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Fibrinolysis in human peritoneum during operation
Fibrinolysis in human peritoneum during operation Lena Holmdahl, MD, PhD, Elsa Eriksson, PhD, Mohammed AI-Jabreen, MD, and Bo Risberg, MD, PhD, Grteborgand Malmb', Sweden
Background. A reduced local flbrinolysis seems to be an important mechanism in the formation of adhesions. Peritonitis may cause adhesions, and the aim of the present study was to determine peritoneal fibrinolytic capacity in inflamed and normal peritoneum. Methods. Biopsy specimens from normal and inflamed human peritoneum were taken at the beginning and end of operation. After extraction plasminogen activator activity (PAA) was determined by using a chromogenic substrate assay in the presence or absence of inhibitory antibodies against tissue-type plasminogen activator (t-PA), urokinase or plasminogen activator inhibitor-1. Results, t-PA exerted 95 % of the PAA. PAA was significantly reduced (p < O.01) during peritonitis (3.0 IU/lzg protein; range, 0.3 to 4.2) compared with normal peritoneum (7.1 IU/lzg protein; range, 0.6 to 18.1). A significant reduction (p < 0.05) in PAA occurred during operation both in normal peritoneum (3.8 IU/Izg protein; range, 0.8 to 8.6) and in peritonitis (0.6 IU/ktg protein; range, 0.16 to 2.1). Values are given as medians. Conclusions. The main PAA in human peritoneum was t-PA. The activity was decreasing during operation and reduced in peritonitis. This reduction in PAA might be a local response to inflammation. (Surgery 1996;119: 701-5.) From the Department of Surgery, Ostra Hospital, University of Grteborg, Grteborg, and Department of Surgery, Malta6 General Hospital, University of Lund, Malmb, Sweden
PERITONEAL FIBRINOLSOSISSEEMS TO BE an i m p o r t a n t d e n o m i n a t o r in the early formation o f postsurgical adhesions.I, 2 According to this hypothesis fibrin is f o r m e d at injured sites either from bleeding or by posttraumatic inflammatory mechanisms. These mechanisms include leakage from the vasculature caused by vasoactive substances (histamine) o r o t h e r mediators released from recruited white b l o o d cells or direct fibrin formation f r o m the peritoneal fluid. These fibrinous adhesions seem to be precursors to fibrous, p e r m a n e n t adhesions a n d can initially be lysed by local fibrinolytic mechanisms. If this local fibrinolysis is insufficient in clearing the fibrin, r e m n a n t s are left and act as guidelines for migrating cells a n d capillary buds a n d eventually organization. Evidence that supports this hypothesis has e m e r g e d from animal experiments where local t r e a t m e n t with fibrinolytic activators streptokinase a n d urokinase (u-PA) has b e e n r e p o r t e d to reduce adhesion formation. 3, 4
Supported by grants from the SwedishMedicalResearchCouncil (proj. 00660), the Gothenburg Medical Society,and the Universityof Grteborg. Accepted for publication Nov. 29, 1995. Reprint requests: Lena Holmdahl, MD, PhD, Department of Surgecr Ostra Hospital, Universityof G6teborg, $416 85 G6teborg, Sweden. Copyright 9 1996 by Mosby-YearBook, Inc. 0039-6060/96/$5.00 + 0 11/56/70920
Recently, h u m a n r e c o m b i n a n t tissue-type plasminogen activator (t-PA) was f o u n d to decrease adhesion formation 59 a n d postoperative inhibition o f fibrinolysis to enhance the formation o f adhesions. ~ Clinically and experimentally, inflammation such as peritonitis is a well-known cause o f i n t r a a b d o m i n a l adhesion formation. 1~ 11 T h e primary aim o f the p r e s e n t study was to test the hypothesis that peritonitis affects the fibrinolytic activity in h u m a n p e r i t o n e u m . Secondary aim was to investigate whether a b d o m i n a l Surgery affected p e r i t o n e a l fibrinolytic capacity.
MATERIAL AND METHODS Study design and patients. To d e t e r m i n e the fibrinolytic capacity in n o r m a l a n d inflamed p e r i t o n e u m during operation plasminogen activator activity (PAA) a n d plasminogen activator inhibitor-1 (PAI-1) activity a n d antigen were m e a s u r e d in serial peritoneal biopsy specimens taken from 26 patients u n d e r g o i n g abdominal operations. T h e fibrinolytic c o m p o n e n t s were normalized to the p r o t e i n content o f the biopsy specimen. I n f o r m e d consent was o b t a i n e d from all patients. Seve n t e e n of the patients u n d e r w e n t clean, elective abd o m i n a l surgery for benign disease and were conside r e d to have a n o r m a l p e r i t o n e u m , a n d nine h a d a purulent or chemical peritonitis from i n t r a a b d o m i n a l infections o r perforations o f the gastrointestinal tract. A total n u m b e r o f 50 biopsy specimens were analyzed, 34 SURGERY
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from patients with normal peritoneum and 16 from patients with peritonitis. Monoclonal antibodies to t-PA, u-PA, and PAl-1 were used to determine the specificity of the fibrinolytic assay system. 9 Biopsy specmens. Peritoneal biopsy specimens were taken immediately after opening of abdominal cavity. Additional biopsy specimens were taken at end of operation but before irrigation of abdominal cavity. Samples were taken from the parietal peritoneum on the anterior abdominal wall adjacent to the incision but remote to application sites of retractors. A free edge of periton e u m was held by pair of surgical forceps, and the thin m e m b r a n e was carefully dissected from the adjacent tissues and removed. The samples were immediately rinsed in room-tempered saline solution until contaminating blood was removed. The rinsing was kept as short as possible. T h e n the biopsy specimens were put in an airtight tube and immediately frozen on dry ice. The biopsy specimens were kept frozen at -70 ~ C until used. Tissue extraction. The frozen specimens were embedded in a medium (Tissue-Tek II O.C.T. compound; Lab-Tek Products, Naperville, Ill.) and cryosectioned in 8 pm thin sections by using a cryostat. The sections were quantitatively transferred with a Pasteur pipette to a test tube placed on ice. Extractions from the cryosections were made by incubating the sections with 0.5 m o l / L sodium thiocyanate for 5 minutes in a shaking water bath at 37 ~ C. After centrifugation the supernatant was frozen in aliquots at -706 C until analyzed. Assay oft-PA activity. The t-PA activity was determined in the supernatant by means of spectrophotometry by using a chromogenic plasmin substrate (S-2251; Pharmacia, Stockholm, Sweden). The determination of t-PA activity has been described in detail by Eriksson. 