Role of protease inhibition in myocardial preservation in prolonged hypothermic cardioplegia followed by reperfusion

J

THoRAc CARDIOVASC SURG

1988;96:314-20

Role of protease inhibition in myocardial preservation in prolonged hypothermic cardioplegia followed by reperfusion Effect of aprotinin in an experimental model The effects of aprotinin on canine myocardium subjected to cardioplegia and global ischemia for 4 hours and then reperfused for 1 hour were investigated. Lysosomal and mitochondrial enzymes and cyclic nucleotides (adenosine cyclic monophosphate and guanosine cyclic monophosphate) were measured in coronary sin~ blood. Aprotinin was given intravenously before cardiopulmonary bypass at total doses of 10 x 1()3 kallikrein units per kilogram (group A, six dogs) and 20 x 1()3 KU/kg (group B, six dogs). In group A, three dogs survived but with poor cardiac function; all dogs in group B survived and had better cardiac function. Lysosomal (N-acetyl-,8-~g1ucosaminidase)and mitochondrial (aspartate aminotransferase) enzymes in coronary sinus blood at 60 minutes of reperfusion were significantly (p < 0.05) lower in group B than in group A. In both groups, guanosine cyclic monophosphate was significantly (p < 0.01) lower during reperfusion than before cardiopulmonary bypass; however, the values were significantly (p < 0.05) higher in group B than in group A. Serum adenosine cyclic monophosphate was lower during reperf~ion than before bypass in both groups, but it recovered during reperfusion in group B. Myocardial adenosine triphosphate was weD preserved in both groups but creatine phosphate was decreased (p < 0.01) in group A. These results suggest that aprotinin at a dose of 20 x 1()3 KU/kg may be effective in preserving myocardial viability and function after prolonged cardioplegia.

Makoto Sunamori, MD, Ryuichi Innami, MD, Jun Amano, MD, Akio Suzuki, MD, and Carlos E. Harrison, MD, PhD, Tokyo. Japan. and Rochester, Minn.

Increased activity of proteases, particularly lysosomal enzymes, in ischemic myocardium leads to biochemical and morphologic damage to the myocardium, I·) including decrease in glycogen stores,' depletion of highenergy phosphate compounds,' ionic imbalances," local From the Department of Thoracic-Cardiovascular Surgery, Tokyo Medical and Dental University School of Medicine, Tokyo, Japan, and the Division of Cardiovascular Diseases and Internal Medicine, Mayo Clinic and Mayo Foundation, Rochester, Minn. Read at the Twelfth Congress of the International Society for Heart Research, Melbourne, Australia, Feb. 9-13, 1986. Received for publication Feb. 4, 1987. Accepted for publication Dec. 16, 1987. Address for reprints: Makoto Sunamori, MD, Department of Thoracic-Cardiovascular Surgery. Tokyo Medical and Dental University School of Medicine, 1-5-45, Yushima, Bunkyo-ku, Tokyo, 113, Japan.

314

release of catecholamine,' accumulation of lactic acid,' and deterioration of cellular and subcellular function." The concentration of lysosomal enzymes increases both in the myocardium in response to ischemia and in the systemic circulation in response to extracorporeal circulation. Cold cardioplegia minimizes the effects of ischemia. There are reports 10.11 that aprotinin, a protease inhibitor, protects the myocardium from the adverse effects of ischemia and reperfusion by suppressing the release of lysosomal enzymes. Lysosomal activity is affected by cyclic nucleotides, particularly guanosine cyclic monophosphate (cGMP).12 This investigation was designed to characterize the changes in lysosomal enzyme concentration, in cyclic nucleotides, and in indices of myocardial damage during and after 4 hours of cardioplegia followed by reperfusion.

