Mechanism of the preserving effect of aprotinin on platelet function and its use in cardiac surgery

Mechanism of the preserving effect of aprotinin on platelet function and its use in cardiac surgery The deficiency of platelet function is the main defect of the hemostatic mechanism during cardiopulmonary bypass, which greatly exacerbates the postoperative bleeding complications. In this study, we assessed, from basic and clinical perspectives, the mechanism of relieving platelet damage by means of aprotinin. In vitro research confirmed that the addition of urokinase (40 U jml) to platelet-rich plasma and the addition of plasmin (0.3 U jml) to washed platelets made ristocetin-induced agglutination decline to 31.6% and 38.5 % of control values, respectively. The extent of decline was positively correlated with the concentration of urokinase and plasmin. In addition, the platelet membrane glycoprotein Ib decreased to 76.4 % of control value. With the addition of urokinase or plasmin to aprotinin-pretreated platelet-rich plasma or washed platelets, the changes in agglutination are not statistically significant and the decrement in glycoprotein Ib is much less marked. Further in vivo research revealed that cardiopulmonary bypass caused a decrease in plasma arantiplasmin, indicating the fibrinolytic system activation. Meanwhile, ristocetin-induced agglutination decreased to 39.6 % and platelet glycoprotein Ib decreased to 50 % of preoperative values. However, with the administration of aprotinin, plasma a2-antiplasmin during cardiopulmonary bypass did not change; platelet agglutination was improved, platelet glycoprotein Ib was preserved, and this consequently resulted in 46 % lower blood loss after the operation. The results showed that fibrinolysis impaired platelet function, and this effect may be associated with the hydrolysis of glycoprotein lb. Fibrinolytic activation occurred during cardiopulmonary bypass and contributed to postoperative platelet dysfunction to a great extent. Aprotinin may inhibit fibrinolysis during cardiopulmonary bypass and thus relieve the platelet damage and improve the postoperative hemostatic mechanism. (J THoRAc CARDIOVASC SURG 1993;106:11-8).

Huiming Huang, PhD, Wenxiang Ding, MD, Zhaokang Su, MD, and Weizhong Zhang, MB, Shanghai, China

Bleeding is one of the major complications after cardiopulmonary bypass (CPB). In pediatric cardiac surgery, this problem is even more serious because the infant and young child have less blood volume and are more sensitive to blood loss. Severe bleeding leads to disorders in many systems, such as the immunologic, metabolic, and endocrinologic systems. Unfortunately, extensive homologous transfusion may transmit viral hepatitis and acquired immunodeficiency syndrome. Platelet dysfunction during CPB, which may contribFrom the Department of Pediatric Thoracocardiac Surgery, Xinhua Hospital, Shanghai Second Medical University, Shanghai, China. Received for publication Jan. 17. 1992. Accepted for publication July 27, 1992. Address for reprints: Huiming Huang. PhD. Department of Pediatric Thoracocardiac Surgery, Xinhua Hospital. Shanghai Second Medical University, 1665 Kong Jiang Road. Shanghai 200092. China. Copyright

1993 by Mosby-Year Book. Inc.

0022-5223/93 $1.00 + .10

12/1/41329

ute to postoperative bleeding, has been reported by many authors. 1-3 Some drugs have been investigated to resolve this problem.v 5 In the past few years, high doses of aprotinin have been used in several cardiac centers, and impressive results were obtained.v '? We report our preliminary experimental results and assess the mechanism of aprotinin's effect on platelet function from basic and clinical perspectives. Materials and methods In vitro study Platelet-rich plasma (PRP). Venous blood of healthy donors was drawn into 3.8% sodium citrate. Nine parts blood were mixed with one part anticoagulant. The blood was centrifuged at 150g for 10 minutes at room temperature, and upper two-thirds platelet-rich plasma (PRP) was collected. The remaining blood was centrifuged at 1500g for another 10 minutes and platelet-poor plasma was collected. Washed platelets. PRP was mixed with an equal volume of washing buffer (Tris, 10 mmol/L; NaC!. 0.15 mol/L; ethylenediamine tetraacetic acid, 2 mmol/L; pH 7.4) and this mix-