12 Briefly, the PAA values were obtained by measuring absorbance at 405 nm, plotted against absorbances of known amounts of t-PA. All determinations were done in duplicates, and the result was expressed as the mean of the two measurements. Assay ofPAI-1. PAI-1 activity was measured in extracts by means of an enzyme-linked immunoabsorbent assay (Functional PAl-l; Novo Nordisk, Bagsvaerd, Denmark) and PAI-1 antigen with an enzyme=linked immunosorbent assay technique (Imulyze PAl-l; Biopool AB, Ume~t, Sweden). Protein content. Protein content was determined in the supernatant and pellet of the biopsy specimen by using a slightly modified m e t h o d according to Lowry. The results of the individual assays were normalized to the total protein content (supernatant + pellet) of the biopsy specimen. Antibodies. The monoclonal antibodies directed
against h u m a n fibrinolytic components were antibody against t-PA (anti-human melanoma t-PA; Biopool AB), u-PA (MUK-4, MUK-1; BiopoolAB), and PAI-1 (MAI-12; Biopool AB). The two different u-PA antibodies react with either single chain (inactive precursor) or double chain u-PA (active compound). The result was expressed as a mean of the two assays. Before usage all am tibodies were reconstituted in phosphate-buffered saline solution at p H 7.4 according to the manufacturers' instructions. If dilution was necessary, the buffer used in the PAA assay (imidazole) was used. All samples were incubated for 30 minutes at r o o m temperature before they were assessed. Specificity of the t-PA assay. To test the specificity of the fibrinolytic assay system monoclonal antibodies to the fibrinolytic components t-PA, u-PA, and PAI-1 were individually added in excess to aliquots of extracts from the initial biopsy specimens from 22 patients (13 with normal peritoneum and 9 with peritonitis). The fibrinolytic activitywith antibodies present was compared with an extract without addition of antibodies (control), and results were expressed as a percentage of the activity in the controls. To eliminate errors in the spectrophotometric assay caused by the addition of the different antibodies values were calculated by comparison with standard curves established with each antibody. Statistics. Data are presented as median and range, and n represents the n u m b e r of patients in each group. Differences were considered significant a t p < 0.05 level. Differences in fibrinolytic activity and protein content between normal and inflamed peritoneum were assessed with the Mann-Whitney Utest. This test was also used for analyzing differences in fibrinolytic activity at different intervals during operation. The nonparametric one-sample sign test was used to assess the change in fibrinolytic activity after addition of different antibodies.
RESULTS Normal peritoneum versus peritonitis. The PAA in normal peritoneum (n = 17) was 7.1 IU/lag protein at the beginning of operation (range, 0.6 to 18.1 IU/lag protein), whereas the PAA was significantly reduced (p< 0.01) in corresponding biopsy specimens from peritonitis (n = 9). The PAA in peritonitis was 3.0 I U / p g protein (range, 0.3 to 4.2 IU/lag protein) (Fig. 1). PAI-1 activity or PAl-1 antigen was not detected in the extracts. During operation. A decline in PAA in peritoneum occurred during abdominal operations both in those patients with normal peritoneum and in those with peritonitis. After 30 minutes or more a statistically significant reduction (p < 0.05) of PAAwas noted in biopsy specimens from normal peritoneum from 7.1 IU/lag protein (range, 0.6 to 18.1 IU/lag protein) to 3.8 IU/lag protein (range, 0.8 to 8.6 IU/lag protein). Similarly, a significant reduction (p < 0.05) in the PAA occurred in
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biopsy specimens from patients with peritonitis from 3.0 IU/lag p r o t e i n (range, 0.3 to 4.2 I U / p g protein) at the start of operation to 0.6 I U / p g p r o t e i n (range, 0.16 to 2.1 IU/lag protein) after 30 minutes or later (Figs. 2 a n d 3). PAI-1 activity or PAI-1 antigen was n o t detected. Specificity o f the t-PA assay. T h e addition of a monoclonal antibody against t-PA r e d u c e d the activity significantly (p < 0.0001) to 4.5% of the control value, whereas the addition of antibodies directed against u-PA a n d PAI-1 did n o t affect the result (102.1, range 76.0 to 189.0 I U / p g protein, p > 0.05 a n d 101.5, range 85.1 to 107.0 I U / p g protein, p > 0.05, respectively). However, when biopsy specimens from n o r m a l perit o n e u m a n d peritonitis were analyzed separately, there was a statistically significantly (p < 0.05) m o r e p r o f o u n d
anti u-PA
anti PAl-1
Fig. 4. PAA in biopsy specimens after quenching w i n antibodies against different components of fibrinolytic system. Results are expressed as percentage of activity in nontreated control tube. Approximately 95% of PAA was exerted by t-PA. Significant difference occurred in PAA in inflamed and normal peritoneum after quenching with anti t-PA. Data are presented as median, 25th, 75th, 10th, and 90th percentiles. [ ] peritonitis; [ ] normal peritoneum.