Volume 96 Number 2

Myocardial preservation

August 1988

Table I. Composition of lidocaine-magnesium cardioplegic solution Lidocaine . HCI (mmoljL) CaCl, (mmoIjL) KCI (mmoljL) Glucose (mmoljL) Mg I-aspartate (mmoljL) Mannitol (mmoljL) NaHCO, Osmolarity (mOsm)

11

0.5

o

247

8 100

to pH 7.4 450

.c ---E CU Q)

C)

~

1.0

C)

.+

~

CJ)

Materials and methods Twelve mongrel dogs weighing between 10 and 17 kg were used in this experiment. All dogs were anesthetized with pentobarbital (approximately 30 mg/kg intravenously) to suppression of corneal reflexes. Respiration was controlled by a positive-pressure ventilator. Arterial and left ventricular pressures and the electrocardiogram were recorded continuously with pressure transducers (Statham P23DB, Statham Instruments, Inc., Los Angeles) and a polygraph (Nihon Kohden Co., Tokyo). Cardiac output was measured, before cardiopulmonary bypass (CPB) and during reperfusion, with a magnetic flowmeter attached to the ascending aorta. These animals received humane care as described in "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences and published by the National Institutes of Health. After left thoracotomy, CPB was instituted with a pediatricsize membrane oxygenator (Terumo Inc., Japan). Venous drainage was conducted via the right atrium with a single catheter, and total perfusion was obtained by clamping the main pulmonary artery. The left ventricle was vented through the left atrium during total perfusion and cross-clamping of the aorta. The priming solution was a mixture of 1000 ml of Ringer's lactate solution and 500 ml of whole blood containing 50 mEq of sodium bicarbonate. Hemodilution to a hematocrit value between 20% and 25% was attained. The perfusion rate during CPB was approximately 100 rnl/rnin/kg: however, the rate was adjusted to maintain the mean arterial pressure between 70 and 100 mm Hg. The pH of arterial blood was maintained within physiologic limits during cross-clamping of the aorta and reperfusion. Systemic hypothermia was provided by a heat exchanger. The aorta was cross-clamped when the myocardial temperature reached 28 ° C, which was achieved in 3 to 5 minutes. Myocardial temperature (midportion of ventricular septum) was monitored with a needle thermistor (Yellow Springs Instrument Co., Yellow Springs, Ohio). Care was taken to prevent spontaneous warming of the myocardium by the light penetrating the thoracotomy. Cardioplegia was induced, immediately after cross-clamping of the aorta, by perfusion of cold (4° to 10° C) cardioplegic solution (Table I) via an 18-gauge needle at a perfusion pressure of 70 to 100 mm Hg. The initial amount was 10 rnl/kg; then 3 mljkg was given every 60 minutes. Myocardial temperature was maintained between 18° and 20° C during the 4-hour period of cross-clamping of the aorta.

~

..

+t

1.5

0.5

t

3

.+

++ <}

4

5

6

3 15

A-PRE, n=6

~ A-REP, n=3

•

o

B-PRE, n=6 B-REP, n=6

~ ~ ~. ¢ J~J P-005

A 7

~JA

8

9

10

LVEDP, mm Hg Fig. 1. Left ventricular stroke work index (LVSWI) plotted against left ventricular end-diastolic pressure (L VEDP) (mean ± standard error). Cardiac function was depressed in both groups after cardioplegia; however, cardiac function was significantly better in group B than in group A. Triangles, group A; circles, group B; solid symbols, before cardioplegia; open symbols. at end of reperfusion. Twenty minutes before reperfusion, rewarming was started gradually with a temperature difference of 6° C between body and perfusion temperatures. The aorta was unclamped when the esophageal temperature reached 28° to 30° C. All dogs resumed sinoatrial and atrioventricular nodal function after unclamping of the aorta during reperfusion. Reperfusion was continued for 60 minutes. The initial phase of reperfusion was supported by CPB (with a left ventricular vent) to an esophageal temperature of 36° C (this required 20 to 30 minutes). Then CPB was terminated gradually. The 12 dogs were grouped as follows. Group A (six dogs) received aprotinin to a total of 10,000 kallikrein units (KU) per kilogram (5000 KU /kg by intravenous drip infusion 30 minutes before CPB and 5000 KU/kg by intravenous continuous infusion during cardioplegia and reperfusion). Group B (six dogs) received a total of 20,000 KU /kg (10,000 KU /kg as a loading dose and 10,000 KU /kg by intravenous continuous infusion as in group A). Both groups were pretreated with the following drugs: (I) nifedipine, 2 rug/kg, intravenous drip infusion 30 minutes before CPB; (2) coenzyme Q (Eisai Company, Tokyo, E0216-018-3),5 mg/kg, intravenous infusion 30 minutes before CPB, then an additional 5 rng /kg continuously infused during cardioplegia and reperfusion; (3) betamethasone, 2 mg/kg, intravenous drip infusion 30 minutes before CPH. At the end of each experiment, tissues from the subendocardium, subepicardium, and septum of the left ventricle were excised while the heart was beating and oxygenated. These tissues were analyzed for myocardial adenosine triphosphate, creatine phosphate, and water content by methods described elsewhere. 13 These tissues also were examined with an electron microscope to evaluate ultrastructural changes.

