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Bradykinin release from high molecular weight kininogen in surgical ICU patients
Immunopharmacoloyy ELSEVIER
lmmunopharmacology33 (1996) 365-368
Bradykinin release from high molecular weight kininogen in surgical ICU patients Tove Sigstad Karlsrud a,*, Laila BuO b, Ansgar O. Aasen b,c, Harald Thidemann Johansen a a Department of Pharmacology, School ofPharmac)', Uniuersity of Oslo, P.O. Box 1068 Blindern, 0316 Oslo, Norway b blstitute of Surgical Research, The National Hospital, Oslo, Norway Department of SuJ~ery B, The National Hospital, Oslo, Norway
Abstract The purpose of this prospective study was to characterize kininogens in plasma from surgical ICU patients. Thirty-five patients, ages 19-79 years, were divided into 2 groups: sepsis (defined by standard criteria) and nonsepsis. Studies of proteolytic degradation of H-kininogen showed degradation in both patient groups compared to healthy controls. Functional quantification of prekallikrein showed a reduction of prekallikrein in plasma from both patient groups. Functional quantification of kininogens by a CPI (cysteine proteinase inhibitor) assay showed no significant differences between the patients and the controls. Immunological levels of H-kininogen and total kininogen were not significantly different from normal plasma. No differences could be detected between the two patient groups in any of the parameters studied. In conclusion, this study supported contact activation taking place in surgical ICU patients, a partial kinin release and a consumption of prekallikrein has taken place in vivo. Keywords: Contact activation; Kininogen; Sepsis; Surgical intensive care
1. Introduction Sepsis is a life-threatening condition caused by the presence of microorganisms or their products in the circulation. Considerable evidence has accumulated that excessive activation of plasma cascade systems, the complement system and the contact
Abbreviations: ICU, intensive care unit; CPI, cysteine proteinase inhibitor; EIA, enzyme immunoassay; H-kininogen. high molecular weight kininogen; L-kininogen, low molecular weight kininogen; PAGE, polyacrylamide gel electrophoresis * Corresponding author. Tel.: +47 (22) 85 75 78. Fax: +47 (22) 85 75 I I; E-mail: [email protected].
system of intrinsic coagulation, with release of biologically active substances (anaphylatoxins and bradykinin), plays a role in the pathogenesis of sepsis (Aasen et al., 1980). Kininogens are multifunctional single-chain proteins, and in human plasma two different kininogens have been described: High molecular weight kininogen (H-kininogen, M r ~ 120,000) and low molecular weight kininogen (L-kininogen, M r ~ 65,000). The vasoactive peptide bradykinin is released from the kininogens by the action of proteolytic enzymes such as kallikreins. The kinins are known to exert a variety of physiological effects, including induction of hypotension, smooth muscle contraction and in-
0162-3109/96/$15.t)0 Copyright ,e) 1996 Elsevier Science B.V. All rights reserved PH S01 62-3 109(96)00087-2
366
T.S. Karlsrud et al. / Immunopharmacology 33 (1996) 365-368
70
crease of vascular permeability. The fact that activation of the bradykinin receptor stimulates synthesis of nitric oxide, brings new focus on this nonapeptide in pathophysiological mechanisms, e.g. in sepsis.
~ 6o 011
!,.~
50 40 '
~
2. P a t i e n t s
3. R e s u l t s
Proteolytic cleavage of H-kininogen was studied by PAG electrophoresis followed by immunoblotting. In non-reduced patient and control plasma samples, three characteristic bands named I ( M r 147,000), II ( M r 104,000) and III ( M r 99,000) were detected. There were significantly lower levels of native H-kininogen (band I) and significantly higher levels of band II + I I I (degraded forms) for both patient groups compared to controls all 3 days considered (Fig. 1). No differences were observed between the two patient groups. Functional quantification of prekallikrein showed significantly lower values in both the sepsis and the nonsepsis groups compared to control plasma on all 3 days considered (Table 1). No differences could be observed between the two patient groups. These prekallikrein values (% of control plasma) were sig-
N
8'
•
°,
3o
o
~ 20
Thirty-five patients (22 male and 13 female, ages 19-79 years; mean 52) treated in the surgical intensive care unit were studied. Sixteen of these patients (11 male and 5 female, ages 19-76 years; mean 46) fulfilled at least four of the following criteria of sepsis: temperature above 38°C, need of respiratory support, leucocyte count above 15 × 109//1 or below 5 × 109//1, a thrombocyte count below 100 X 109//1, positive blood cultures, or an obvious septic focus. Citrated plasma samples from the day of diagnosis (day 1) and the following 2 days (day 2 and day 3) were studied in the sepsis group. Samples from the first 3 days of ICU treatment were studied in the nonsepsis patient group (11 male and 8 female; ages 2 4 - 7 9 years; mean 56). Plasma collected from 9 healthy persons (4 female and 5 male; ages 3 0 - 6 0 years; mean 43) served as controls.
