2 production by the laser radiation (Table II). Since both superoxide dismutase and catalase are known to destroy superoxide anions, the effect of the laser-induced stimulation of TXAz formation in platelet was mediated apparently through the generation of these anions in the plasma.

Effects of superoxide dismutase and catalase on the production of TXA2 by the laser-exposed PRP. The re-

phologic

sult described above indicated that the stimulation of TXAz production in platelets by laser radiation was mediated through plasma. In an effort to understand the nature of the mediator(s) generated in plasma because of the exposure to laser radiation, PRP was treated with various concentrations of either superoxide dismutase or catalase before the platelet suspensions were exposed to the radiation. The presence

any cellular damage in comparison with the control platelets (Fig. 3, A and B). However, when the PRP was irradiated with laser for 300 seconds, noticeable

ation (Table I). It was found that the addition

of aslittle as 0.7 unit superoxide dismutase or 0.5 unit

catalase in PRP completely

blocked the stimulation

Morphologic characteristics of platelets exposed to laser radiation. Although the exposure of PRP to la-

ser radiation for less than 5 seconds stimulated the production of TXAz in the absence of any aggregating agents, electron microscopic studies of the mor-

characteristics

of the platelets

exposed to

the radiation for as long as 200 seconds did not show

damage of platelet structure became apparent (Fig.

3, C). The plasma membrane became discontinuous and cytoplasma appeared watery. Even in damaged platelets, however, the granules and other subcellular organelles apparently remained intact. In addition, when PRP was exposed to the laser radiation for 300 seconds, large, finely granular aggregates of ap-

360

Arora, Mueller, and Sinha

February 1993 Heart Journal

Amencan

Table I. Effect of’ laser-exposed of’ TXA2 1,~ (;FP

plasma on the pro(lu(,ti~ln

I’latelet

AdditiUrl

preparation _____.

PRP PRP (laser exposed) GFP GFP (laser

ipmol/ll~’

None Kane

T.4 :I1 f~lnti~l~~t~~

dj.r, :\5..5

”

:<.:I

None

None

exposed) GFP GFP (laser exposed)

Platelet-free plasma (laser exposed) Platelet-free plasma

Either PRP or GFP (0.5 ml each) was exposed to 28-second pulses of laser radiation at 3.5 W as described in the Methods section. The platelet-free plasma (0.5 ml) was added to GFP (0.5 ml) immediately (<5 seconds) after it was exposed to laser radiation for 38 seconds. In control experiments the platelet-free plasma was similarly added to GFP. The results shown here are the mean i SEM of four experiments. 20

.

v I;-;

0

IO

20

EXPOSURE

30

40

TIME

50

60

I

70

( SECOND 1

Fig. 2. Relationship between production of TXAz and

exposuretime of PRP to laser radiation. PRP (0.5 ml) was exposedto laserradiation for various times asindicated. At various intervals, ethylenediaminetetraacetic acid (1.0 mmol/L final) was added to PRP to stop the reaction. TXA2 was determined as thromboxane Bz (TXB2) by radioimmunoassay. parently proteinaceous materials were seen in the surrounding medium. None of these changes were detected either in control platelets or in PRP exposed to the laser for 30-, loo-, or 200-second pulses. These proteinaceous aggregates were presumably derived from platelets that had been destroyed by the laser radiation. DISCUSSION

These studies showed that the exposure of PRP to laser radiation makes these platelets hypersensitive to aggregating agents and simulates the production of TXAz. This enhanced formation of TXAz by laser in platelets occurred without any apparent cellular damage. Whereas the stimulation of TXAZ formation in PRP by laser radiation could be demonstrated within 5 seconds of radiation (Fig. 2), the platelet suspension had to be irradiated for at least 300 seconds before damage to the cell structure became noticeable (Fig. 3). The hypersensitivity of the laser-