q u e n c h i n g o f PAA by addition of inhibitory antibodies against t-PA in the biopsy specimens from n o r m a l perit o n e u m (3.7%, range 1.3% to 19.4%) c o m p a r e d with inflamed p e r i t o n e u m (7.6%, range 3.5% to 12.3%). T h e addition o f m o n o c l o n a l antibodies against u-PA or P A I d d i d n o t significantly change the activity either in n o r m a l p e r i t o n e u m or d u r i n g peritonitis. Data are summarized in Fig. 4. P r o t e i n c o n t e n t in b i o p s y specimens. To ensure that differences in fibrinolytic activity between n o r m a l a n d inflamed p e r i t o n e u m were n o t merely d u e to increased levels o f p r o t e i n in inflamed tissue, the p r o t e i n c o n t e n t of samples was compared. T h e m e d i a n p r o t e i n c o n t e n t
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in normal peritoneum was 140.0 lag (range, 37.5 to 321.7 pg) and in inflamed peritoneal tissue 148.7 pg (range, 74.1 to 583.3 pg). This difference was not statistically significant (p = 0.644). DISCUSSION
In the present study h u m a n peritoneum was f o u n d to possess PAA in biopsy specimens from both normal peritoneum and peritonitis. The main plasminogen activator was t-PA, responsible for 95 % of the plasminogen conversion. However, the activity varied during operation and in different conditions, suggesting that local fibrinolysis could be modulated. PAl-1 was not detected, either functionally or immunologically. Determinations of compounds in tissue can be confounded in several ways, One possibility is to express the levels in relation to total DNA. In the present study DNA measurements failed, probably because of interference by the extraction buffer in the DNA assay. Previous investigators have related the fibrinolytic activity to surface area of the biopsy specimens. 13, 14 However, specimens are three-dimensional, and the depth of the biopsy specimen is difficult to control. Therefore we chose to use protein content. A confounding factor by doing so may be that protein content in inflammed and normal tissue may vary, thereby affecting results. In the present study no significant differences in protein content were observed, indicating that changes in fibrinolytic activity recorded were accurate. Moore et al. 15 demonstrated in dogs that peritoneum has both powerful coagulation a n d fibrinolytic capacities during normal conditions. It has also been shown in animals that the fibrinolytic capacity after operation was inversely correlated to the formation of adhesions and that perturbation of the peritoneum such as diathermy coagulation, ischemia, free peritoneal grafting, z or infection TM reduced peritoneal fibrinolysis. It was suggested that a reduced local fibrinolysis was a central c o m m o n pathway leading to adhesion formation. ] The formation of fibrin in peritoneal cavity requires the coincidence of two events, a supply of the substrate fibrinogen and the formation of thrombin. Fibrinogen is present in plasma and escapes into wounded areas. Furthermore, fibrinogen is also present in peritoneal fluid.17, 18 The role of the fibrinolytic system is to remove fibrin by degradation. Fibrinolysis is generally plasmin mediated, but at the local level lysis of fibrin can, at least to some extent, be non-plasmin mediated and moderated by leukocyte elastase) 9 The plasmin-mediated fibrinolysis involves the activation of plasminogen to plasmin. This conversion is balanced by activators: t-PA and u-PA and inhibitors and PAls. Several types of PAl s have been described, but PAl-1 is considered the dominating inhibitor in plasma.
Surgery June 1996 Recently t-PA has been identified in many tissues. T h o m p s o n e t a l . 13 also demonstrated that t-PA and PAl were present in standardized biopsy specimens from h u m a n peritoneum a n d that the t-PA antigen levels were decreased in peritonitis when normalized to peritoneal surface area. 13 In this quantitative assay system a significant reduction occurred in the fibrinolytic capacity in peritonitis compared with normal conditions, confirming the previous findings with the fibrin plate technique by previous investigators. 13' 14 Moreover, a significant reduction occurred in the PAA in peritoneum during operation both in patients with normal peritoneum and in those with peritonitis. Plasminogen activation is counterbalanced by a rapid inhibition by PAl, which complex binds to t-PA. The reduction of PAA in peritonitis can be due to either a decrease in t-PA synthesis/release or a complex formation to PAI-1. In endothelial cell cultures challenged with endotoxin, tumor necrosis factor-a, or interleukin-1 the PAI-1 release increased, z~ Recently, it has been shown that the concentrations of these cytokines were elevated in peritoneal drain fluid after abdominal surgery and that the concentration correlated with peritoneal bacterial count and the length of the operation, zl A possible explanation of the reduced fibrinolytic capacity therefore seems to be that tumor necrosis factor-c~ and interleukin-1 induce the release of PAl-l, which may reduce PAA. However, this remains to be shown. In the present study the addition of inactivating antibodies directed against PAl to the extracts did not influence the plasminogen activation, indicating that no active PAl was present in the extracts. Vipond et al)4 were able to detect PAl immunologically in extracts from homogenized h u m a n peritoneum. In the present study tissue samples were homogenized differently and extracted with another buffer. Lack of effect of anti-PAl antibodies might be due to a preexisting complex formarion or to an inadequacy of PAl extraction from the specimens. The reason for this is currently u n d e r investigation in the laboratory. Interestingly, it was not possible to completely abolish the PAA in extracts even after addition of anti-t-PA in excess, neither in normal nor in inflamed peritoneum. Further, a difference was noted in the ability to quench t-PA activity between normal and inflamed peritoneum, indicating that some other factor(s) influenced, either directly by being able to convert plasminogen to plasmin or indirectly by affecting the activity of t-PA. Because the addition of antibodies against u-PA and PAl-1 indicated that these components were inactivated if present, these factors seem not to be responsible. Thus the remaining fibrinolytic activity might be due to other local serine proteases.