• 0 0
10 0 Control Day 1 Day2
Day3
Fig. 1. Relative amounts of band II + III of H-kininogen in control plasma (0), plasma from the sepsis ((3) and nonsepsis (0) groups. The differences between the patient and control plasmas were significant for both patient groups all 3 days considered. nificantly lower than the corresponding values of total protein for both patient groups ( p < 0.005). The median values of functional kallikrein inhibition on day 1 were lower in the sepsis ( p = 0.063) and nonsepsis ( p = 0.036) groups compared to controls. No further differences could be detected. Significantly lower CPI (cysteine proteinase inhibitor) activities compared to the control group were observed both in the sepsis and nonsepsis groups on all 3 days considered (Fig. 2). No differences could be observed between the patient groups. In the nonsepsis group the CPI activity was slightly 43.5 32.5,6
21.510.50
t
t"4
t'~
"1 Control
$e' ~sis
Nonse 9sis
Fig. 2. Plasma samples were acid treated and appropriately diluted before the CPI activity was measured as described in (Karlsrud et al., submitted). The bars show the median values, 10th and 90th percentiles are indicated.
367
T.S. Karlsrud et al./ lmmunopharmacology 33 (1996) 365-368
Table 1 Quantification of plasma kininogens, total protein, prekallikrein, and kallikrein inhibition n H-kininogen~' Total kininogen a Totalprotein b Sepsis
Nonsepsis
Day 1 Day2 Day 3 Day 1 Day2 Day3
Control
7 12 8 11 8 9 9
59 * (22-78) 66 * (17-99) 76 (17-119) 78 * (22-96) 28 (21-59) 65(34-123) 101 (78-116)
57 *~ (20 80) 49 (25 97) 49 (26-95) 73 (33-97) 40 *~ (8 73) 88(38 113) 101 (81-129)
37 ** (23-48) 38 ** (30-48) 42 (29-55) 40 (22-57) 41 * (34-57) 37 "* (31-46) 49 (46-56)
Prekallikreina
Kallikrein inhibitiona
42 * * (20-60) 41 ~* (28 65) 57 * * (23-92) 44 ** (27-78) 41 ** (13 49) 53 * (35-73) 122 (62-173)
96(58-102) 103 (72-123) 97 (83-126) 89 * (77-109) 108 (80-126) 116(91-127) 117 (20-149)
% of pool plasma; b g/l; ' p < 0.05, * ~ p < 0.005. increasing from day 1 to day 3 ( p = 0.08), no such increase could be seen in the sepsis group ( p = 0.51). Quantification of H-kininogen and total kininogen in patient and control plasma was performed by EIA methods. The results show significantly lower levels of H-kininogen and total kininogen in both patient groups on day 1 and 2 compared to the control group (Table 1). Day 3 shows lower level of total kininogen in the sepsis group. There were no significant differences observed between the two patient groups. Total protein measurements gave values about 7 5 - 8 5 % of control plasma in both patient groups (Table 1). Significantly lower levels compared to the controls were observed in the sepsis group on day 1 and 2 and in the nonsepsis group on day 2 and 3. No significant differences between the patient groups were observed.
4. Discussion In this study a significant degradation of Hkininogen was found in plasma from critically ill patients treated in the surgical ICU. There were no significant differences between septic and nonseptic patients. Proteolytic cleavage of H-kininogen has been described in several diseases, e.g. during attacks of hereditary angioedema (Cugno et al., 1993), but few studies have focused on this in ICU patients. Band II is produced by kinin liberation from the intact H-kininogen, while band III is formed by kallikrein-induced degradation of the light chain of H-kininogen from the native 56 kDa to 45 kDa. This bradykinin release might be of pathophysiological importance in terms of a stimulator of nitric oxide synthesis.