11

The Journal of Thoracic and Cardiovascular Surgery July 1993

I 2 Huang et al.

ture was centrifuged at 1500g for 10 minutes at room temperature. The platelet pellet was then resuspended and washed an additional three times. The pellet was finally suspended in the same buffer at a concentration of 150 X 109 jL. Effect of urokinase and aprotinin on ristocetin-induced agglutination ofplatelets in PRP. PRP was divided into three tubes. Urokinase (Shanghai Biological Pharmaceutical Factory, Shanghai, China) was dissolved in Tris-buffered saline solution, added to tube 2, and incubated at room temperature for 10 minutes. Final concentration was 40 U jml. The same concentration of urokinase was added to tube 3, which had been previously incubated with aprotinin (250 KIU jm!) for 5 minutes. Tube I served as the control and was handled in a similar fashion without the addition of urokinase and aprotinin. Platelet agglutination was initiated by an additional 50 JLl of ristocetin (Chrono-Log 380; Chrono-Log Inc., Havertown, Pa.) and the sample was stirred at 37° C in a Chrono-Log 560 dual-channel aggregometer. The final concentration of ristocetin was 1.0 mgjml. Plasminogen, plasmin, fibrin(ogen) degradation products (FDPs), and von Willebrand factor (vWF) were measured with immunodiffusion, a spectrophotometer at 600 nm, reverse hemogglutination, and rocket immunoelectrophoresis, respectively. All the research reagents were donated by Shanghai Research Institution of Biological Products. Effect ofplasmin and aprotinin on ristocetin-induced agglutination ofwashed platelets. Washed platelets were divided into four tubes. Tube I served as the control. Plasmin (0.3 U jml; Boehringer Inc., Mannheim, Germany) was added to tube 2 and incubated for 10 minutes at room temperature. The same concentration of plasmin was also added to tube 3 and tube 4, which had been incubated with aprotinin (250 KIU jm!) before and after plasmin treatment, respectively. Agglutination was initiated by the addition of ristocetin (1.0 mg/rnl) and factor VIII-vWF (I U jm!), which was sufficient to ensure agglutination. We further divided another sample of washed platelets into nine tubes. Tube I served as the control. Plasmin, ranging from 0.15 Ujml to 0.50 Ujml, was added to tubes 2 through 9. Agglutination was initiated by the same method as previously mentioned. Effect of urokinase and aprotinin on ristocetin-induced agglutination of washed platelets. Washed platelets were divided into three tubes. Tube I served as the control. Urokinase (400 U jm!) was added to tube 2 and incubated for 10 minutes at room temperature. The same concentration of urokinase was added to tube 3, which had been incubated with aprotinin (250 KIU jm!) previously. Ristocetin-induced agglutination was initiated by the same method. Effect ofurokinase and aprotinin on platelet membrane glycoprotein lb. Citrated blood was divided into three tubes. Tube I served as the control. Urokinase (40 U jm!) was added to tube 2 and the same concentration of urokinase was added to tube 3, which had been incubated with aprotinin (250 KIU jm!) previously. After 10 minutes of incubation with urokinase, the blood was mixed with an equal volume of 0.4% glutaraldehyde. The fixed blood containing 2.5 X 106 platelets was then collected and washed with washing buffer (BSA, 3.5 gmjL; NaCl, 138 mmoljL; NaHC03, 29 mmoljL; glucose, 1.0 gmjL; NaH2P04, 0.36 mrnol/L; pH 7.4). The pellet was then mixed with 50 JLl of iodine-labeled monoclonal antibody SZ-2, donated by Dr. C. G. Ruan of Suzou Medical College, and total counts per minute was measured. After 30 minutes of incubation, the blood was then washed with the same washing buffer

