The effects of aprotinin on hemostatic function during cardiac surgery

The Effects of Aprotinin

on Hemostatic

Function

During Cardiac Surgery

G. Marx, MD, H. Pokar, MD, H. Reuter, V. Doering, MD, and V. Tilsner, MD The mechanism of action by which large doses of aprotinin decrease blood loss during cardiac surgery is not completely understood. In a prospective, controlled study, 30 patients undergoing cardiac surgery were given high-dose aprotinin in accordance with a commonly used regimen. Twenty untreated but otherwise comparable patients served as the control group. The effects of aprotinin therapy during cardiopulmonary bypass on coagulation parameters, the kallikreinkinin system, fibrinolysis, platelet stimulation, and the release of elastase from neutrophils were studied. The fibrinolysis parameters were the only measurements that showed clear and significant differences between the two

I

NTRAOPERATIVE and postoperative bleeding continue to play an important role in cardiac surgery. Even though surgical problems may be the most common cause of hemorrhagic complications, cardiopulmonary bypass (CPB) always disrupts the coagulation system and can be responsible for an increased bleeding tendency.’ An important factor is the activation and increased turnover of plasma and cellular components caused by contact between blood and the foreign surface of the oxygenator.2-5 This results in a reduction in the number of platelets and in their ability to function, ‘-I6 induction of increased fibrinolysis w’-*~ activation of coagulation and fibrinolysis via the kailikrein-kinin system,“~z5~27 and the release of proteases from activated granulocytes.28329 Various blood products and drugs have been used in an attempt to prevent or alleviate the negative effects of CPB on hemostasis. Aprotinin was tried in the 1960s for this purpose,‘o,31 and has been an established part of therapy since the early 1980s in some centers.3z35 However, prophylactic use of aprotinin did not become a widespread practice until the publications by Royston et al’ and van Oeveren et aP6 in 1987, which documented a convincing reduction in blood loss and the need for blood transfusion following high-dose aprotinin. The beneficial effect on intraoperative blood loss has been described in more recent publications as we11.37-4o The aim of this investigation was not to provide additional documentation of the clinical effect of aprotinin, but to clarify the mechanism of action that produces this effect. Therefore, a number of laboratory parameters were investigated in an attempt to determine the effect of aprotinin on the above-mentioned functional systems. MATERIALS

AND METHODS

Fifty patients scheduled for surgery consented to be enrolled in this prospective, randomized study. The study protocol had previously been approved by the ethical committee. Thirty patients were given an aprotinin preparation (Trasylol; Bayer, Leverkeusen, Germany); 20 were not treated and served as a control group. The patients were anesthetized with a standardized regimen of fentanyl, etomidate, and pancuronium. The oxygenator was an EXCEL disc-membrane oxygenator (COBE Laboratories Inc, Lakewood, CO) that was primed with 2 L of Ringer’s lactate solution, 100 mL of 20% mannitol, 12.5 mEq of sodium hydrogen carbonate, and 2,500 U of heparin. The target hematocrit value during perfusion was 22%, and the flow rate was 2.0 L/minim2 at

Journal of Cardiothoracic and VascularAnesthesia,

groups. Aprotinin almost completely inhibited the formation of fibrin and fibrinogen degradation products. It is assumed that inhibition of systemic fibrinolysis and suppression of local fibrinolysis contribute to the hemostatic action of aprotinin. The study did not demonstrate a significant protective effect of aprotinin on platelets. In addition, the dose of aprotinin administered did not affect the kallikrein-kinin system or elastase. Therefore, these data suggest that the previously demonstrated hemostatic effects of aprotinin derive primarily from its antifibrinolytic action. Copyright o 199 1 by W. B. Saunders Company