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M a c r o p h a g e s a r e c a p a b l e o f s e c r e t i n g a wide variety o f c o m p o u n d s i n c l u d i n g activators a n d i n h i b i t o r s o f fibrinolysis. 22 It h a s p r e v i o u s l y b e e n s h o w n t h a t m a c r o p h a g e s a r e r e c r u i t e d to i n j u r e d a r e a s i n a b d o m i n a l cavity23 a n d t h e s e cells h a v e i n vitro b e e n s h o w n to m o d u late f i b r i n o l y t i c activity. 24, 25 I n a p r e l i m i n a r y s t u d y w i t h immunohistochemistry (fluorescein isothiocyanate-lab e l e d C D 6 8 a n t i b o d i e s ) we f o u n d t h a t a s i g n i f i c a n t increase occurred in the density of macrophages both at t h e p e r i t o n e a l surface a n d i n t h e s u b m e s o t h e l i a l tissue i n p e r i t o n i t i s c o m p a r e d w i t h n o r m a l p e r i t o n e u m . However, n o s u b s t a n t i a l c h a n g e was d e t e c t e d i n s p e c i m e n s o b t a i n e d a t t h e b e g i n n i n g a n d e n d o f o p e r a t i o n (unp u b l i s h e d o b s e r v a t i o n ) . F r o m this it c a n b e d e d u c t e d that peritoneal macrophages cannot be the only modu l a t o r o f tissue f i b r i n o l y t i c capacity. Abdominal surgery represents an ongoing trauma. It is k n o w n f r o m e x p e r i m e n t a l a n d clinical studies t h a t surgical t r a u m a r e d u c e s tissue fibrinolysis b o t h locally i n p e r i t o n e u m 1, 26 a n d at r e m o t e sites. 27 T h e p r e s e n t s t u d y i n d i c a t e d t h a t this p h e n o m e n o n was a p p l i c a b l e i n h u m a n b e i n g s . L o c a l p r e o p e r a t i v e t r a u m a as a c a u s e o f t h e f i b r i n o l y t i c s h u t d o w n c o u l d b e e x c l u d e d b e c a u s e all biopsy s p e c i m e n s w e r e t a k e n r e m o t e f r o m local t r a u m a areas, s u c h as a p p l i c a t i o n sites o f r e t r a c t o r s . I n a n i m a l m o d e l s a r e d u c t i o n i n fibrinolysis s e e m e d to b e a n imp o r t a n t f a c t o r i n t h e series o f e v e n t s l e a d i n g to p o s t o p erative a d h e s i o n f o r m a t i o n . I n t h e p r e s e n t s t u d y a similar p a t t e r n o f d e c r e a s e d f i b r i n o l y t i c activity a t t h e tissue level was s e e n d u r i n g b o t h a b d o m i n a l s u r g e r y a n d peritonitis. T h i s r e d u c e d f i b r i n c l e a r i n g capacity m a y b e a n i m p o r t a n t step in t h e early f o r m a t i o n o f a d h e s i o n s , We t h a n k Lotta Falkendahl, Susanne Melander, Mafia Tylman, Ingrid Ovesson-Frisk, a n d Anneli S6derbom for technical assistance. REFERENCES
1. Buckman RF, Woods M, Sargent L, Gervin AS. A unifying pathogenetic mechanism in the etiology of intraperitoneal adhesions. J Surg Res 1976;20:1-5. 2. Raftery AT. Effect of peritoneal trauma on peritoneal fibrinolytic activity and intraperitoneal adhesion formation. Eur Surg Res 1981;13:397-401. 3. James DCO, Ellis H, Hugh TB. The effect of streptokinase on experimental intraperitoneal adhesion formation. J Pathol Bacteriol 1965;90:279-87. 4. Gervin AS, Puckett CL, Silver D. Serosal hypofibrinolysis. Am J Surg 1973;125:80-8. 5. Doody KJ, Dunn RC, Buttram VC. Recombinant tissue plasminogen activator reduces adhesion formation in a rabbit uterine horn model. Fertil Steril 1989;51:509-12. 6. D6rr PJ, Vetoer HM, Brommer EJP, Willemsen WNP, Veldhuizen RW, Rolland R. Prevention of postoperative adhesions by tissuetype plasminogen activator (t-PA) in the rabbit. EurJ Obstet Gynecol Reprod Biol 1990;37:287-91.
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7. Menzies D, Ellis H. Intraabdominal adhesions and their prevention by topical tissue plasminogen activator.J R Soc Med 1989;82: 534-5. 8. Orita H, Fukasawa M, Girgis W, diZerega GS. Inhibition of postsurgical adhesions in a standardized rabbit model: intraperitoheal treatment with tissue plasminogen activator. Int J Fertil 1991;36:172-7. 9. Holmdahl L, A1-Jabreen M, Risberg B. The role of fibrinolysis in the formation of postoperative adhesions. Wound Rep Reg 1994;7:171-6. 10. Rubin J, Herrera GA, Collins D. An autopsy study of the peritoneal cavity from patients on continuous ambulatory peritoneal dialysis. AmJ Kidney Dis 1991;18:97-102. 11. O'Leary DP, CoakleyJB. The influence of suturing and sepsis on the development of postoperative peritoneal adhesions. Ann R Coll Surg Engl 1992;74:134-7. 12. Eriksson E. Tissue plasminogen activator and inhibitor in blood and tissue [doctoral dissertation]. Grteborg, Sweden: University of Grteborg, 1989. 49 p. 13. Thompson JN, Paterson Brown S, Harbourne T, Whawell SA, Kalodiki E, Dudley HAF. Reduced h/anan peritoneal plasminogen activating activity: possible mechanism of adhesion formation. BrJ Surg 1989;76:382-4. 14. Vipond MN, Whawell SA, Thompson JN, Dudley HA. Peritoneal fibrinolytic activity and intra-abdominal adhesions. Lancet 1990;335:1120-2. 15. Moore KL, Bang NU, Broadie TA, Mattler LE, Marks CA. Peritoneal fibrinolysis: evidence for the efficiently of the tissue-type plasminogen activator. J Lab Clin Med 1983;101:921-9. 16. Hau T, Payne WD, Simmons RL. Fibrinolyfic activity of the peritoneum during experimental peritonitis. Surg Gynecol Obstet 1979;148:415-8. 17. Bouckaert PXJM, Van Wersch JWJ, Schellekens LA, EversJLH, Rolland R. Hamaeostatic and fibrinolytic properties of peritoneal fluid in the menstrual cycle. BrJ Obstet Gynaecol 1984;91:256-9. 18. Baxter GM, Parks AIt, Prasse KW. Effects of exploratory laparotomy on plasma and peritoneal coagulation/fibrinolysis in horses. AmJ Vet Res 1991;52:1121-7. 19, Plow EF. The major fibrinolytic proteases in human leucocytes. Biochim Biophys Acta 1980;630:47-56. 20. Emeis JJ, Kooistra T. Interleukin-1 and lipopolysaccharides induce an inhibitor of tissue-type plasminogen activator in vivo mad in cultured endothelial cells. J Exp Med 1986;163:1260-6. 21. Tsukada K, Katoh H, Shiojima M, Suzuki T, Takenoshita S, Hagamachi Y. Concentrations of cytokines in peritoneal fluid after surgery. EurJ Surg 1993;159:475-9. 22. Takemura R, Werb Z. Secretory products of macrophages and their physiological functions. Am J Physiol 1984;246:C1-9. 23. Raftery AT. Regeneration of parietal and visceral peritoneum: an electron microscopical study. J Anat 1973;115:375-92. 24. Orita H, CampeauJD, Gale JA, Nakamura RM, diZerega GS. Differential secretion of plasminogen activator activity by postsurgical activated macrophages. J Surg Res 1986;41:569-73. 25. Rodgers KE, Ellefson D, Girgis W, diZerega GS. Protease and protease inhibitor secretion by postsurgical macrophages following in vitro exposure to tolmetin. Agents Actions 1992;36:248-57. 26. Raftery AT. Regeneration of peritoneum: a fibrinolytic study. J Anat 1979;129:659-64. 27. Ljungner H, Bergqvist D, von Hebel I, Isacson S. Influence of major surgery on plasminogen activator activity in superficial hand veins. Eur Surg Res 1983;15:161-7.