exposed platelets to aggregating agents might be a consequence of the laser-induced stimulation of TXA2 synthesis in these cells.13Although the direct irradiation of GFP failed to stimulate the TXAz production, platelet-free plasma previously exposed to the radiation and immediately added to the suspension of isolated platelets increased TXAZ production compared with control platelets. The stimulatory effect of the laser-exposed plasma on TXAz production in GFP platelets was transient in nature. No effect of the laser-exposed plasma could be demonstrated if it was added to the isolated platelets 5 minutes after the exposure (Table I). These results showed that the stimulation of production of TXA2 in PRP by laser radiation was due not to the direct effect of the radiation on platelets but to the generation of short-lived mediators in plasma. Because the addition of superoxide dismutase or catalase to PRP completely blocked the stimulated production of TXA2 by laser radiation, the short-lived mediators agents generated in plasma by the radiation were apparently superoxide anions. It could be conjectured that the effect of laser radiation on platelets was mediated through thromboxane receptors; however, our preliminary results do not indicate that TXA2 receptors were involved in the process. As discussed above, the use of laser radiation to evaporate an atherosclerotic lesion by intravascular delivery over a flexible optical fiber is potentially a valuable technique. However, in animal model experiments, the endovascular damage caused by laser radiation has been related to the enhanced thrombotic potential in the study postoperative peri0d.s.“’ Unlike these studies, where the effects of laser radi-

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Number

2, Pert

Effects of laseron platelet

1

thromboxane

361

Table II. Effect of superoxide dismutaseand catalaseon laser-inducedstimulation of TXAt production in PRP TXAz

Addition None Superoxide dismutase Catalase (0.5 unit/ml)

(0.7 unit/ml)

(pmol/l@

platelets)

35.5 +- 3.5 <0.5 <0.5

Bovine kidney superoxide dismutase or bovine liver catase was added to PRP (0.5 ml) before the platelet suspension was irradiated for 28 seconds by argon laser. The results shown here are the mean + SEM of four experiments.

ation, described above, were directly due to the endothetical cell injury, our studies showed that the hypersensitivity of platelets to aggregating agents

and to enhanced formation

of TXAs in PRP exposed

to laser radiation is not necessarily related to the direct effect of the radiation on the cells. The

production

of TXA2 has been related to thrombotic

phenomenon and is known to cause vascular ische-

mia.14> l5 The effect of laser radiation on plasma that subsequently stimulated the production of TXAx in platelets would be expected to be more pervasive in its action than the direct effects of the radiation on endothelial cells in the site of injury. The inhibitory effect of aspirin on the laser-stimulated production of TXAs in platelets indicated that the compound could be potentially useful in controlling the formation of the prostanoid in laser endarterectomy. We sincerely thank Dr. H. M. Dembitzer for the electron micropic studies, Dr. D. M. Chutorion for his interest, and James Dainty for technical assistance. REFERENCES

1. Abela GS, Normann SJ, Cohen DM, Franzini D, Feldman RL, Crea F, Fenech A, Pepine CJ, Conti CR. Laser recanalization of occluded atherosclerotic arteries in vivo and in vitro. Circulation 1985;71:403-7. 2. Cumberland DC, Sanborn TA, Tayler DI, Moore DJ, Welsh CL, Greenfield AJ, Guben JK, Ryan JJ. Percutaneous laser thermal angioplasty: initial clinical results with a laser probe in total peripheral artery occlusions. Lancet 1986;1:1457-9. 3. Choy DSJ, Stertzer SH, Myler RR, Marco J, Fournial G. Human coronary laser recanalization. Clin Cardiol1984;7:377-81. 4. Crea F, Abela GS, Fenech A, Smith W, Pepine CJ, Conti CR. Transluminal laser irradiation of coronary arteries in live dogs: an angiographic and morphological study of acute effects. Am J Cardiol 1985;57:171-5. 5. Weichert W, Paulkis V, Breddin HK. Laser-induced thrombi in rat mesentric vessels and antithrombotic drugs. Haemostasis 1983;13:61-71. 6. Treat MR, Weld FM, White JV. Effect of COs laser on the luminal surface of blood vessels in vivo. Lasers Surg Med 1983;3:247-54. 7. Choy DSJ, Stertzer SH, Rollerdam HZ, Bruno MS. Laser coronary angioplasty: experience with 9 cadaver hearts. Am J Cardiol 1982;50:1209-11. 8. McVicker JH, Day AL, Sevage DF. Laser endarterectomy: a comparison of thrombotic potential following COz laser vs surgical endarterectomy. Stroke 1986;17:266-9.

Fig. 3. Electron microscopic pictures after exposure of PRP to laser radiation for different periods of time. A, Control PRP. B, PRP exposedto laser radiation for 200 seconds.

C, PRP

irradiated

for

300 seconds.