three more times and then bound counts per minute was measured. We calculated the molecular number of platelet membrane glycoprotein Ib (GPIb). The changes of GPIb were expressed as percentage of control value. In vivo study. We selected 30 patients undergoing CPB for this trial. Congenital heart disease was diagnosed in all patients. They were randomized into two groups: 15 patients served as the control group, and the other 15 patients were assigned to aprotinin treatment. The mean ages of the control and aprotinin groups were 5.8 ± 2.3 and 5.9 ± 3.2 years, and the mean weights were 16.2 ± 4.8 and 16.8 ± 5.2 kg, respectively. No statistical difference existed between the two groups for either age or body weight (Table 0. Conventional methods of anesthetic treatment and CPB were used in both groups. Anesthesia was induced with fentanyl citrate (7.5 JLgjkg). Heparin was administered at an initial concentration of 2 mgjkg through the right atrial appendage and 20 mg through the prime. Activated clotting time was maintained for more than 450 minutes throughout CPB. A membrane oxygenator (AL-08; Fudang Inc., Shanghai; China) and roller pump (Sarns-7400; Sarns Inc.j3M Health Care, Ann Arbor, Mich.) were used in the extracorporeal circuit. Blood flow was maintained at 80 to 120 mljkg. Multidose cold crystalloid cardioplegic solution was used for myocardial preservation. Systemic hypothermia (24° to 26° C) was maintained during aortic crossclamping. After discontinuation of CPB, heparin was neutralized with protamine sulfate. The remaining blood in the bypass circuit was returned to the patient through the external jugular vein. CPB lasted 87.8 ± 18.3 minutes in the untreated group versus 106.7 ± 35.7 minutes in the aprotinin-treated group, and the aortic crossclamping durations were 43.1 ± 16.2 minutes versus 53.8 ± 24.1 minutes, respectively. No significant difference was found between the two groups for either bypass duration or aortic crossc1amping duration. After induction of anesthesia, an infusion of aprotinin (3.5 to 5.0 X 104 KIU jkg) was given to treated patients for 20 minutes. Then, a continuous infusion of 3.5 to 5.0 X 103 KIU jkg per hour was maintained until the end of the operation. Another bolus of 65 to 75 X 104 KIU was added to the prime solution before the start of CPB. Blood samples were taken periodically: after induction of anesthesia, before CPB (injected with heparin), 30 minutes after the start of CPB, at termination of CPB, 20 minutes after protamine administration, and 2 hours after operation. Hematocrit values and platelet counts were measured with a Cell-Dyn 900 (DuPont Company, Wilmington, Del.) blood cell analyzer. The platelet agglutination and the platelet membrane GPIb were determined by the same method as was used in the in vitro experiment. Plasma llC2-antiplasmin was measured by ACL-81 0 automatically with the use of a chromogenic substrate (IL Inc., Lexington, Mass.) Statistical analysis. Statistical analysis was performed with Student's t test in all matched groups. Regression statistics were used to confirm plasmin dose-dependent platelet agglutination. A p value of less than 0.05 was considered significant.

Results In vitro study Effect of urokinase and aprotinin on ristocetin-induced agglutination of platelets in PRP. To study the effect of fibrinolysis on platelet function, we added uroki-

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Huang et at.

I3

Table I. Clinical characteristics of patients with and without aprotinin No.

Sex

Age

I 2 3 4 5 6 7 8 9 10 11 12 13 14 15

M M

IOyr2rna 2 yr 2 rna 4 yr 8 rna 2 yr 5 rna 4 yr 5 rna 8 yr 8 rna 8 yr 9 rna 4 yr 7 rna 9 yr 7 rna 4 yr 8 rna 4 yr 2 rna 2 yr 10 rna 3 yr 0 rna 6 yr 0 rna 3 yr 6 rna