Background. A reduced local flbrinolysis seems to be an important mechanism in the formation of adhesions. Peritonitis may cause adhesions, and the aim of the present study was to determine peritoneal fibrinolytic capacity in inflamed and normal peritoneum. Methods. Biopsy specimens from normal and inflamed human peritoneum were taken at the beginning and end of operation. After extraction plasminogen activator activity (PAA) was determined by using a chromogenic substrate assay in the presence or absence of inhibitory antibodies against tissue-type plasminogen activator (t-PA), urokinase or plasminogen activator inhibitor-1. Results, t-PA exerted 95 % of the PAA. PAA was significantly reduced (p < O.01) during peritonitis (3.0 IU/lzg protein; range, 0.3 to 4.2) compared with normal peritoneum (7.1 IU/lzg protein; range, 0.6 to 18.1). A significant reduction (p < 0.05) in PAA occurred during operation both in normal peritoneum (3.8 IU/Izg protein; range, 0.8 to 8.6) and in peritonitis (0.6 IU/ktg protein; range, 0.16 to 2.1). Values are given as medians. Conclusions. The main PAA in human peritoneum was t-PA. The activity was decreasing during operation and reduced in peritonitis. This reduction in PAA might be a local response to inflammation. (Surgery 1996;119: 701-5.) From the Department of Surgery, Ostra Hospital, University of Grteborg, Grteborg, and Department of Surgery, Malta6 General Hospital, University of Lund, Malmb, Sweden
PERITONEAL FIBRINOLSOSISSEEMS TO BE an i m p o r t a n t d e n o m i n a t o r in the early formation o f postsurgical adhesions.I, 2 According to this hypothesis fibrin is f o r m e d at injured sites either from bleeding or by posttraumatic inflammatory mechanisms. These mechanisms include leakage from the vasculature caused by vasoactive substances (histamine) o r o t h e r mediators released from recruited white b l o o d cells or direct fibrin formation f r o m the peritoneal fluid. These fibrinous adhesions seem to be precursors to fibrous, p e r m a n e n t adhesions a n d can initially be lysed by local fibrinolytic mechanisms. If this local fibrinolysis is insufficient in clearing the fibrin, r e m n a n t s are left and act as guidelines for migrating cells a n d capillary buds a n d eventually organization. Evidence that supports this hypothesis has e m e r g e d from animal experiments where local t r e a t m e n t with fibrinolytic activators streptokinase a n d urokinase (u-PA) has b e e n r e p o r t e d to reduce adhesion formation. 3, 4
Supported by grants from the SwedishMedicalResearchCouncil (proj. 00660), the Gothenburg Medical Society,and the Universityof Grteborg. Accepted for publication Nov. 29, 1995. Reprint requests: Lena Holmdahl, MD, PhD, Department of Surgecr Ostra Hospital, Universityof G6teborg, $416 85 G6teborg, Sweden. Copyright 9 1996 by Mosby-YearBook, Inc. 0039-6060/96/$5.00 + 0 11/56/70920
Recently, h u m a n r e c o m b i n a n t tissue-type plasminogen activator (t-PA) was f o u n d to decrease adhesion formation 59 a n d postoperative inhibition o f fibrinolysis to enhance the formation o f adhesions. ~ Clinically and experimentally, inflammation such as peritonitis is a well-known cause o f i n t r a a b d o m i n a l adhesion formation. 1~ 11 T h e primary aim o f the p r e s e n t study was to test the hypothesis that peritonitis affects the fibrinolytic activity in h u m a n p e r i t o n e u m . Secondary aim was to investigate whether a b d o m i n a l Surgery affected p e r i t o n e a l fibrinolytic capacity.
MATERIAL AND METHODS Study design and patients. To d e t e r m i n e the fibrinolytic capacity in n o r m a l a n d inflamed p e r i t o n e u m during operation plasminogen activator activity (PAA) a n d plasminogen activator inhibitor-1 (PAI-1) activity a n d antigen were m e a s u r e d in serial peritoneal biopsy specimens taken from 26 patients u n d e r g o i n g abdominal operations. T h e fibrinolytic c o m p o n e n t s were normalized to the p r o t e i n content o f the biopsy specimen. I n f o r m e d consent was o b t a i n e d from all patients. Seve n t e e n of the patients u n d e r w e n t clean, elective abd o m i n a l surgery for benign disease and were conside r e d to have a n o r m a l p e r i t o n e u m , a n d nine h a d a purulent or chemical peritonitis from i n t r a a b d o m i n a l infections o r perforations o f the gastrointestinal tract. A total n u m b e r o f 50 biopsy specimens were analyzed, 34 SURGERY
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from patients with normal peritoneum and 16 from patients with peritonitis. Monoclonal antibodies to t-PA, u-PA, and PAl-1 were used to determine the specificity of the fibrinolytic assay system. 9 Biopsy specmens. Peritoneal biopsy specimens were taken immediately after opening of abdominal cavity. Additional biopsy specimens were taken at end of operation but before irrigation of abdominal cavity. Samples were taken from the parietal peritoneum on the anterior abdominal wall adjacent to the incision but remote to application sites of retractors. A free edge of periton e u m was held by pair of surgical forceps, and the thin m e m b r a n e was carefully dissected from the adjacent tissues and removed. The samples were immediately rinsed in room-tempered saline solution until contaminating blood was removed. The rinsing was kept as short as possible. T h e n the biopsy specimens were put in an airtight tube and immediately frozen on dry ice. The biopsy specimens were kept frozen at -70 ~ C until used. Tissue extraction. The frozen specimens were embedded in a medium (Tissue-Tek II O.C.T. compound; Lab-Tek Products, Naperville, Ill.) and cryosectioned in 8 pm thin sections by using a cryostat. The sections were quantitatively transferred with a Pasteur pipette to a test tube placed on ice. Extractions from the cryosections were made by incubating the sections with 0.5 m o l / L sodium thiocyanate for 5 minutes in a shaking water bath at 37 ~ C. After centrifugation the supernatant was frozen in aliquots at -706 C until analyzed. Assay oft-PA activity. The t-PA activity was determined in the supernatant by means of spectrophotometry by using a chromogenic plasmin substrate (S-2251; Pharmacia, Stockholm, Sweden). The determination of t-PA activity has been described in detail by Eriksson. 