27°C. Prior to CPB, patients were given 300 U/kg of heparin, a further 100 U/kg, 40 minutes later, and subsequently 50 U/kg every 50 minutes for the duration of CPB. A predetermined heparin regimen was used instead of the more common monitoring via the activated clotting time (ACT). At the end of CPB, the heparin was neutalized with 3 mg/kg of protamine chloride. A further 20 to 30 mg were administrated where clinically necessary. Aprotinin was administered as follows: prior to the start of CPB 2 million kallikrein inactivator units (KIU) were given over 15 minutes, followed by 0.5 million KIU/h via a continuous infusion until administration of protamine at the end of CPB. The pump prime also contained :2 million KIU of aprotinin. Samples of blood were taken from the patients at five stages: (1) after induction of anesthesia, immediately before the initial infusion of aprotinin; (2) 10 minutes after the infusion of 2 million KIU of aprotinin; (3) 20 to 30 minutes after the start of the CPB; (4) shortly before the end of the CPB; and (5) 10 minutes after neutralization with protamine. At each time, 9 mL of blood was collected into a plastic syringe containing 1.0 mL of 0.11 sodium citrate; 4.5 mL of blood was collected in a siliconized glass tube containing 1,000 U of heparin and 1,000 KIU of aprotinin for measurement of fibrinopeptide A (FPA). The latter was placed on ice immediately after collection. All blood samples were centrifuged for 20 minutes at 3,000 rpm at 4°C. Factors XI and XII were determined using control plasma obtained from Immuno AG (Vienna, Austria). High molecular weight kininogen (HMWK) was determined using control plasma from Behringwerke AG (Marburg, Germany). Prekallikrein, kallikrein-like activity, and kallikrein inhibitor activity were determined using the chromogenic substrate S-2302 (AB Kabi Diagnostica, Molndal, Sweden). Plasminogen, cY,-antiplasmin, and antithrombin III (AT-III) were determined with the chromogenic substrates HD-Nva-CHA-Lys-pNA and HD-CHA-But-Arg-pNA (Behringwerke AG) in a Chromotimer (Behringwerke AG) using the kinetic method. Thrombin-AT-III-complex (TAT) was determined using an enzyme-linked immunosorbent assay (ELISA) from Behringwerke AG, and D-dimers using an ELISA from Boehringer Mannheim GmbH (Mannheim, Germany). For FPA measurement, fibrinogen was completely removed by repeated bentonite absorption; FPA was measured using a rabbit FPA

From the Departments of Blood Coagulation Disorders, Anesthesiology, and Cardiovascular Surgery, University Hospital Eppendod Hamburg Germany. Address reprint requests to Dr G. Malx, Abteilung fiir Blutgetinnungsstiincngen, Universitiitskrankenhaus Eppendolf; Martinistrape 52,200O Hamburg 20, Germany. Copyright o 1991$ W.B. Saunders Company 1053-0770191/0505-0009$03.00/0

Vol 5, No 5 (October), 1991:

pp 467-474

467

MARX E r A:

468

antibody

GmbH).

from a commercial ELISA kit (Boehringer Mannheim Elastase (PMN-elastase-ol,-proteinase inhibitor complex)

was determined

using an ELISA

from Merck

(West Point,

PA).

Activity of tissue plasminogen activator (t-PA:C) was determined photometrically using the chromogenic substrate S-2251 (AB Kabi Diagnostica):

antigen

Imco (Stockholm,

(t-PA:Ag)

Sweden),

activity (PAI) using the method chromogenic

substrate

using a monoclonal

and plasminogen described

from

and platelet

factor

determined using an ELISA from Behringwerke were counted using a phase-contrast microscope. The distribution

of most of the variables

hand tail, and logarithmic the

approximation

statistical formed, geometric

were performed

mean

levels

Nontransformed

data

AG.

showed

Platelets

a long rightto improve

the corresponding

with data logarithmically

of these

(standard

Behring-

4 (PF 4) were

was required

Therefore,

means (Z,). The distribution

&, x (SDF)”

deviation

to normality.

analyses and

transformation

deviation

from

inhibitor

by Stief et alJ”” with the

HD-Nva-CHA-Lys-pNA

werke AC. P-Thromboglobulin

antibody

activator

variables

are

trans-

expressed

as

is then given as the interval: factor

= antilog

are given as arithmetic

d&).

mean

? standard

(jt % SD).

Data were analyzed by a multivariate technique of profile analysis.“’ It was tested whether the profiles were parallel (H,,), the treatment different

levels were equal (H,,,), and the response means sampling times did not change by time (H,,);

parallelism

hypothesis

was not accepted,

at the if the

the last test was carried

out separately for the two groups (H,,, for the control group, H,,,, for the aprotinin group). Multiple comparisons between pairs of means were done by computing Scheffk intervals on the corresponding contrasts; regarding repeated measurements, adjacent means and the difference

between

the final value and baseline

package,

program

version S.0.4*

RESULTS

The demographic details of the two groups were similar (Table 1). Due to some missing samples, the number of values is reduced for some tests. The number of measurements for each test in the two groups is referred to in the following text as n, = control, n2 = aprotinin if all samples were not present.

Table 1. Demographic Details of the Two Groups

Control Group

Aprotinin Group

20

30

Coronary artery bypass

16

26

Organic heart defect

A

4

Male

15

24

Female

5

6

No

Factors XI and XII, HMWK, and prekallikrein did not differ between the two treatment groups, and there was a high level of agreement between their profiles over time (Figs 1 and 2). Factor XI decreased almost at the beginning of the operation and independently from treatment with aprotinin. During CPB, the activities of factor XI, factor XII, and HMWK decreased almost to zero because of the pronounced heparin effect. At the end of the operation there was a distinct increase in the measured values, although they did not reach their baseline values. In contrast to the near-complete abolition of activity of factor XI, factor XII, and HMWK, the prekallikrein determined using a chromogenic substrate dropped during CPB to about half of the baseline value (Fig 2). Kallikrein and Kallikrein Inhibitor (Kallikrein inhibitor: n, = 19; n, = 30) Although the graphic representation of mean kallikreinlike activity suggests that the profiles of the two treatment groups were different, it was not possible to demonstrate this effect conclusively because the measured values were widely distributed. Overall, however, it can be said that kallikrein-like activity decreased during surgery. In contrast, the therapy had a distinct effect on measured kallikrein-inhibitor activity, which increased following administration of aprotinin (Fig 2).