I 2 3 4 5 6 7 8 9 10 II 12 13 14 15

F F

Diagnosis

Weight

Operation

Control group

F

M F F

M M M F

M M M M M

M M M M M M M M M F

M M F

26 kg 12 kg 18 kg 23 kg II kg 19 kg 21 kg 15 kg 20.5 kg 12 kg II kg 13 kg 12 kg 14 kg 15 kg Aprotinin-treated group

8 yr 8 rna 7 yr 8 rna 3 yr 5 rna 6 yr I rna 8 yr 0 rna 10 yr 3 rna 5 yr I rna 7 yr 6 rna 2 yr 0 rna 6 yr 5 rna 3 yr 5 rna 7 yr II rna 2 yr 6 rna 5 yr 5 rna 4 yr 6 rna

19 kg 17 kg II kg 21 kg 29 kg 22 kg 15 kg 17 kg 9 kg 16 kg 13 kg 21.5 kg II kg 15.5 kg 15 kg

SV VSD,PH TOF TOF VSD, PH TOF TOF TOF TOF TOF TOF TOF TOF TOF TOF

BD shunt Repair Repair Repair Repair Repair Repair Repair Repair Repair Repair Repair Repair Repair Repair

TOF VSD (sip) TOF VSD, DCRV TOF TOF, PA TOF TOF TOF SV TOF VSD, AI (sip) TOF TOF SV

Repair Reoperation Repair Repair Repair Homograft Repair Repair Repair

Fontan Repair

Rcoperation Repair Repair Fontan

SV. Single ventricle; RD, bidirectional; VSD, ventricular septal defect; PH, pulmonary hypertension; TOF, tetralogy of Fa llot: sip, status post; DORV, double outlet right ventricle; PA, pulmonary atresia; AI, aortic insufficiency.

Table II. Effect ofplasmin and aprotinin on fibrinolysis and vWF Items Pg (rng/L) Prn(U) FDPs (",g/rnl) vWF(%)

Tube A 186.6 ± 18.l ± 1.4 ± 89.2 ±

68.8 5.3 0.4 31.2

Tube B 157.9 ± 60.6* 21.7 ± 4.6*** 32.9 ± 19.8** 115.1±41.7*

Tube C 173.3 17.5 3.2 70.0

± ± ± ±

60.0 5.0 5.3 28.1

Pg. Plasminogen, Pm, plasmin.

nase to PRP. The addition of extrinsic urokinase (40 U Iml) made the ristocetin-induced agglutination decline to 31.6% of control value, which was accompanied by parallel changes of fibrinolytic parameters: Plaminogen decreased by 16%, plasmin increased by 20%, and FDPs increased to about 24 times the control value. However, when we added the same concentration of urokinase to aprotinin-pretreated PRP, the agglutination and fibrinolytic parameters did not change statistically (Table II, Fig. I).

We have further observed the change of vWF after urokinase and aprotinin treatment to see whether the vWF deficiency existed. As a result, the plasma vWF in the urokinase-treated group increased by 30%, whereas that in the aprotinin-pretreated group did not differ statistically from control value (Table II). Effect ofplasmin and aprotinin on ristocetin-induced agglutination of washed platelets. The addition of plasmin (0.3 U 1m!) to washed platelets caused the ristocetininduced platelet agglutination to decline to 38.5% of the

The Journal of Thoracic and Cardiovascular Surgery July 1993

Huang et al.

I4

80

.

~

...

co

.-..

70

120

T

T

60

.. .... ~

60

-....... .. -.

80

....

20

II

•••

30

f

20

~ ~

10

Group A

Group B

Group C

Fig. 1. Effectof urokinaseand aprotinin on ristocetin-induced platelet agglutination. PRP was divided into three groups of tubes, notedas Group A, Group B, and Group C. Tube A served as control;tube B wasincubated with urokinase( 40 U jml); tube C was incubated with same concentration of urokinasebut was previously treated with aprotinin (250 KIU jm!). Each value represents mean of 12 determinations. ***p < 0.001.