Another indication of contact activation is present by the significantly lower levels of prekallikrein in the patient samples, very likely due to a consumption of this proenzyme. Activation of the plasma kallikrein-kinin system has been repeatedly described in patients with septicemia (Aasen et al., 1980, 1983; Kalter et al., 1985), and may be partly responsible for the haemodynamic changes resulting in oedema frequently observed in these patients. A 10-fold increase in blood plasma cysteine proteinase activity has been reported in septic shock compared to healthy controls (Assfalg-Machleidt et al., 1988). Similar increases were not found in ICU patients, but the observed degradation of H-kininogen did not seem to reduce the total CPI activity. This confirms the observation that bradykinin release does not affect the CPI function of purified Lkininogen (Vogel et al., 1988). Although reduced levels of kininogens were found compared to control plasma, this difference disappears when relating to total concentration of proteins. This points to decreased production by the liver, increased clearance, extravasation, a n d / o r hemodilution as likely causes to the reduction, a general reduction probably affecting all plasma proteins. The functional consequences, however, could be of importance, since inhibitor control over proteolytic activation on cell surfaces still may be disturbed.
References Aasen AO, Smith-Erichsen N, Gallimore MJ, Amundsen E. Studies on components of the plasma kallikrein-kinin system in plasma samples from normal individuals and patients with septic shock. In: Schumer W, Spitzer JJ, Marshall BE, Eds.
368
T.S. Karlsrud et al. / lmmunopharmacology 33 (1996) 365-368
Avd. in Shock Res. Vol 4. New York: A.R. Liss. Inc., 1980; 1-10. Aasen AO, Smith-Eriksen N, Amundsen E. Plasma kallikrein-kinin system in septicemia. Arch Surg 1983; 118: 343-345. Assfalg-Machleidt I, Jochum M, Klaubert W, Inthorn D, Machleidt W. Enzymatically active cathepsin B dissociating from its inhibitor complexes is elevated in blood plasma of patients with septic shock and some malignant tumors. Biol Chem Hoppe-Seyler 1988; 369(suppl): 263-269. Cugno M, Hack CE, de Boer JP, Eerenberg AJM, Agostoni A,
Cicardi M. Generation of plasmin during attacks of hereditary angioedema. J Lab Clin Med 1993; 121: 38-43. Kalter ES, Daha MR, ten Cate JW, Verhoef J, Bouma BN. Activation and inhibition of Hageman factor-dependent pathways and the complement system in uncomplicated bacteremia or bacterial shock. J Infect Dis 1985; 151: 1019-1027. Vogel R, Assfalg-Machleidt I, Esterl A, Machleidt W, MtillerEsterl W. Proteinase-sensitive regions in the heavy chain of low molecular weight kininogen map to the inter-domain junctions. J Biol Chem 1988; 263: 12661-12668.
lmmunopharmacology33 (1996) 365-368
Bradykinin release from high molecular weight kininogen in surgical ICU patients Tove Sigstad Karlsrud a,*, Laila BuO b, Ansgar O. Aasen b,c, Harald Thidemann Johansen a a Department of Pharmacology, School ofPharmac)', Uniuersity of Oslo, P.O. Box 1068 Blindern, 0316 Oslo, Norway b blstitute of Surgical Research, The National Hospital, Oslo, Norway Department of SuJ~ery B, The National Hospital, Oslo, Norway
Abstract The purpose of this prospective study was to characterize kininogens in plasma from surgical ICU patients. Thirty-five patients, ages 19-79 years, were divided into 2 groups: sepsis (defined by standard criteria) and nonsepsis. Studies of proteolytic degradation of H-kininogen showed degradation in both patient groups compared to healthy controls. Functional quantification of prekallikrein showed a reduction of prekallikrein in plasma from both patient groups. Functional quantification of kininogens by a CPI (cysteine proteinase inhibitor) assay showed no significant differences between the patients and the controls. Immunological levels of H-kininogen and total kininogen were not significantly different from normal plasma. No differences could be detected between the two patient groups in any of the parameters studied. In conclusion, this study supported contact activation taking place in surgical ICU patients, a partial kinin release and a consumption of prekallikrein has taken place in vivo. Keywords: Contact activation; Kininogen; Sepsis; Surgical intensive care
1. Introduction Sepsis is a life-threatening condition caused by the presence of microorganisms or their products in the circulation. Considerable evidence has accumulated that excessive activation of plasma cascade systems, the complement system and the contact
Abbreviations: ICU, intensive care unit; CPI, cysteine proteinase inhibitor; EIA, enzyme immunoassay; H-kininogen. high molecular weight kininogen; L-kininogen, low molecular weight kininogen; PAGE, polyacrylamide gel electrophoresis * Corresponding author. Tel.: +47 (22) 85 75 78. Fax: +47 (22) 85 75 I I; E-mail: [email protected].