12 Briefly, the PAA values were obtained by measuring absorbance at 405 nm, plotted against absorbances of known amounts of t-PA. All determinations were done in duplicates, and the result was expressed as the mean of the two measurements. Assay ofPAI-1. PAI-1 activity was measured in extracts by means of an enzyme-linked immunoabsorbent assay (Functional PAl-l; Novo Nordisk, Bagsvaerd, Denmark) and PAI-1 antigen with an enzyme=linked immunosorbent assay technique (Imulyze PAl-l; Biopool AB, Ume~t, Sweden). Protein content. Protein content was determined in the supernatant and pellet of the biopsy specimen by using a slightly modified m e t h o d according to Lowry. The results of the individual assays were normalized to the total protein content (supernatant + pellet) of the biopsy specimen. Antibodies. The monoclonal antibodies directed
against h u m a n fibrinolytic components were antibody against t-PA (anti-human melanoma t-PA; Biopool AB), u-PA (MUK-4, MUK-1; BiopoolAB), and PAI-1 (MAI-12; Biopool AB). The two different u-PA antibodies react with either single chain (inactive precursor) or double chain u-PA (active compound). The result was expressed as a mean of the two assays. Before usage all am tibodies were reconstituted in phosphate-buffered saline solution at p H 7.4 according to the manufacturers' instructions. If dilution was necessary, the buffer used in the PAA assay (imidazole) was used. All samples were incubated for 30 minutes at r o o m temperature before they were assessed. Specificity of the t-PA assay. To test the specificity of the fibrinolytic assay system monoclonal antibodies to the fibrinolytic components t-PA, u-PA, and PAI-1 were individually added in excess to aliquots of extracts from the initial biopsy specimens from 22 patients (13 with normal peritoneum and 9 with peritonitis). The fibrinolytic activitywith antibodies present was compared with an extract without addition of antibodies (control), and results were expressed as a percentage of the activity in the controls. To eliminate errors in the spectrophotometric assay caused by the addition of the different antibodies values were calculated by comparison with standard curves established with each antibody. Statistics. Data are presented as median and range, and n represents the n u m b e r of patients in each group. Differences were considered significant a t p < 0.05 level. Differences in fibrinolytic activity and protein content between normal and inflamed peritoneum were assessed with the Mann-Whitney Utest. This test was also used for analyzing differences in fibrinolytic activity at different intervals during operation. The nonparametric one-sample sign test was used to assess the change in fibrinolytic activity after addition of different antibodies.
RESULTS Normal peritoneum versus peritonitis. The PAA in normal peritoneum (n = 17) was 7.1 IU/lag protein at the beginning of operation (range, 0.6 to 18.1 IU/lag protein), whereas the PAA was significantly reduced (p< 0.01) in corresponding biopsy specimens from peritonitis (n = 9). The PAA in peritonitis was 3.0 I U / p g protein (range, 0.3 to 4.2 IU/lag protein) (Fig. 1). PAI-1 activity or PAl-1 antigen was not detected in the extracts. During operation. A decline in PAA in peritoneum occurred during abdominal operations both in those patients with normal peritoneum and in those with peritonitis. After 30 minutes or more a statistically significant reduction (p < 0.05) of PAAwas noted in biopsy specimens from normal peritoneum from 7.1 IU/lag protein (range, 0.6 to 18.1 IU/lag protein) to 3.8 IU/lag protein (range, 0.8 to 8.6 IU/lag protein). Similarly, a significant reduction (p < 0.05) in the PAA occurred in
Surgery Volume 119, Number 6
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tis at beginning of operation. Data are presented as median, 25th, 75th, 10th, and 90th percentiles.
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Fig. 2. PAA in normal peritoneum at beginning of abdominal operation compared with after 30 minutes or later. Significant decline occurred in PAA during operation. Data are presented as median, 25th, 75th, 10th, and 90th percentiles.
biopsy specimens from patients with peritonitis from 3.0 IU/lag p r o t e i n (range, 0.3 to 4.2 I U / p g protein) at the start of operation to 0.6 I U / p g p r o t e i n (range, 0.16 to 2.1 IU/lag protein) after 30 minutes or later (Figs. 2 a n d 3). PAI-1 activity or PAI-1 antigen was n o t detected. Specificity o f the t-PA assay. T h e addition of a monoclonal antibody against t-PA r e d u c e d the activity significantly (p < 0.0001) to 4.5% of the control value, whereas the addition of antibodies directed against u-PA a n d PAI-1 did n o t affect the result (102.1, range 76.0 to 189.0 I U / p g protein, p > 0.05 a n d 101.5, range 85.1 to 107.0 I U / p g protein, p > 0.05, respectively). However, when biopsy specimens from n o r m a l perit o n e u m a n d peritonitis were analyzed separately, there was a statistically significantly (p < 0.05) m o r e p r o f o u n d
anti u-PA
anti PAl-1
Fig. 4. PAA in biopsy specimens after quenching w i n antibodies against different components of fibrinolytic system. Results are expressed as percentage of activity in nontreated control tube. Approximately 95% of PAA was exerted by t-PA. Significant difference occurred in PAA in inflamed and normal peritoneum after quenching with anti t-PA. Data are presented as median, 25th, 75th, 10th, and 90th percentiles. [ ] peritonitis; [ ] normal peritoneum.