measure-

ment were compared. Significance was accepted at P < 0.05 level. The calculations and graphics were carried out using the SYSTAT statistics

Factors of Contact Activation and the Kallikrein-Kimn System (FXI. II, = 15: II, = 20; Prekallikrein; n, = 20: n, = 29)

Operation

Plasminogen and uZ-Antiplasmin (Plasrninogen: n, = 20: n, = 29) Plasminogen activity was reduced immediately after the initial infusion of aprotinin; there was no discernible difference in the untreated group. Thereafter the curves ran largely parallel to each other. Aprotinin greatly increased the measured activity of qantiplasmin, which remained at a high level throughout the operation. On the other hand, in the control group there was a decrease in qantiplasmin (HOc, rejected; Fig 3). FPA and D-dimers There were striking differences between FPA and D-dimers in the aprotinin and control groups (Fig 4). Both increased considerably without aprotinin, whereas the effect was almost completely suppressed by therapy with aprotinin. The main increase in both laboratory parameters occurred in the second half of CPB, although FPA reacted at an earlier stage than DD. t-PA:C, t-PA:Ag, and PA1

Sex

Mean age (vr) Range Mean weight (kg) Range Mean duration of CPB (min) Range

60

61

45-75

44-77

76

73

51-102

51-106

96

108

50-155

21-220

Distinct differences in these results were also found between the two treatment groups. In the control group, t-PA:C and t-PA:Ag increased and at the end of the operation were still higher than their baseline values. In the aprotinin group, there was a slight decrease immediately after the initial infusion, and the expected increase at the start of CPB did not occur. Both activity and concentration were highly correlated for the control group and for the

EFFECTS OF APROTININ IN CARDIAC SURGERY

469

HMWK

Factor XII

Factor XI 140 %

140

120

120

100

100

% T

100

T

0 Control ??Aprotinin

%

T

80

80

80

6o

TT

___-60

80 1+5*=*

40

/ 20

I _ XL-

4

**-

??

0

5

HOa : P > 0.05 / Hoc: P < 0.001 HOb : t’> 0.05

H0a : P > 0.05 / Hoc: P < 0.001 t-tab: f’> 0.05

H0a : P > 0.05 / Hoc: P < 0.001 HOb : P > 0.05

Fig 1. Factors of contact activation and HMWK at different sampling times (as defined in the text). Plotted are mean f standard deviation, geometric mean x (standard deviation factor)“, if data have been logarithmically transformed for statistical analysis. Hypotheses tested: H,., profile parallelism; H,, equal treatment effect; H,, changes by time (H,, for control; H, for aprotinin, if H,, is rejected). Asterisks between two values indicate significant differences between treatment levels respectively between adjacent sampling times (at the bottom of the graph, if curves are parallel); asterisks associated with “1 + 5” indicate significant difference of sample 5 from sample 1 (baseline value). ?? P < 0.05; ?? *P < **P -< 0.001. Dashed lines indicate lower limits of normal range. 0.01; ??

aprotinin group at sampling time 4. Treatment with aprotinin brought about a considerable increase in PAI, whereas there was a slight drop during the course of the operation without aprotinin (Fig 5). No correlation was found between t-PA and PAI activity for the control group, or for the aprotinin group at sampling time 4.

at the start of CPB and increased again at the end of the operation, although not back to its baseline value. The TAT concentration increased continually, with the greatest increase in the second half of CPB (Fig 6). During CPB there was a marked increase in elastase, without any significant difference between the two treatment groups.

AT-III and TAT (AT III: n, = 15; n, = 21)

Platelet Count, f3-Thromboglobulin, and PF 4

These laboratory parameters showed no differences between the two treatment groups. AT-III activity decreased

At no point was there a significant difference between the platelet counts recorded in the two groups. There was a Kallikrein-inhibitor

Kallikrein-like activity

Prekallikrein 225

0

XGx(.SDF)*’

%

Control

?? Aprotinin

I

180

80 60

ii,r(SDF)*’

Fig 2. Prekallikrein, kallikreinlike activii, and kallikrein inhibitor at different sampling times. Statistical details are defined in the legend to Fig 1. Dashed lines indicate lower limits of normal range.