3 16

80 70 ..J

2

60

~

50

U

cD

40

E

30

:::!;

2 Q)

en

The Journal of Thoracic and Cardiovascular

Sunamori et al.

80

l>.A. n e S

• B. n~6

70 ..J

2

~E

10

.

5

8,000

60

l>.A,n=3

• B. n~6

50

7.000

40 30

;2

en

20

~

5.000

!5

4.000

10 0

60

5

60

Reperfusion, min

Reperfusion, min

80

~ 6,000

E

2 Q)

20

0

Surgery

Fig. 2. Changes in cardiac enzymes (mean ± standard error). Left, Serum creatine kinase MB (MB-CK) in coronary sinus. Right, Serum mitochondrial aspartate aminotransferase (m-AAT) in coronary sinus. Release of MB-CK into the coronary sinus increased without significant difference between groups. Serum m-AA T increased significantly during reperfusion in both groups but not as much in group B as in group A.

enCD

~

70

:::>

60

5t Z

E :>

3.000

CD

o 1L-...L...----'-_--l....--'I Control

o

5

en

50

40 30

20 10

60

o L..-'-----'_ _.......- -' Control o 5 60 Reperfusion, min

Reperfusion, min

Fig. 3. Release of lysosomal enzymes into the coronary sinus (mean ± standard error). Left, Serum /3-G. Right, Serum NAG./3-G and NAG both increased during reperfusion. At 0, 5, and 60 minutes of reperfusion, NAG was significantly lower in group B than in group A.

Table II. Effect of pretreatment on hemodynamic values* Group LVEDP (mm Hg)

A B

Heart rate (beats/min)

A

Blood pressure, systolic (mm Hg) Cardiac index (rnl/kg/rnin) LVSW1 (gm . m/kg/beat)

A

B B A

B A

B

Before pretreatment 4J 4.6 160 147 123 129 112 121 1.19 1.40

± ± ± ± ± ± ± ± ± ±

0.1 0.1 6 8 9 9 4 8 0.09 0.09

After pretreatment Raw

p

± ± ± ± ± ± ± ± ± ±

<0.05 <0.05 NS NS <0.01 <0.01 <0.01 <0.01 <0.01 <0.01

2.8 3.3 142 133 70 85 70 85 0.62 0.84

OJ 0.3 6 7 8 9 8 9 0.10 0.07

Adjusted]

p

± ± ± ± ± ± ± ± ± ±

NS NS NS NS <0.05 <0.05 NS NS NS NS

4.3 4.6 144 133 93 103 125 129 l.15 1.31

0.1 0.1 6 7 5 6 10 6 0.09 0.06

LVEDP. Left ventricular end-diastolic pressure; LVSWI. left ventricular stroke work index; NS. not significant. • Data shown as mean ± standard error. tMeasured at the same LVEDP as before pretreatment.

Immediately before CPB (after pretreatment) and at 5 and 60 minutes of reperfusion, blood was withdrawn from the aorta and the coronary sinus through a catheter placed in the coronary sinus from the left internal jugular vein.ji-Glucuronidase (/3-G), N-acetyl-/3-D-glucosaminidase (NAG), MB fraction of creatine kinase, mitochondrial aspartate aminotransferase, adenosine cyclic monophosphate (cAMP), and cGMP in serum were measured." All data were analyzed by Student's paired or unpaired t test; p < 0.05 was accepted as showing statistical significance.