9. Doerger PT, Blueck HI, Bel-Kah J, Taylor comparative effects of the argon, Nd: YAG, lasers on human platelets and erythrocytes Surg Med 1985;5:457-68.

A, Goldman argon-pumped in vitro.

L. The dye Lasers

Am-a, Mueller,

and Sinha

American

10. Kim L, Day A, Agela G. Laser vs. surgical endarterectomy in atherosclerotic rabbits: relationship between thrombotic potential and prostacyclin biosynthesis [Abstract]. Circulation 1986;74(2): 203. 11. Sinha AK, Shaltil SJ, Colman RW. Cyclic AMP metabolism in cholesterol-rich platelets. J Biol Chem 1986;252:3310-4. 12. Lages MC, Holmsen H. Studies on gel-filtered human platelets: isolation and characterization in a medium containing no added Ca2+, Mg”+, or K. J Lab Clin Med 1975;85:811-22. 13. Hamberg M, Svensson J, Samuelsson B. Thromboxanes: a new group of biologically active compounds derived from pros-

February 1993 Heart Journal

taglandin endoperoxides. I’roc Nat1 Acad Sci USA 197&Y?: 2994-8. 14. L RI, Wiener L, Walinsky P, Lefer AM, Silver JS, Smith JB. Thromboxane release during pacing-induced angina pectoris: possible vasoconstrictor influence on coronary vasculature. Circulation 1980;61:1165-‘il. 15. Hirsh PD, Hillis LD, Campbell WB, Firth BG, Willerson 57’. Release of prostaglandins into the coronary circulation in patients with ischemic heart disease. N Engl d Med 1981;304:68591.

Percutaneous transluminal venous angioplasty in occlusive iliac vein thrombosis resistant to thrombolysis Systemic thrombolysis is less than optimal in total occlusions of the iliac vein in which patency is 20% or less. We describe an interventional therapeutic procedure that may be effective in such cases. We selected 18 patients (average age, 29.5 years; range, 16 to 71 years) with complete iliac vein occlusion that persisted after 24 to 48 hours of systemic thrombolysis (streptokinase 100,000 Ulhr). The ipsilateral femoral vein was punctured, and a guide wire was gently advanced through the thrombus into the inferior vena cava. Multiple inflations were performed with a balloon catheter that was advanced on the wire. A temporary vena cava filter was placed as a protection against possible embolic migration. Systemic thrombolysis was administered for 24 to 48 hours. Control venography and pulmonary angiography were performed. Venography showed good recanaliration in seven cases, incomplete recanalization in five cases, and failure in six cases. Patency was maintained for a long time (15.6 months). In conclusion, (1) percutaneous transluminal venous angioplasty is a valuable adjunct to systemic thrombolysis when the latter alone fails; (2) segmental flow and mechanical obstruction were the critical factors, since the pharmaceutical factors were held constant, and (3) a more aggressive incremental interventional strategy warrants consideration. (AM HEART J 1993;125:362.)

Philippe Marache, MD, Philippe Asseman, MD, Jean Louis Jabinet, MD, Alain Prat, MD, Jean Jacques Bauchart, MD, Jean Charles Aisenfarb, MD, Martine Lesenne, MD, Brigitte Jude, MD, and Claude Thery, MD Lil le, France

Deep phlebitis is responsible for long-term venous sequelae.l, 2 Such complications are more frequent in cases of proximal phlebitis.3 Systemic thrombolytic therapy in the acute phase restores better venous patency than does administration of heparin.4* 5 From Service de Soins HBpital Cardiologique, Received Reprint ologique,

for publication requests: Boulevard

Intensifs et Service Lille, France. May

12, 1992;

de Radiologie accepted

Claude Thery, Service de Soins du Pr Leclercq, 59000 Lille,

Aug.

Cardiovasculaire.

METHODS

14, 1992.

Intensifs, France.

Hopital

Nonetheless, 80% of totally occluded iliac clots are resistant to thrombolytic therapy, and the majority of patients with such clots have venous sequelae during follow-up.6 Our interventional therapeutic technique combines percutaneous venous angioplasty with thrombolysis in patients with iliac veins in which occlusions persist after thrombolysis.

Cardi-

In our unit, patients with proximal deep venous thrombosisof recent onset without contraindication to throm(‘opyright

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