Sample: 1

2

3

4

5

H0s : P > 0.05 / Hoc: P < 0.001 Hot, : s=’ > 0.05

1

2

H0s : P > 0.05 tiob: P> 0.05

3

/ Hoc:

4

I

5

P < 0.001

Hoa : P < 0.001

I HOc,

Hob

/ HOc2

:P

< 0.001

:P :P

< 0.01 < 0.001

MARX Ei

470

AL

ayAntiplasmin

Plasminogen

0 Control ??Aprotinin 150

100 60

50

/ XGx(SDF)*'

XGx (SDF) *’

L

Sample:

1

2

3

4

5

HOa : P < 0.001 / HOc, : P < 0.001 H,,b : P < 0.001 / Hoc2 : P < 0.001

01

1

2

3

4

5 Fig 3. Plasminogen and wantiplasmin at different sampling times. Statistical details are defined in the legend to Fig 1. Dashed lines indicate lower limits of normal range.

HOa : P < 0.001 / HOC,: P < 0.01 H,,b : P < 0.001 / HOc2: P < 0.001

DISCUSSION

decrease in the platelet count only between the first two intervals, and thereafter the counts remained constant. Between the first two measurements, p-thromboglobulin showed the opposite tendency in both groups. Thereafter, the profiles were similar, and the values increased during CPB. The latter observation also applies to PF 4, which, in contrast to B-thromboglobulin, had decreased considerably again by the end of theoperation (Fig 7).

The most striking differences between the patients treated with aprotinin and those not given aprotinin are in the fibrinolytic system. The antiplasmin potential increased considerably when aprotinin was administered, as reflected in the increased q-antiplasmin activity.‘3 Therefore, it is understandable that the expected increase in plasmin Fibrinopeptide

A

D-dimers 3

10

??Aprotinin

f

:

0 Control

8 2 6

XG x (SDF)*’

Sample: Fig 4. FPA and D-dimers at different sampling times. Statistical details are defined in the legend to Fig 1. Dashed lines indicate upper limits of normal range.

1

2

Xc, x (SDF)”

3

HOa: P < 0.001 HOb : P < 0.001

4

5

/ HOC, : P < 0.001 / HOc2: P < 0.001

HOa:P < 0.001 HOb : P < 0.001

i HOC, :P < 0.001 / Hoc*: P < 0.05

EFFECTS OF APROTININ IN CARDIAC SURGERY

471

t-PA:C 2.5 !!J ml

t-PA:Ag

-

PAI 14 u

2c 0

Control

“c

??Aprotinin

ml

mI

12

6

Fig 5. t-PA:C, t-PA:Ag, and PAI at different sampling times. Statistical details are defined in

Qx(SDF)*l

(“.:‘““““O Sample: 1 2

the legend to Fig 1. Dashed lines indicate upper (.-.-.-.-.I and lower (____________ ) limits of normal

Hoa:

3

4

12

5

P < 0.001 /HOC,: P < 0.01

I

I

/

3

4

5

H0s: P < 0.001 I Hoc, :P < 0.01

HO,,: P < 0.001 / HOc2: P < 0.001

0.001/ HOc2:P<

tiob:P<

0.001

Hoa Hob

:P :P

< 0.001 / Hoc,: P < 0.01 < 0.001 I HOc2: P < 0.001

range.

activity in the initial phase of CPB did not occur.“3zo This was also demonstrated by the decrease in plasminogen activity, which was probably due to inhibition of plasminogen activation by aprotinin. The changes in vivo may be smaller than shown here because the assay used can be influenced by aprotinin. However, the aprotinin that interferes with plasmin generated in vitro originates from the circulating blood and, therefore, can be expected to have similar effects in vivo. The authors were more surprised by the differences between the t-PA curves. Because both activity and protein concentration behaved in exactly the same way, aprotinin must affect the release of t-PA from endothelial cells rather than its function. Apart from a slight decrease in t-PA immediately after the initial dose, aprotinin appeared mainly to inhibit the increase in t-PA that occurred in the initial phase of CPB in patients not given aprotinin. Himmelreich et al got a similar result in a study of aprotinin during orthotopic liver transplantation.44 TAT

Antithrombin Ill 120

60

40

20

1000

Control

??Aprotinin

100

80

Elastase

40 0

%

PA1 activity was almost a mirror image of the t-PA curves; one striking feature is the great increase immediately following the first dose of aprotinin. Because there was no correlation between t-PA and PAI activity, decrease of t-PA was not the reason for the high PAI levels. This result might be an in vitro effect of aprotinin. The curves of the FPA and D-dimers over time illustrate the primary clinical effect of aprotinin therapy: almost total inhibition of the fibrinogenolysis and fibrinolysis, which were considerably activated during CPB in the control group.36,39.45 D-dimers are thought to be generated by plasmin-mediated cleavage of fibrin, whereas FPA derives from fibrinogen split by thrombin. However, in vivo these mechanisms are not activated independently, and the antibodies used in the test systems are not totally specific.46’47 The present results agree with the findings and hypotheses put forward by Gram et al, who suggested that CPBrelated hemorrhage is likely to be due to local hyperfibrino-

z

2

TT

800

30

---I +it -...