80

60 40

o

0. 16

O. 20

O. 26

O. 30

0. 36

O. 40 O. 46 O. 60 Plasmin (u/mll

Fig. 3. Correlation between plasmin concentration and ristocetin-induced platelet agglutination. Washed platelets were dividedinto ninetubes.Tube I servedas control;tubes 2 through 9 were incubated with plasmin (from 0.15 to 0.50 U jml). Each point represents mean of two determinations,expressed as percentage of control value. Correlation coefficient is 0.92 (p < 0.001).

control (data not shown). After the concentration was increased to 400 V Iml, agglutination declined to 65.4% of the control value and that of the aprotinin-pretreated group declined slightly to 93.4% of control value. (Fig. 4) .

70

.. 60 -... 60 -.". 40 f-. 2030

~

.. II

co

" 40

~

~ 100

co

•••

•••

~ ~

.... 10 Group A

Group B

Group C

Group D

Fig. 2. Effect of plasmin and aprotinin on ristocetin-induced washed platelet agglutination. Washed platelets were divided into four tubes. Tube A servedas control;tube B was incubated with plasmin (0.3 U jm!); tubes C and 0 were also incubated with same concentrations of plasmin but were treated with aprotinin (250 KIU jm!) before and after plasmin incubation, respectively. Each value representsmean of five determinations. ***p < 0.001. control value. Adding the same concentration of plasmin to aprotinin-pretreated washed platelets reduced agglutination to 88.5% of the control value. However, with the addition of aprotinin to washed platelets after plasmin treatment, agglutination still declined remarkably to about 45.9% of the control value (Fig. 2). Moreover, the decrement of plasmin-induced agglutination was dose dependent (Fig. 3). The correlation coefficient was 0.92 (p < 0.001).

Effect of urokinase and aprotinin on ristocetin-induced agglutination of washed platelets. By adding urokinase (40 V /rnl) to washed platelets, the ristocetininduced agglutination was almost the same as that of

Effect of urokinase and aprotinin on platelet GPlb. After incubation with urokinase (40 Uyrnl), platelet membrane GPIb decreased to 76.4% ± 10.2% of the control value, whereas the GPlb decreased to 85.7% ± 8.2% ofthe control value when pretreated with aprotinin (Fig. 5). In vivo study Hematocrit values. Hematocrit values decreased after the start of CPB because of hemodilution. No significant differences between two groups were found during CPB or at the end of CPR However, the hematocrit values of aprotinin-treated patients were higher than those of untreated patients after protamine administration and 2 hours after the operation (Fig. 6). Platelets. Platelet counts decreased in both groups during CPB because of hemodilution, as did hematocrit values. After the termination of CPB, the platelet count increased gradually, but no significant difference could be found between the two groups in either matched sample (Fig. 7). Ristocetin-induced platelet agglutination. Platelet agglutination induced by ristocetin declined remarkably in the untreated group during CPB and declined to a greater degree after protamine administration and 2 hours after the operation. In the aprotinin-treated group, agglutination was significantly higher than that in the untreated group during CPB, although a slight decrease still existed, and returned to the preoperative value 2 hours after the operation (Fig. 8).

The Journal of Thoracic and Cardiovascular Surgery Volume 106, Number 1

Huang et al.

80

80

~ 70 co 60

70

.... a

....

::

T

T

r

60

';'40 .,

*

co

.

30

==

Group B

120 .... 100

...

T

80

T

...

...

Pre-II'

Hepar I.

I7B 100Ia

EDd-17B

ProiEla Pod-II' 2i

Fig. 6. Hematocrit value before,during, and after CPB. Thirty patients were randomized into two groups. Fifteen cases served as control whereas the other 15 cases were assigned to aprotinin treatment. Conventional methods of anesthetic treatment and CPB were used in both groups, and high-dose aprotinin was administered to patients in treated group. Pre-op, Preoperative; CPS, cardiopulmonary bypass; h, hours. *p < 0.05; **p < 0.01.