system of intrinsic coagulation, with release of biologically active substances (anaphylatoxins and bradykinin), plays a role in the pathogenesis of sepsis (Aasen et al., 1980). Kininogens are multifunctional single-chain proteins, and in human plasma two different kininogens have been described: High molecular weight kininogen (H-kininogen, M r ~ 120,000) and low molecular weight kininogen (L-kininogen, M r ~ 65,000). The vasoactive peptide bradykinin is released from the kininogens by the action of proteolytic enzymes such as kallikreins. The kinins are known to exert a variety of physiological effects, including induction of hypotension, smooth muscle contraction and in-
0162-3109/96/$15.t)0 Copyright ,e) 1996 Elsevier Science B.V. All rights reserved PH S01 62-3 109(96)00087-2
366
T.S. Karlsrud et al. / Immunopharmacology 33 (1996) 365-368
70
crease of vascular permeability. The fact that activation of the bradykinin receptor stimulates synthesis of nitric oxide, brings new focus on this nonapeptide in pathophysiological mechanisms, e.g. in sepsis.
~ 6o 011
!,.~
50 40 '
~
2. P a t i e n t s
3. R e s u l t s
Proteolytic cleavage of H-kininogen was studied by PAG electrophoresis followed by immunoblotting. In non-reduced patient and control plasma samples, three characteristic bands named I ( M r 147,000), II ( M r 104,000) and III ( M r 99,000) were detected. There were significantly lower levels of native H-kininogen (band I) and significantly higher levels of band II + I I I (degraded forms) for both patient groups compared to controls all 3 days considered (Fig. 1). No differences were observed between the two patient groups. Functional quantification of prekallikrein showed significantly lower values in both the sepsis and the nonsepsis groups compared to control plasma on all 3 days considered (Table 1). No differences could be observed between the two patient groups. These prekallikrein values (% of control plasma) were sig-
N
8'
•
°,
3o
o
~ 20
Thirty-five patients (22 male and 13 female, ages 19-79 years; mean 52) treated in the surgical intensive care unit were studied. Sixteen of these patients (11 male and 5 female, ages 19-76 years; mean 46) fulfilled at least four of the following criteria of sepsis: temperature above 38°C, need of respiratory support, leucocyte count above 15 × 109//1 or below 5 × 109//1, a thrombocyte count below 100 X 109//1, positive blood cultures, or an obvious septic focus. Citrated plasma samples from the day of diagnosis (day 1) and the following 2 days (day 2 and day 3) were studied in the sepsis group. Samples from the first 3 days of ICU treatment were studied in the nonsepsis patient group (11 male and 8 female; ages 2 4 - 7 9 years; mean 56). Plasma collected from 9 healthy persons (4 female and 5 male; ages 3 0 - 6 0 years; mean 43) served as controls.
• 0 0
10 0 Control Day 1 Day2
Day3
Fig. 1. Relative amounts of band II + III of H-kininogen in control plasma (0), plasma from the sepsis ((3) and nonsepsis (0) groups. The differences between the patient and control plasmas were significant for both patient groups all 3 days considered. nificantly lower than the corresponding values of total protein for both patient groups ( p < 0.005). The median values of functional kallikrein inhibition on day 1 were lower in the sepsis ( p = 0.063) and nonsepsis ( p = 0.036) groups compared to controls. No further differences could be detected. Significantly lower CPI (cysteine proteinase inhibitor) activities compared to the control group were observed both in the sepsis and nonsepsis groups on all 3 days considered (Fig. 2). No differences could be observed between the patient groups. In the nonsepsis group the CPI activity was slightly 43.5 32.5,6
21.510.50
t
t"4
t'~
"1 Control
$e' ~sis
Nonse 9sis
Fig. 2. Plasma samples were acid treated and appropriately diluted before the CPI activity was measured as described in (Karlsrud et al., submitted). The bars show the median values, 10th and 90th percentiles are indicated.