600

:

I :

:

1+5***

20

,’ ,’

:

10

XfSD

12

Hoa :P >0.05

H0b

: P> 0.05

3

4

5

/Hoc: P ~0.001

1 HOa:P>0.05

H0b

: P> 0.05

I Hoc:P


Hoa:

2

3

4

P > 0.05 I H,k:P

H0b:P>0.05

5 < 0.001

Fig 6. AT-III, TAT, and PMNelastase-u,-proteinase inhibitor complex at different sampling times. Statistical details are defined in the legend to Fig 1. Dashed lines indicate upper (.-.-.-.-.I and lower (____________) limits of normal range.

MARX ET A!.

472

b-Thromboglobulin

Platelet count 210 0 Control ??Aprotlnin

inI 180

I

PF4

125

100

u ml

!!J ml

100

80

150 60

120 90 60 25 30

x,x(SDF) t’

0 Sample: 1

.fl_ *s.- **I)

0

2

3

4

5

HOa : P > 0.05 / HOc: P < 0.001 HOb : P > 0.05

1

2

3

4

5

1

HOa : P < 0.01 / HOC, : P < 0.001 Hob : P > 0.05 / Hoc2 : P < 0.001

lysis.48 The demonstrable changes that take place with the fibrinolysis factors in the circulating blood occur mainly in the first half of CPB, whereas the most marked increase in degradation products does not occur until later.“” Local activation of fibrinolysis is more likely to be due to opening the thorax than to operating on the heart because the heart does not contain fibrinolysis activators.@ However, the changes in the fibrinolytic enzymes border on the normal. Thus, it is impossible to demonstrate either marked systemic hyperfibrinolysis in the control group or complete suppression of intrinsic fibrinolysis in the aprotinin gro~p.~ Because thrombin formation is inhibited by heparin, intraoperative hemostasis is reduced during CPB, and enhanced local fibrinolysis shifts the balance in the direction of a greater bleeding tendency. The inhibition of this in situ hyperflbrinolysis by aprotinin may reestablish the balance in hemostasis and thus reduce blood loss during and after operation. The well-known in vitro effect of aprotinin on the kallikrein-kinin system” was not observed at the particular measurements in this study. The kallikrein inhibitor potential did increase considerably when aprotinin was given, but it was not possible to demonstrate a reduction in kallikreinlike activity. Thus, it is unlikely that the coagulation system, fibrinolysis, or the complement cascade are affected in this way. The aprotinin concentration produced by this therapeutic regimen may be too low to have such an effect.5’-53 This investigation also confirmed that elastase was released during CPB, indicating distinct stimulation of neutrophils.

2

HOa P > 0.05 Hot,. P > 0.05

3

4

5

/ HOc: P < 0 001

Fig 7. Platelet count, B_thromboglobulin, and PF 4 at diierent sampling times. Statistical details are defined in the legend to Fig 1. Dashed lines indicate upper (.-.-.-.-.) and lower (-----------) limits of normal range.

This effect was not prevented by the dosage of aprotinin used in this study.40,54 It was also demonstrated that, at the dose given and considering its short half-life time, aprotinin did not reverse or outlast the action of heparin. Therefore, the positive effect on hemostasis is not likely to be achieved at the expense of a greater risk of thrombotic complications, such as an enhanced rate of coronary reocclusions. This type of complication has not been reported in clinical studies 37,38.40,55 Recent studies have aimed to explain the beneficial action of aprotinin primarily in terms of its stabilizing effect on platelets. Aprotinin is thought to prevent excessive stimulation of platelets without impairing their function at the site of bleeding.i6~‘7~55~57 However, the present study found only a slight effect on the release of P-thromboglobulin at a very early stage of the operation. This agrees with a finding of Reuter,j6 who assumed that platelets are affected by the anesthesia drugs. There was subsequently no discemible difference, a finding confirmed by the PF 4 curve. The present study was unable to demonstrate that aprotinin causes any relevant inhibition of platelet stimulation to the extent that this is reflected by an increase in PF 4 and P-thromboglobulin. A study by van Oeveren et al has shown that aprotinin inhibits the plasmin-related degradation of the GP lb receptor in platelets.” This would suggest that the possible effect of aprotinin on platelets is due to its antifibrinolytic action.