~18

-

-.

40 20 Group A

Group B

. - - Control ....-- Treated

~H

~12 :;: 10 co .. 8

co ., 60

.,

20

20 , . . . - - - - - - - - - - - - - - - - - - . , ;::; 18

~

....,

40

Group C

Fig. 4. Effectof urokinaseand aprotinin on ristocetin-induced washed platelet agglutination. Washed platelets were divided intothree tubes. Tube A served as control; tube 2 was incubated with urokinase(400 U Iml); tube C was incubated with same concentration of urokinase but was previously treated with aprotinin (250 KIU Iml). Each value representsfive determinations. *p < 0.05.

co

60

10 Group A

... ...

~Treated

Ii

.. .... 10

....

-Control

10

.... ao ~

.: 20

.:.,

..

~

I5

Group C

Fig. 5. Effect of urokinase and aprotinin on platelet membraneGPlb (for procedure see Fig. I). After fixation with glutaraldehyde, GPlb was determined with monoclonal antibody SZ-2 by means of radioimmunoassay. Each value represents mean of five determinations expressedas percentage of control value. *p < 0.05.

Platelet membrane GPIb. We detected a 50% reduction of molecular number of GPIb in samples obtained from untreated patients during CPB, and it remained lower after protamine administration and 2 hours after the operation. The GPIb in aprotinin-treated patients remained unchanged in all samples. Significant differences existed between the two groups either during CPB or soon after the operation (Fig. 9). 112 -Antiplasmin. lXz-Antiplasmin concentration decreased remarkably during CPB in untreated patients. After termination of CPB, it increased gradually but remained lower than the preoperative value. In aprotinintreated patients, it remained unchanged during and soon after CPB. Differences were statistically significant between the two groups (Fig. 10).

.:

6

~ ....

2

4 Pre-II'

Hepar 10 I7B biD

lad-CPR

ProtEID P08i-1I' Ii

Fig. 7. Platelet count before, during, and after CPB (for procedure and for abbreviations see Fig. 6).

Postoperative bleeding. Drainage in the first 3 postoperative hours was 7.56 ± 4.68 mljkg in untreated patients and 3.63 ± 2.77 mljkg in aprotinin-treated patients; the total drainage in 18 postoperative hours was 17.42 ± 6.97 mljkg and 9.62 ± 6.03 mljkg, respectively. As the data show, the bleeding of aprotinin-treated patients was much less than that of untreated patients (Fig. II).

Discussion The bleeding complication after CPB is mostly attributed to platelet dysfunction, which is the main defect of hemostatic mechanism caused by CPB. I-3 CPB is a nonphysiologic procedure, and many factors may contribute to platelet injury. The contact of the platelet with non biologic materials, shearing stress, suction of the surgical field, and activation of the complement system during CPB all might injure the platelet to some

I6

The Journal of Thoracic and Cardiovascular Surgery July 1993

Huang et al.

160 r - - - - - - - - - - - - - - - - - - - ,

100

. ..-

~

co

-.:

-=

90 80 70 60

--Control ~Treated

*

~ 60 :: 40

H

**

*

~ 20 ... 10

IIep.rin

CPB albin

£nd-CPB

60 40

- - - Control -Treated

Pre-Ill

IIeparln CPB 81bln £nd-CPB

Prot.ln Pod-(JI 211

Fig. 10. Plasma alpha 2-antiplasmin before, during, and after CPB (for procedure and abbreviations see Fig. 6). ***p < 0.001.

Oil

-Control ~Treated

..

:;. 26

"

A

.... 16 ...1:. 10 -... 6 ::. 20

i

h

:- 100 80 lOa 80 ~

.:

~

!

30,....-------------------,

200 ~ 180 ~ 160 .~ 140 -: 120

~

80

Prot.ln POd-lI' 211

Fig. 8. Ristocetin-induced platelet agglutination before, during, and after CPB (for procedure and abbreviations see Fig. 6). *p < 0.05; **p < 0.01.