Results Hemodynamic results. Arterial pressure, left ventricular end-diastolic pressure, cardiac index, and left

ventricular stroke work index were significantly depressed after pretreatment compared with before pretreatment, although heart rate remained unchanged (Table II). There was no difference between the two groups. When preload after pretreatment was adjusted to the same level as before pretreatment, all variables in both groups, except heart rate, increased to their pretreatment level. Cardiac function curves were expressed as the relationship of left ventricular stroke work index to enddiastolic pressure (Fig. 1). Cardiac function in group A was si'gnificantly depressed at 60 minutes of reperfusion compared to group B. All dogs in group B survived reperfusion but three of the six dogs in group A died.

Volume 96 Number 2 August 1988

Myocardial preservation

I:>. A

30

• B

€ (5 E a.

a: ~ '5 E

2

cJl

·P
3I7

. A , n=3 ~B. n=6

20 c:

ea.

'Qj

01

E 10 C,

::I.. 0.:-

10

~

0 Control

5·

c:

30

E

a.

a: ~

2

G>

*P
ea. ~ 10

C,

::I..

a: o

20

0

ra

E

*

'Qj

I:>. A • B ·P
€ (5

20

60

Reperfusion. min

10

CIJ

60

Control

Reperfusion, min

Fig. 4. Serum concentrations of cyclic nucleotides in the coronary sinus (mean ± standard error). Top. AMP (normal range, 18.5 to 40 pmol/rnl). Bottom. GMP (normal range, 10 to 15 pmol/rnl). The increase in serum cAMP in group B after pretreatment but before CPB is not significant. No significant difference was noted between groups. cAMP was lower during reperfusion than before CPB; it was significantly higher in group B than in group A at 5 and 60 minutes of reperfusion. cGMP in group B decreased significantly after pretreatment and during reperfusion in both groups; however, it remained significantly higher in group B than in group A during reperfusion.

Myocardial isoenzymes. Serum creatine kinase MB increased significantly during reperfusion after 4 hours of cardioplegia; however, no significant difference was noted between the groups (Fig. 2, left). Serum mitochondrial aspartate aminotransferase increased significantly during reperfusion in both groups, but it was significantly lower in group B than in group A at 60 minutes of reperfusion (Fig. 2, right). Coronary sinus tJ-G and NAG. Serum {j-G increased significantly during reperfusion after cardioplegia; however, there was no significant difference between the two groups (Fig. 3, left). Serum NAG increased during reperfusion after cardioplegia, but the increase was

EPI

END

SEP

Fig. 5. Myocardial high-energy stores (mean ± standard error). Top, Myocardial adenosine triphosphate (ATP) content; bottom, myocardial creatine phosphate (ep) content. EPl, Subepicardial layer of left ventricle; END. subendocardial layer of left ventricle; SEP. ventricular septum. Myocardial CP was higher in group B than in group A.

significantly less in group B than in group A at 60 minutes of reperfusion (Fig. 3, right). The concentrations of {j-G and NAG were slightly higher in the coronary sinus than in arterial blood in both groups; thus the possibility of a systemic effect is excluded. Serum cAMP and cGMP. Serum cAMP decreased during reperfusion after cardioplegia in both groups; however, the serum concentration of cAMP in group B was greater than that in group A, and at 5 minutes of reperfusion cAMP was significantly higher in group B than in group A (normal range, 18.5 to 40 pmoljml) (Fig. 4, top). Serum cGMP was significantly higher in group B than in group A throughout the experiment; however, the values during reperfusion were less than those before cardioplegia (normal range, 0 to 15 pmolj ml) (Fig. 4, bottom). Myocardial high-energy stores. Myocardial adenosine triphosphate content remained between 50% and 70% of normal in tissue in the left ventricle; no significant difference was noted between two groups (Fig. 5, top). Myocardial creatine phosphate content was markedly lower (approximately 20% of the normal tissue value) in group A than in group B (30% to 50% of normal) (Fig. 5, bottom).