REFERENCES 1. Royston D, Taylor KM, Bidstrup BP, et al: Effect of aprotinin on need for blood transfusion after repeat open heart surgery. Lancet 2:1289-1291, 1987 2. Dale J, Lilleaasen P, Erikssen J: Hemostasis after open heart surgery with extreme or moderate hemodilution. Eur Surg Res 19:339-347, 1987 3. Harker LA: Bleeding after cardiopulmonary bypass. N Engl J Med 22:1446-1447, 1986 4. Kalmar P, Krebber HJ, Pokar H, et al: Bioadhesives in

cardiac and vascular surgery. Thorac Cardiovasc Surg 30:230-231. 1982 5. Tilsner V, Reuter H, Kalmar P, et al: Beeinflussung der Blutgerinnung durch Fremdobefilchen bei extrakorporaler Zirkulation, in Bliimel G, Haas S (eds): Mikrozirkulation und Prostaglandinstoflivechsel. Stuttgart, Germany, Schattauer, 1981, pp 199-202 6. Bagge L, Lilienberg G, Nystrom SO, et al: Coagulation, fibrinolysis and bleeding after open heart surgery. Stand J Thorac Cardiovasc Surg 20:151-160, 1986

EFFECTS OF APROTININ

IN CARDIAC SURGERY

7. Broonstra PW, van Imhoff GW, Eysman L, et al: Reduced platelet activation and improved hemostasis after controlled cardiotomy suction during clinical membrane oxygenator perfusions. J Thorac Cardiovasc Surg 89:900-906,198s 8. Cella G, Vittadello 0, Gallucci V, et al: The release of 8-thromboglobulin and platelet factor 4 during extracorporeal circulation for open heart surgery. Eur J Clin Invest 11:165-169, 1981 9. Davies GC, Sobel M, Salzman EW: Elevated plasma fibrinopeptide A and thromboxane B, levels during cardiopulmonary bypass. Circulation 61:808-814,198O 10. Harker LA, Malpass TW, Branson HE, et al: Mechanism of abnormal bleeding in patients undergoing cardiopulmonary bypass: Acquired transient platelet dysfunction associated with selective alpha-granule release. Blood 56:824-834, 1980 11. Holloway SD, Summaria L, Sandesara J, et al: Decreased platelet number and function and increased fibrinolysis contribute to postoperative bleeding in cardiopulmonary bypass patients. Thromb Haemost 59:62-67,1988 12. McKenna R, Bachmann F, Whittaker B, et al: The hemostatic mechanism after open heart surgery. II. Frequency of abnormal platelet function during and after extracorporeal circulation. J Tborac Cardiovasc Surg 70:298-308,1975 13. Mezzano D, Aranda E, Urzua J, et al: Changes in platelet 8-thromboglobulin, fibrinogen, albumin, 5_hydroxyttyptamine, ATP, and ADP during and after cardiopulmonary bypass surgery. Am J Hematol22:133-142,1986 14. Mohr R, Martinowitz U, Golan M, et al: Platelet size and mass as an indicator for platelet transfusion after cardiopulmonary bypass. Circulation 74:153-158,1986 15. Wenger RK, Lukasiewicz H, Mikuta B, et al: Loss of platelet fibrinogen receptors during clinical cardiopulmonary bypass. Thorat Cardiovasc Surg 97:235-239,1989 16. Zilla P, Faso1 R, Groscurth P, et al: Blood platelets in cardiopulmonary bypass operations. J Thorac Cardiovasc Surg 97:379-389,1989 17. Dietrich W, Barankay A, Wendt P, et al: Fibrinolysis caused by cardio-pulmonary bypass and shed mediastinal blood retransfusion-Is it of clinical relevance? Adv Exp Med Biol 240:405-410, 1988 18. Bick RL: Hemostasis defects associated with cardiac surgery, prosthetic devices, and other extracorporeal circuits. Semin Thromb Hemost 11:249-280,1985 19. Kladetzky RG, Popov-CeniC S, Biittner W, et al: Studies of fibrinolytic and coagulation factors during open heart surgery. I. Fibrinolytic problems during open heart surgery with ECC. Thromb Res 7:579-588,1975 20. Kongsgaard UE, Smith-Erichsen N, Geiran 0, et al: Changes in the coagulation and fibrinolytic systems during and after cardiopulmonary bypass surgery. Thorac Cardiovasc Surg 37:158162,1989 21. Kucuk 0, Kwaan HC, Frederickson J, et al: Increased fibrinolytic activity in patients undergoing cardiopulmonary bypass operation. Am J Hematol23:223-229,1986 22. Mammen EF, Koets MH, Washington BC, et al: Hemostasis changes during cardiopulmonary bypass surgery. Semin Thromb Hemost 11:281-292,1985 23. Stibbe J, Kluft C, Brommer EJP, et al: Enhanced fibrinolytic activity during cardiopulmonary bypass in open-heart surgery in man is caused by extrinsic (tissue-type) plasminogen activator. Eur J Clin Invest 14:375-382,1984 24. Tilsner V, Pokar H, Doring V, et al: Nichtkardiale Risikofaktoren in der Herzchirurgie-Gerinnungssystem. Z Kardiol 79:H7, 1990 (suppl 1)