...

-

***

-;. 100

;: 20 Pre-lI'

***

a 120

...::

. : 30

-.

...

~ 140

o §

cont ro l treat ed

~

40

~

20 Pre-lI'

IIepar in

CPB albin

£nd-CPB

Prot.ln Poit-lI' III

POI t-OP 3h

POlt-OP 18h

Fig. 9. Platelet GPlb before, during, and after CPB (for procedureand abbreviations see Fig. 6). *p < 0.05; **p < 0.01.

Fig. 11. Drainage of two groups after CPS (for procedure and abbreviations see Fig. 6). *p < 0.05.

extent. The fibrinolysis is another significant damaging factor. Many authors I3, 14 have studied fibrinolysis during CPB and considered it a primary hyperfibrino(geno)lytic syndrome. During CPB, the level of the plasma tissue plasminogen activator, a potent plasminogen activator, increased remarkably. I5, 16 Furthermore, the factor XII pathway of fibrinolysis was also activated, 17 Consequently, a large amount of plasminogen was converted into plasmin, and FDPs were produced. However, some studies achieved different results, and severe fibrinolysis during CPB could not be detected in their experiments.s 18 cx2-Antiplasmin is an important plasma factor in the regulation of the fibrinolytic system. As the fibrinolytic system activated, the amount of plasmin in plasma increased rapidly. Plasmin is unstable and easily combined with cxrantiplasmin. The plasmin-antiplasmin component has no biologic activity, and thus the damaging effect of excessive plasmin was alleviated. At the same time, plasma cxrantiplasmin was consumed and decreased. The concentration of plasma cx2-antiplasmin can be used as a very sensitive indicator of fibrinolytic activity. In our results, plasma cx2-antiplasmin decreased

to about 30% of its preoperative value during CPB, whereas hemodilution was about 50%, which indicated that the fibrinolysis surely exists, to some extent, during CPB. The initial physiologic response to vessel damage is platelet adhesion, which plays an important role in primary hemostasis. In addition to the exposure of subendothelial tissue, the plasma vWF and platelet GPIb also were known to be involved in platelet adhesion. 19-21 Deficiency of either factor leads to platelet dysfunction. Use of ristocetin permits quantitative study of the GPlbvWF reaction and identification of binding domains. Antibodies that block ristocetin-induced binding also block GPIb-vWF-dependent adhesion in the perfusion system, indicating that ristocetin-induced binding is similar to shear-dependent adhesion.F' 23 To explore the mechanism of the effect of fibrinolysis on platelet adhesion, we designed an in vitro experimental model to eliminate the interference of other damaging factors caused by CPB. First, we incubated PRP of normal subjects with urokinase, a plasminogen activator. As a result, the plasminogen level decreased while the plasmin and FDPs levels increased, indicating that the