3I8

The Journal of Thoracic and Cardiovascular Surgery

Sunamori et al.

Fig. 6. Myocardial ultrastructure (endocardial layer). Top, With aprotinin at 10,000 KU /kg, there is destruction of cristae and swelling of mitochondria; also lysosomes are apparent. Bottom, At aprotinin dosage of 20,000 KU/kg, mitochondria are better preserved in membranes and cristae. Lysosomes are present. Myofibrils are well maintained in both groups. (XI2,OOO.)

Myocardial water content. There was no significant difference between the groups in myocardial water content in the surviving animals; however, myocardial water content did increase significantly in both groups compared with normal myocardium. Myocardial ultrastructure. Mitochondrial changes were minimal in the group treated with aprotinin at 20,000 KU jkg. On the other hand, severe damage was

observed in cristae and membranes of mitochondria in the group treated with the lower dose of aprotinin. Myofibrils were well preserved in both groups. Representative photomicrographs are shown in Fig. 6. Discussion

Yokoyama" and others I 1,12. 15-20 have reported significant beneficial effect of combined pretreatment with

Volume 96 Number 2 August 1988

nifedipine, betamethasone, and coenzyme Q for myocardial preservation in the ischemia-reperfusion model. Therefore, in this investigation, the animals in both groups received nifedipine, betamethasone, and coenzyme Q before ischemia. The method of myocardial protection in this study is different from the common method in clinical practice with respect to both formula and frequency of injection of cardioplegic solution. This study was conducted as a prospective animal experiment, not using placebo; however, the data were analyzed in a blind fashion, particularly with regard to biochemical and ultrastructural changes. Lysosomal enzyme activity increases as the ischemic interval becomes prolonged. Therefore, a lysosomal enzyme inhibitor, aprotinin, at 10,000 KU jkg was used in Yokoyama's experiment" in our laboratory (3 hours ofischemia followed by reperfusion) in a previous study. We2I.2 2 also have been using aprotinin almost routinely at 10,000 KUjkg in clinical cardiac operations, with satisfactory myocardial preservation. Thus a higher dose, 20,000 KUjkg, was used for prolonged ischemia in this study. It has been reportedv" that aprotinin exerts various effects on tissue when used in a tissue culture model at extremely high concentrations, 300 to I,OOO KU jml. An advantageous effect of aprotinin on organ preservation has also been reportedv"; however, the optimal dose of aprotinin varied in each report. Further study is mandatory to determine optimal dose of aprotinin for myocardial preservation. All dogs treated with aprotinin at 20,000 KU jkg survived 4 hours of cardioplegia followed by I hour of reperfusion although the cardiac function of these animals was moderately depressed. On the other hand, 50% of the dogs given aprotinin at 10,OOO KUjkg did not survive 60 minutes of reperfusion, and even the surviving dogs had significantly poorer cardiac function. These results suggest that aprotinin at a dose of 20,000 KU jkg has a beneficial effect on preservation of cardiac function in cardioplegia followed by reperfusion. Because aprotinin is a protease inhibitor, the beneficial effect on cardiac function is likely mediated through maintaining biochemical and morphologic integrity of the ischemic-reperfused myocardium. Aprotinin has various actions: (I) inhibition of plasmin and kallikrein," (2) inhibition of trypsin and chymotrypsin," (3) suppression of release of lysosomal enzymes (by reducing rate of ischemic necrosis), 30 (4) enhancement of the total antiprotease capacity of human plasma (especially in conditions associated with an increase of lysosomal degradation from granulocytes), (5) inhibition of the decrease in kininogen activity after cardiopulmonary bypass," and (6) inhibition of the uptake of adenosine by cardiac cells."