473

25. Klauser RJ: Induction of Hageman factor dependent pathway in human plasma by polyanions. Haemostasis 18:S2,1988 26. Philapitsch A, Popov-CeniC S: Pilotuntersuchungen von Inhibitoren, Inhibitionswirkungen und Aktivierbarkeiten in Plasmen wlhrend der Herz-Lungen-Maschine, in Bliimel G, Haas S (eds): Mikrozirkulation und Prostaglandinstotfwechsel. Stuttgart, Germany, Schattauer, 1981, pp 221-231 27. Wachtfogel YT, Harpel PC, Edmunds LH, et al: Formation of Ci, -CT-inhibitor, kallikrein-CT-inhibitor, and plasmin-cu,antiplasmin-inhibitor complexes during cardiopulmonary bypass. Blood 73:468-471,1989 28. Hind CRK, Griffin JF, Pack S, et al: Effect of cardiopulmonary bypass on circulating concentrations of leukocyte elastase and free radical activity. Cardiovasc Res 22:37-41, 1988 29. Riegel W, Spillner G, Schlosser V, et al: Release of granulocyte proteins during cardiopulmonary bypass: Effect of different pharmacological interventions. Adv Exp Med Biol240:391397,1988 30. Mammen EF: Natural proteinase inhibitors in extracorporeal circulation. Ann NY Acad Sci 146:754-762, 1968 31. Gans H, Subramanian V, John S, et al: Theoretical and practical (clinical) considerations concerning proteolytic enzymes and their inhibitors with particular reference to changes in the plasminogen-plasmin system observed during assisted circulation in man. Ann NY Acad Sci 146:721-736,1968 32. Hack G, Kirchhoff PG, Popov-CeniC S, et al: Aprotinin bei Operationen am offenen Herzen. Medwelt 34:726-731,1983 33. Popov-CeniC S, Urban A, Kulzer R: Untersuchungen zur Blutungsursache wiihrend und nach Operationen mit der HerzLungen-Maschine bei Kindern mit zyanotischen und azyanotisthen angeborenen Herzfehlern und deren prophylaktische Behandlung, in Bltimel G, Haas S (eds): Mikrozirkulation und Prostaglandinstoffwechsel. Stuttgart, Germany, Schattauer, 1981, pp 203-210 34. Popov-CeniC S, Murday H, Kirchhoff PG, et al: Anlage und zusammenfassendes Ergebnis einer klinischen Doppelblindstudie bei aorto-koronaren Bypass-Operationen, in Dudziak R, Reuter HD, Kirchhoff PG, Schumann F (eds): Proteolyse und Proteinaseninhibition in der Herz- und Gefapchirurgie. Stuttgart, Germany, Schattauer, 1985, pp 171-186 35. Urban AE, Brecher AM, Popov-CeniC S: Blutungen nach intrakardialen operativen Eingriffen im Sauglingsalter: Klinische Relevanz und perioperative Therapie, in Bliimel G, Haas S (eds): Mikrozirkulation und Prostaglandinstolbvechsel. Stuttgart, Germany, Schattauer, 1981, pp 275-278 36. van Oeveren W, Jansen NJG, Bidstrup BP, et al: Effects of aprotinin on hemostatic mechanism during cardiopulmonary bypass. Ann Thorac Surg 44:640-645,1987 37. Bidstrup BP, Royston D, Taylor KM: Reduction in blood loss and blood use after cardiopulmonary bypass with high dose aprotinin (Trasylol). J Thorac Cardiovasc Surg 97:364-372, 1989 38. Dietrich W, Barankay A, Dilthey G, et al: Reduction of homologous blood requirement in cardiac surgery by intraoperative aprotinin application-Clinical experience in 152 cardiac surgical patients. Thorac Cardiovasc Surg 37:92-98, 1989 39. Dietrich W, Jochum M, Schramm W, et al: Reduction of homologous blood requirement in cardiac surgery using high dose aprotinin. Anesthesiology 71:A7,1989 (suppl) 40. Fraedrich G, .Weber C, Bernard C, et al: Reduction of blood transfusion requirement in open heart surgery by administration of high doses of aprotinin-Preliminary results. Thorac Cardiovasc Surg 37:89-91, 1989 40a. Stief TW, Lenz P, Becker U, et al: Determination of plasminogen activator inhibitor (PAI) capacity of human plasma