The Journal of Thoracic and Cardiovascular Surgery Volume 106, Number 1

conversion of plasminogen into plasmin did occur. Also, ristocetin-induced platelet agglutination declined remarkably, showing that the vWF-GPlb reaction had been blocked by urokinase; however, the question remains as to whether the urokinase directly affected the platelets and blocked the reaction. To assess this process, we further incubated washed platelets with plasmin and urokinase to eliminate the interference of the plasma factors. After treatment with plasmin, ristocetin-induced agglutination of washed platelets declined remarkably, and it correlated negatively with the concentration of plasmin, which is similar to the effect of urokinase on PRP. But no such change could be found in washed platelets treated with the same concentration urokinase as in PRP (40 U jm\). As the concentration of urokinase increased to 10 times its original level (400 U jm!), agglutination began to decline, but the extent of decrement was still less marked. These results showed that plasmin reduced the GPlb-vWF reaction directly and the effect of urokinase on PRP may also be caused mainly by plasmin, which increased in PRP after incubation with urokinase. Aprotinin is a proteinase inhibitor. Plasmin, kallikrein, trypsin, and other proteinases are known to be inhibited by aprotinin. 24. 25 The first success with high doses of aprotinin in ePB to reduce postoperative hemorrhage was reported by van Oeveren in 1987. 16 Several authors'': 7. 9 have since reported the same results; the published experiences have been impressive, but the mechanism remains unclear. Generally, the theory that aprotinin protects the circulating platelet during ePB is accepted. In an in vitro study, we incubated PRP and washed platelets with aprotinin and then with urokinase and plasmin, respectively. Ristocetin-induced agglutination was initiated, and no statistically significant changes could be found in either group of samples. The levels of plasminogen, plasmin, and FDPs in PRP also did not differ from control values. These results showed that the conversion of plasminogen into plasmin, which could be induced by urokinase, had been inhibited and the activity of extrinsic plasmin had also been restrained by aprotinin pretreatment. Because of the inhibition of fibrinolytic activity,the platelet adhesive function was preserved perfectly. Furthermore, we assessed whether aprotinin had the same preserving effect on plasmin-pretreated platelets. When aprotinin was added to washed platelets that had been previously incubated with plasmin, agglutination stilldeclined remarkably, in a manner almost identical to that in the group treated with plasmin alone. It seems that the damaging effect of fibrinolysis on platelet function is irreversible and aprotinin has no preserving effect on injured platelets.

Huang et at.

I7

The main effect of aprotinin appears to be related directly or indirectly to platelet preservation at the molecularlevel. 8. 10 After the activation ofthe fibrinolytic system by urokinase, the concentration of vWF in plasma did not decrease but increased, and the amount of vWF that was added to washed platelets was also excessive. Therefore, the reduction of vWF-GPlb reaction is surely not caused by deficiency ofvWF, but it may have some association with the proteinolysis of adhesive receptor GPlb, which can easily be cleaved in the presence of some proteinolytic enzymes. 24-27 Platelet GPlb plays an essential role in platelet adhesion, which is the first step in formation of the platelet hemostatic plug in response to vessel injury. The level of GPlb decreased greatly after treatment with urokinase, and it could be relieved by previous incubation with aprotinin. Aprotinin may prevent GPIb from attaining complete hydrolysis by inhibiting the activity of the fibrinolytic system and consequently improving platelet function. Our in vivo experiment further confirmed that aprotinin has the same effect on Cl'B, During ePB and the early postoperative period in patients treated with aprotinin, the plasma cx2-antiplasmin level was much higher, ristocetin-induced platelet agglutination was much better, and the reduction of platelet membrane GPIb was much less than those in untreated patients. We postulated that platelet membrane GPIb might be cleaved by the plasmin that increased during ePB and aprotinin prevented GPlb from damage by inhibiting the fibrinolysis, thus leading to a better hemostatic mechanism and consequently reducing postoperative blood loss, stabilizing hemodynamics and reducing blood requirement. However, the change of GPlb in our in vitro experiment was not parallel to the degree of reduction of agglutination. We found by chance that platelet GPlb levels increased several times after it was washed with hypotonic washing buffer. As seen under the electron microscope, many hollows exist at the platelet surface, which is the open canalicular system with a diameter of about 50 nm. We can measure molecular numbers of only the GPlb on platelet surface, with radioimmunoassay with monoclonal antibody whereas the GPIb in open canalicular system may not be detected with this method. We postulate that after treatment with urokinase or plasmin, the platelets might be activated and swollen and GPlb in the open canalicular system might be redistributed to the platelet surface after cleavage of GPIb from the platelet surface. Therefore, the measured GPlb molecular number may simply reflect the hydrolytic tendency of GPlb but cannot be considered to be its quantitative equivalent. Further research on the effect of fibrinolysis and aprotinin on platelet GPlb will require the analysis of plasma glycocalicin, a hydrolytic segment of GPlb.

The Journal of Thoracic and Cardiovascular Surgery July 1993

1 8 Huang et al.

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