Myocardial preservation

3I9

Inhibition of kallikrein seems to be important in CPB because the kinin not only increases histamine release from mast cells, thereby increasing vascular permeability, but also stimulates release of cyclic nucleotides from granulocytes.v" Cyclic nucleotides are known to be closely linked to the action of the lysosomal systern. 10. 12 Ischemia induces liberation of lysosomal enzymes':'; with prolonged ischemia in our experiment, there was a significant increase in lysosomal enzyme release into the coronary sinus. Ignarro, Krassikoff, and Slywka 12 reported that cGMP unstabilizes the lysosomal membrane and cAMP stabilizes it. The results reported by Carr and Goldfarb" support the thesis of Ignarro's group." Our data suggest that release of NAG is suppressed by aprotinin, consistent with the data of Ignarro, Krassikoff, and Slywka." In our experiments, myocardial adenosine triphosphate content was relatively well preserved during cardioplegia and reperfusion. All dogs receiving the high dose of aprotinin survived, whereas survival rate was very poor in the low-dose group. Myocardial subcellular structure was less injured in the high-dose group, whereas severe destruction of mitochondria was observed in the low-dose group. The use of pretreatment with nifedipine, betamethasone, and coenzyme Q seemed to maintain high-energy stores in the myocardium in both groups as suggested by our previous studies." We wish to express our gratitude to the Eisai Company, Tokyo, for supplying coenzyme 010' REFERENCES I. Wildenthal K. Lysosomal alterations in ischemic myocar-

2. 3.

4.

5. 6.

dium: resultor causeof myocellular damage? (Editorial). J Mol Cell Cardiol 1978;10:595-603. Decker RS, Wildenthal K. Sequential lysosomal alterations during cardiac ischemia. II. Ultrastructural and cytochemical changes. Lab Invest 1978;38:662-73. Fox AC, Hoffstein S, Weissmann G. Lysosomal mechanisms in production of tissue damage during myocardial ischemia and the effects of treatment with steroids. Am Heart J 1976;91 :394-7. Cornblath M, Randle PJ, Parmeggiani A, Morgan HE. Regulation of glycogenolysis in muscle: effects of glucagon and anoxia on lactate production, glycogen content, and phosphorylase activity in the perfused isolated rat heart. J Bioi Chern 1963;238: 1592-7. Case RB. Ion alterations during myocardial ischemia. Cardiology 1971;56:245-62. Wollenberger A, Shahab L. Anoxia-induced release of noradrenaline from the isolated perfused heart. Nature 1965;207;88-9.

7. Wollenberger A, Krause E-G. Metabolic control charac-

The Journal of Thoracic and Cardiovascular Surgery

3 2 0 Sunamori et al.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

tensncs of the acutely ischemic myocardium. Am J Cardiol 1968;22:349-59. Ogletree ML, Lefer AM. Influence of nonsteroidal antiinflammatory agents on myocardial ischemia in the cat. J Pharmacol Exp Ther 1976;197:582-93. Jennings RB. Relationship of acute ischemia to functional defects and irreversibility. Circulation 1976;53(Pt 2): 126-9. Busuttil RW, George WJ. Myocardial ischemia, cyclic nucleotides, and lysosomal enzymes. Adv Cyclic Nucleotide Res 1978;9:629-45. Carr FK, Goldfarb RD. Ischemia-induced canine myocardial lysosome labilization: the role of endogenous prostaglandins and cyclic nucleotides. Exp Mol Pathol 1980;33:36-42. Ignarro LJ, Krassikoff N, Slywka J. Release of enzymes from a rat liver lysosome fraction: inhibition by catecholamines and cyclic 3',5' -adenosine monophosphate, stimulation by cholinergic agents and cyclic 3',5' -guanosine monophosphate. J Pharmacol Exp Ther 1973;186:86-99. Sunamori M, Amano J, Okamura T, Suzuki A. Superior action of magnesium-lidocaine-I-aspartate cardioplegia to glucose-insulin-potassium cardioplegia in experimental myocardial protection. Jpn J Surg 1982;12:372-80. Yokoyama M. The effect of pharmacological pretreatment on the long-term globally ischemic heart: lysosome, cyclic nucleotide and myocardial injury. J Jpn Surg Soc 1987;88:109-18. Clark RE, Christlieb IY, Henry PD, et al. Nifedipine: a myocardial protective agent. Am J Cardiol 1979;44:82531. Sunamori M, Harrison CE Jr. Effect of betamethasone on mitochondrial oxidative phosphorylation in ischemic canine myocardium. Mayo Clin Proc 1980;55:377-82. Sunamori M. Protective effect of betamethasone on the subendocardial ischemia after the cardiopulmonary bypass. J Cardiovasc Surg (Torino) 1978;19:291-310. Okamura T, Sunamori M, Amano J, Ozeki M, Suzuki A. Protective effect of coenzyme QIO in reperfused ischemic myocardium. Jpn Ann Thorac Surg 1982;2:827-34. Nayler WG. The use of coenzyrne., to protect ischemic heart muscle. In: Yamamura Y, Folkers K, Ito Y, eds. Biomedical and clinical aspects of coenzyme Q. Vol 2. Amsterdam: Elsevier Science Publishers, 1980;409-25. Ozawa T. Mode of CoQlO action on biomembrane. In: Asano K, Manabe H, Suzuki A, eds. Coenzyme QIO in surgery. Tokyo: Ishiyaku-Shuppan, 1983:1-17. Sunamori M, Amano J, Kameda T, Okamura T, Ozeki M, Suzuki A. Additive protection of aprotinin, protease