q u e n c h i n g o f PAA by addition of inhibitory antibodies against t-PA in the biopsy specimens from n o r m a l perit o n e u m (3.7%, range 1.3% to 19.4%) c o m p a r e d with inflamed p e r i t o n e u m (7.6%, range 3.5% to 12.3%). T h e addition o f m o n o c l o n a l antibodies against u-PA or P A I d d i d n o t significantly change the activity either in n o r m a l p e r i t o n e u m or d u r i n g peritonitis. Data are summarized in Fig. 4. P r o t e i n c o n t e n t in b i o p s y specimens. To ensure that differences in fibrinolytic activity between n o r m a l a n d inflamed p e r i t o n e u m were n o t merely d u e to increased levels o f p r o t e i n in inflamed tissue, the p r o t e i n c o n t e n t of samples was compared. T h e m e d i a n p r o t e i n c o n t e n t
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in normal peritoneum was 140.0 lag (range, 37.5 to 321.7 pg) and in inflamed peritoneal tissue 148.7 pg (range, 74.1 to 583.3 pg). This difference was not statistically significant (p = 0.644). DISCUSSION
In the present study h u m a n peritoneum was f o u n d to possess PAA in biopsy specimens from both normal peritoneum and peritonitis. The main plasminogen activator was t-PA, responsible for 95 % of the plasminogen conversion. However, the activity varied during operation and in different conditions, suggesting that local fibrinolysis could be modulated. PAl-1 was not detected, either functionally or immunologically. Determinations of compounds in tissue can be confounded in several ways, One possibility is to express the levels in relation to total DNA. In the present study DNA measurements failed, probably because of interference by the extraction buffer in the DNA assay. Previous investigators have related the fibrinolytic activity to surface area of the biopsy specimens. 13, 14 However, specimens are three-dimensional, and the depth of the biopsy specimen is difficult to control. Therefore we chose to use protein content. A confounding factor by doing so may be that protein content in inflammed and normal tissue may vary, thereby affecting results. In the present study no significant differences in protein content were observed, indicating that changes in fibrinolytic activity recorded were accurate. Moore et al. 15 demonstrated in dogs that peritoneum has both powerful coagulation a n d fibrinolytic capacities during normal conditions. It has also been shown in animals that the fibrinolytic capacity after operation was inversely correlated to the formation of adhesions and that perturbation of the peritoneum such as diathermy coagulation, ischemia, free peritoneal grafting, z or infection TM reduced peritoneal fibrinolysis. It was suggested that a reduced local fibrinolysis was a central c o m m o n pathway leading to adhesion formation. ] The formation of fibrin in peritoneal cavity requires the coincidence of two events, a supply of the substrate fibrinogen and the formation of thrombin. Fibrinogen is present in plasma and escapes into wounded areas. Furthermore, fibrinogen is also present in peritoneal fluid.17, 18 The role of the fibrinolytic system is to remove fibrin by degradation. Fibrinolysis is generally plasmin mediated, but at the local level lysis of fibrin can, at least to some extent, be non-plasmin mediated and moderated by leukocyte elastase) 9 The plasmin-mediated fibrinolysis involves the activation of plasminogen to plasmin. This conversion is balanced by activators: t-PA and u-PA and inhibitors and PAls. Several types of PAl s have been described, but PAl-1 is considered the dominating inhibitor in plasma.
Surgery June 1996 Recently t-PA has been identified in many tissues. T h o m p s o n e t a l . 13 also demonstrated that t-PA and PAl were present in standardized biopsy specimens from h u m a n peritoneum a n d that the t-PA antigen levels were decreased in peritonitis when normalized to peritoneal surface area. 13 In this quantitative assay system a significant reduction occurred in the fibrinolytic capacity in peritonitis compared with normal conditions, confirming the previous findings with the fibrin plate technique by previous investigators. 13' 14 Moreover, a significant reduction occurred in the PAA in peritoneum during operation both in patients with normal peritoneum and in those with peritonitis. Plasminogen activation is counterbalanced by a rapid inhibition by PAl, which complex binds to t-PA. The reduction of PAA in peritonitis can be due to either a decrease in t-PA synthesis/release or a complex formation to PAI-1. In endothelial cell cultures challenged with endotoxin, tumor necrosis factor-a, or interleukin-1 the PAI-1 release increased, z~ Recently, it has been shown that the concentrations of these cytokines were elevated in peritoneal drain fluid after abdominal surgery and that the concentration correlated with peritoneal bacterial count and the length of the operation, zl A possible explanation of the reduced fibrinolytic capacity therefore seems to be that tumor necrosis factor-c~ and interleukin-1 induce the release of PAl-l, which may reduce PAA. However, this remains to be shown. In the present study the addition of inactivating antibodies directed against PAl to the extracts did not influence the plasminogen activation, indicating that no active PAl was present in the extracts. Vipond et al)4 were able to detect PAl immunologically in extracts from homogenized h u m a n peritoneum. In the present study tissue samples were homogenized differently and extracted with another buffer. Lack of effect of anti-PAl antibodies might be due to a preexisting complex formarion or to an inadequacy of PAl extraction from the specimens. The reason for this is currently u n d e r investigation in the laboratory. Interestingly, it was not possible to completely abolish the PAA in extracts even after addition of anti-t-PA in excess, neither in normal nor in inflamed peritoneum. Further, a difference was noted in the ability to quench t-PA activity between normal and inflamed peritoneum, indicating that some other factor(s) influenced, either directly by being able to convert plasminogen to plasmin or indirectly by affecting the activity of t-PA. Because the addition of antibodies against u-PA and PAl-1 indicated that these components were inactivated if present, these factors seem not to be responsible. Thus the remaining fibrinolytic activity might be due to other local serine proteases.