474

in presence of oxidants:A novel principle. ‘Thromb Res SO:S5Y-573, 1988 41. Morrison DF: Multivariate statistical analysis (ed 2). New York, NY, McGraw-Hill International Editions, 1978, pp 205-212 42. Wilkinson L: SYSTAT: The System for Statistics. Evanston, IL. SYSTAT. 1989, pp 254-280 43. Harke H. Rahman S: The effect of aprotinin administration on the intraoperative histamine release and haemostatic disorders. Adv Exp Med Biol240:509-514, 1988 44. Himmelreich G, Muser M, Steffen R, et al: Different aprotinin applications influencing hemostatic changes in orthotopit liver transplantation (OLT). Presented at the 35th Annual Meeting of the GTH (Gesellschaft fiir Thromboseund Haemostaseforschung), Gottingen, Germany 1991. Al 18 45. Popov-Cenic S, Kirchhoff PG, Hack G, et al: Prophylaktische Behandlung mit Antiplasmin (Aprotinin) vor, wahrend und nach Operationen am offenen Herzen bei Envachsenen. Klinische Bedeutung und ein neues Behandlungskonzept, in Bliimel G, Haas S (eds): Mikrozirkulation und Prostaglandinstoffwechsel. Stuttgart, Germany, Schattauer, 1981, pp 211-219 46. Mombelli G. Monotti R, Haeberli A. et al: Relationship between fibrinopeptide A and fibrinogen/fibrin fragment E in thromboembolism, DIG and various non-thromboembolic diseases. Thromb Hemost 5X:758-763. 1987 47. Woodhams BJ. Kernoff PBA: Problems with antisera specificity in immunoassays for human fibrinopeptide A. Thromb Res 42:689-693. 19% 48. Gram J, Janetzko T, Jespersen J, et al: Fibrinolysis during extracorporeal circulation, in Bruhn HD (ed): 6. KongreB der Gesellschaft fur Thromboseund Hamostaseforschung. Stuttgart, Germany, Schattauer. 1990, pp 158-161 49. Greul W: Untersuchungen zur Bedeutung der lokalen Fibrinolyse fur die Entstehung hamorrhagischer Pericardergisse nach kardiochirurgischen Eingriffen. Habilitationsschrift an der Medizinischen Fakultat Hamburg, 1977. pp 93-95 50. Minale C. Schmitz H, Behrendt W, et al: Hyperfibrinolyse wahrend Operationen mit extrakorporaler Zirkulation, in Dudziak R, Reuter HD, Kirchhoff PG, Schumann F (eds): Proteolyse und Proteinaseninhibition in der Herz- und GefaBchirurgie. Stuttgart, Germany, Schattauer, 1985, pp 4X-57

MARX Fi Ai

5 I. Fritz If, Wunderer G: Biochcmratr\ and application< I*I aprotinin, the kallikrein inhibitor from hovine organs. Drug Rex 33479494, I983 52. Philapitsch A. Murday H. Ecrpov-C’emc S: Das Kalhkrcrn System: Aktivierungs- und Inhibitorpotential wahrend des extrakorporalen Kreislaufs in ciner Placebo-Aprotinin-Cl-Inhibitor Studie. in Dudziak R. Reuter HD. Kirchhotf PG. Schumann F (eds): Proteolyse und Proteinaseninhibition m der Iterr- und Gefapchirurgie. Stuttgart. Germany. Schattauer, 19X5, pp 201-30X 53. Fuhrer G, Heller W. Hoffmeister H: Das Verhalten con Plasma-Prakallikreini-Kallikrein hei Patienten mit aorto-koronarer Bypass-Operation unter Anwendung zweier verachiedencr Aprotinin-Dosierungsschemata. in Dudziak R. Reuter HD. Kirchhoff PC, Schumann F (eds): Proteolysr und Proteinaseninhibition in der Herz- und Geflpchirurgie. Stuttgart. Germany, Schattauer. 1985, pp 255-25’) 54. Lestienne P, Bieth JG: The inhibition of human leukocyte elastase by basic pancreatic trypsin inhibitor. Arch Biochem Biophys lYO:?iX-360, lY7X 55. Hannekum A. Reuter HD. Dalichau H. et al: Anlagc und zusammenfassendes Ergebnis einer klinischen Doppelblindstudie bei Operationen am offenen Herzen. Einfluf3 von Aprotinin auf Thrombozytenzahl und -funktion. in Dudziak R, Reuter HI>. Kirchhoff PC;, Schumann F (eds): Proteolyse und Proteinaseninhibition in der lierz- und Gefafichirurgie. Stuttgart. Germany. Schattauer, 1985, pp 222-233 56. Reuter HD: Zum Verhalten der Thrombozyten in Gcgenwart von Aprotinin. in Dudziak R, Reuter HD, Kirchhotf PCi. Schumann F (eds): Proteolyse und Proteinaseninhibition in dcr Herz- und Gefapchirurgie. Stuttgart. Germany. Schattauer, 1985. pp 236-242 57. Popov-CeniC S, Murday H: Das Verhalten der Thronbozyten bei Herzoperationen unter Aprotininund Cl-EsteraseInhibitor-Therapie, in Dudziak R. Reuter HD, Kirchhoff PG. Schumann F (eds): Proteolyse und Proteinaseninhibition in der Herz- und Gefapchirurgie. Stuttgart. Germany, Schattauer. 1985. pp 1X7-193 5X. van Oeveren W, Eijsman L. Roozendaal KJ. et al: Platelet presevation by aprotinin during cardiopulmonary bypass. Lancet 2~644. 1988