22.

23.

24.

25.

26. 27.

28.

29.

30.

31.

32.

33.

34.

inhibitor to cold cardioplegia from ischemic myocardium. Jpn Circ J 1980;44:771-5. Sunamori M, Tanaka H, Okamura T, et al. Pharmacological myocardial protection in addition to cold cardioplegia in coronary artery revascularization: effect of protease-inhibitor on intraoperative myocardial protection. In: Dudziak R, Reuter HD, Kirchhoff PG, Schumann F, eds. Proteolyse und Proteinaseninhibition in der Herz- und Gefaesschirurgie. Stuttgart: Schattauer, 1985;281-8. Davis H, Gascho C, Kiernan JA. Effects of aprotinin on organ cultures of the rat's kidney. In Vitro 1976;12: 192-7. Davis H, Gascho C, Kiernan JA. Action of aprotinin on the survival of adult cerebellar neurons in organ culture. Acta Neuropathol (Berl) 1975;32:359-62. Guthrie R, Rizzi GT, McCabe RE Jr. Experimental study of the effect of a proteinase inhibitor (Trasylol) on organ preservation. Am J Surg 1966;112:835-8. Godfrey AM, Salaman JR. Trasylol (aprotinin) and kidney preservation. Transplantation 1978;25:167-8. Hoyer J, Garbe L, Delpierre S, Prieur A, MacquartMoulin G, Noirclerc M. Functional evaluation of the transplanted lung after long-term preservation. Respiration 1980;39:323-32. Wiman B. On the reaction of plasmin or plasminstreptokinase complex with aprotinin or Q'rantiplasmin. Thromb Res 1980;17:143-152. Balldin G, Ohlsson K. Demonstration of pancreatic proteaseantiprotease complexes in the peritoneal fluid of patients with acute pancreatitis. Surgery 1979;85:451-6. Glenn TM, Tauber PF, Miller AG, Golfarb RD, Mustafa SJ. Alterations of the hemodynamic and biochemical sequelae associated with acute coronary ligation by aprotinin. In: Cantin M, Haberland GL, Schnells G, Selye H, eds. New aspects of trasylol therapy. Vol 8. Stuttgart: Schattauer, 1975;329-45. Nagaoka H, Katori M. Inhibition of kinin formation by a kallikrein inhibitor during extracorporeal circulation in open-heart surgery. Circulation 1975;52:325-32. Mustafa SJ. Effect of aprotinin on the metabolism of adenosine in cultured heart cells. Biochem Pharmacal 1979;28:340-3. Abell CW, Monahan TM. The role of adenosine 3',5'cyclic monophosphate in the regulation of mammalian cell division. J Cell BioI 1973;59:549-58. Katoh K. The effect of chemical mediators (bradykinin, histamine) on cyclic AMP levels of murine lymphocytes subpopulation. J Nippon Med Sch 1979;46:234-40.