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M a c r o p h a g e s a r e c a p a b l e o f s e c r e t i n g a wide variety o f c o m p o u n d s i n c l u d i n g activators a n d i n h i b i t o r s o f fibrinolysis. 22 It h a s p r e v i o u s l y b e e n s h o w n t h a t m a c r o p h a g e s a r e r e c r u i t e d to i n j u r e d a r e a s i n a b d o m i n a l cavity23 a n d t h e s e cells h a v e i n vitro b e e n s h o w n to m o d u late f i b r i n o l y t i c activity. 24, 25 I n a p r e l i m i n a r y s t u d y w i t h immunohistochemistry (fluorescein isothiocyanate-lab e l e d C D 6 8 a n t i b o d i e s ) we f o u n d t h a t a s i g n i f i c a n t increase occurred in the density of macrophages both at t h e p e r i t o n e a l surface a n d i n t h e s u b m e s o t h e l i a l tissue i n p e r i t o n i t i s c o m p a r e d w i t h n o r m a l p e r i t o n e u m . However, n o s u b s t a n t i a l c h a n g e was d e t e c t e d i n s p e c i m e n s o b t a i n e d a t t h e b e g i n n i n g a n d e n d o f o p e r a t i o n (unp u b l i s h e d o b s e r v a t i o n ) . F r o m this it c a n b e d e d u c t e d that peritoneal macrophages cannot be the only modu l a t o r o f tissue f i b r i n o l y t i c capacity. Abdominal surgery represents an ongoing trauma. It is k n o w n f r o m e x p e r i m e n t a l a n d clinical studies t h a t surgical t r a u m a r e d u c e s tissue fibrinolysis b o t h locally i n p e r i t o n e u m 1, 26 a n d at r e m o t e sites. 27 T h e p r e s e n t s t u d y i n d i c a t e d t h a t this p h e n o m e n o n was a p p l i c a b l e i n h u m a n b e i n g s . L o c a l p r e o p e r a t i v e t r a u m a as a c a u s e o f t h e f i b r i n o l y t i c s h u t d o w n c o u l d b e e x c l u d e d b e c a u s e all biopsy s p e c i m e n s w e r e t a k e n r e m o t e f r o m local t r a u m a areas, s u c h as a p p l i c a t i o n sites o f r e t r a c t o r s . I n a n i m a l m o d e l s a r e d u c t i o n i n fibrinolysis s e e m e d to b e a n imp o r t a n t f a c t o r i n t h e series o f e v e n t s l e a d i n g to p o s t o p erative a d h e s i o n f o r m a t i o n . I n t h e p r e s e n t s t u d y a similar p a t t e r n o f d e c r e a s e d f i b r i n o l y t i c activity a t t h e tissue level was s e e n d u r i n g b o t h a b d o m i n a l s u r g e r y a n d peritonitis. T h i s r e d u c e d f i b r i n c l e a r i n g capacity m a y b e a n i m p o r t a n t step in t h e early f o r m a t i o n o f a d h e s i o n s , We t h a n k Lotta Falkendahl, Susanne Melander, Mafia Tylman, Ingrid Ovesson-Frisk, a n d Anneli S6derbom for technical assistance. REFERENCES
1. Buckman RF, Woods M, Sargent L, Gervin AS. A unifying pathogenetic mechanism in the etiology of intraperitoneal adhesions. J Surg Res 1976;20:1-5. 2. Raftery AT. Effect of peritoneal trauma on peritoneal fibrinolytic activity and intraperitoneal adhesion formation. Eur Surg Res 1981;13:397-401. 3. James DCO, Ellis H, Hugh TB. The effect of streptokinase on experimental intraperitoneal adhesion formation. J Pathol Bacteriol 1965;90:279-87. 4. Gervin AS, Puckett CL, Silver D. Serosal hypofibrinolysis. Am J Surg 1973;125:80-8. 5. Doody KJ, Dunn RC, Buttram VC. Recombinant tissue plasminogen activator reduces adhesion formation in a rabbit uterine horn model. Fertil Steril 1989;51:509-12. 6. D6rr PJ, Vetoer HM, Brommer EJP, Willemsen WNP, Veldhuizen RW, Rolland R. Prevention of postoperative adhesions by tissuetype plasminogen activator (t-PA) in the rabbit. EurJ Obstet Gynecol Reprod Biol 1990;37:287-91.
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7. Menzies D, Ellis H. Intraabdominal adhesions and their prevention by topical tissue plasminogen activator.J R Soc Med 1989;82: 534-5. 8. Orita H, Fukasawa M, Girgis W, diZerega GS. Inhibition of postsurgical adhesions in a standardized rabbit model: intraperitoheal treatment with tissue plasminogen activator. Int J Fertil 1991;36:172-7. 9. Holmdahl L, A1-Jabreen M, Risberg B. The role of fibrinolysis in the formation of postoperative adhesions. Wound Rep Reg 1994;7:171-6. 10. Rubin J, Herrera GA, Collins D. An autopsy study of the peritoneal cavity from patients on continuous ambulatory peritoneal dialysis. AmJ Kidney Dis 1991;18:97-102. 11. O'Leary DP, CoakleyJB. The influence of suturing and sepsis on the development of postoperative peritoneal adhesions. Ann R Coll Surg Engl 1992;74:134-7. 12. Eriksson E. Tissue plasminogen activator and inhibitor in blood and tissue [doctoral dissertation]. Grteborg, Sweden: University of Grteborg, 1989. 49 p. 13. Thompson JN, Paterson Brown S, Harbourne T, Whawell SA, Kalodiki E, Dudley HAF. Reduced h/anan peritoneal plasminogen activating activity: possible mechanism of adhesion formation. BrJ Surg 1989;76:382-4. 14. Vipond MN, Whawell SA, Thompson JN, Dudley HA. Peritoneal fibrinolytic activity and intra-abdominal adhesions. Lancet 1990;335:1120-2. 15. Moore KL, Bang NU, Broadie TA, Mattler LE, Marks CA. Peritoneal fibrinolysis: evidence for the efficiently of the tissue-type plasminogen activator. J Lab Clin Med 1983;101:921-9. 16. Hau T, Payne WD, Simmons RL. Fibrinolyfic activity of the peritoneum during experimental peritonitis. Surg Gynecol Obstet 1979;148:415-8. 17. Bouckaert PXJM, Van Wersch JWJ, Schellekens LA, EversJLH, Rolland R. Hamaeostatic and fibrinolytic properties of peritoneal fluid in the menstrual cycle. BrJ Obstet Gynaecol 1984;91:256-9. 18. Baxter GM, Parks AIt, Prasse KW. Effects of exploratory laparotomy on plasma and peritoneal coagulation/fibrinolysis in horses. AmJ Vet Res 1991;52:1121-7. 19, Plow EF. The major fibrinolytic proteases in human leucocytes. Biochim Biophys Acta 1980;630:47-56. 20. Emeis JJ, Kooistra T. Interleukin-1 and lipopolysaccharides induce an inhibitor of tissue-type plasminogen activator in vivo mad in cultured endothelial cells. J Exp Med 1986;163:1260-6. 21. Tsukada K, Katoh H, Shiojima M, Suzuki T, Takenoshita S, Hagamachi Y. Concentrations of cytokines in peritoneal fluid after surgery. EurJ Surg 1993;159:475-9. 22. Takemura R, Werb Z. Secretory products of macrophages and their physiological functions. Am J Physiol 1984;246:C1-9. 23. Raftery AT. Regeneration of parietal and visceral peritoneum: an electron microscopical study. J Anat 1973;115:375-92. 24. Orita H, CampeauJD, Gale JA, Nakamura RM, diZerega GS. Differential secretion of plasminogen activator activity by postsurgical activated macrophages. J Surg Res 1986;41:569-73. 25. Rodgers KE, Ellefson D, Girgis W, diZerega GS. Protease and protease inhibitor secretion by postsurgical macrophages following in vitro exposure to tolmetin. Agents Actions 1992;36:248-57. 26. Raftery AT. Regeneration of peritoneum: a fibrinolytic study. J Anat 1979;129:659-64. 27. Ljungner H, Bergqvist D, von Hebel I, Isacson S. Influence of major surgery on plasminogen activator activity in superficial hand veins. Eur Surg Res 1983;15:161-7.