367
T.S. Karlsrud et al./ lmmunopharmacology 33 (1996) 365-368
Table 1 Quantification of plasma kininogens, total protein, prekallikrein, and kallikrein inhibition n H-kininogen~' Total kininogen a Totalprotein b Sepsis
Nonsepsis
Day 1 Day2 Day 3 Day 1 Day2 Day3
Control
7 12 8 11 8 9 9
59 * (22-78) 66 * (17-99) 76 (17-119) 78 * (22-96) 28 (21-59) 65(34-123) 101 (78-116)
57 *~ (20 80) 49 (25 97) 49 (26-95) 73 (33-97) 40 *~ (8 73) 88(38 113) 101 (81-129)
37 ** (23-48) 38 ** (30-48) 42 (29-55) 40 (22-57) 41 * (34-57) 37 "* (31-46) 49 (46-56)
Prekallikreina
Kallikrein inhibitiona
42 * * (20-60) 41 ~* (28 65) 57 * * (23-92) 44 ** (27-78) 41 ** (13 49) 53 * (35-73) 122 (62-173)
96(58-102) 103 (72-123) 97 (83-126) 89 * (77-109) 108 (80-126) 116(91-127) 117 (20-149)
% of pool plasma; b g/l; ' p < 0.05, * ~ p < 0.005. increasing from day 1 to day 3 ( p = 0.08), no such increase could be seen in the sepsis group ( p = 0.51). Quantification of H-kininogen and total kininogen in patient and control plasma was performed by EIA methods. The results show significantly lower levels of H-kininogen and total kininogen in both patient groups on day 1 and 2 compared to the control group (Table 1). Day 3 shows lower level of total kininogen in the sepsis group. There were no significant differences observed between the two patient groups. Total protein measurements gave values about 7 5 - 8 5 % of control plasma in both patient groups (Table 1). Significantly lower levels compared to the controls were observed in the sepsis group on day 1 and 2 and in the nonsepsis group on day 2 and 3. No significant differences between the patient groups were observed.
4. Discussion In this study a significant degradation of Hkininogen was found in plasma from critically ill patients treated in the surgical ICU. There were no significant differences between septic and nonseptic patients. Proteolytic cleavage of H-kininogen has been described in several diseases, e.g. during attacks of hereditary angioedema (Cugno et al., 1993), but few studies have focused on this in ICU patients. Band II is produced by kinin liberation from the intact H-kininogen, while band III is formed by kallikrein-induced degradation of the light chain of H-kininogen from the native 56 kDa to 45 kDa. This bradykinin release might be of pathophysiological importance in terms of a stimulator of nitric oxide synthesis.
Another indication of contact activation is present by the significantly lower levels of prekallikrein in the patient samples, very likely due to a consumption of this proenzyme. Activation of the plasma kallikrein-kinin system has been repeatedly described in patients with septicemia (Aasen et al., 1980, 1983; Kalter et al., 1985), and may be partly responsible for the haemodynamic changes resulting in oedema frequently observed in these patients. A 10-fold increase in blood plasma cysteine proteinase activity has been reported in septic shock compared to healthy controls (Assfalg-Machleidt et al., 1988). Similar increases were not found in ICU patients, but the observed degradation of H-kininogen did not seem to reduce the total CPI activity. This confirms the observation that bradykinin release does not affect the CPI function of purified Lkininogen (Vogel et al., 1988). Although reduced levels of kininogens were found compared to control plasma, this difference disappears when relating to total concentration of proteins. This points to decreased production by the liver, increased clearance, extravasation, a n d / o r hemodilution as likely causes to the reduction, a general reduction probably affecting all plasma proteins. The functional consequences, however, could be of importance, since inhibitor control over proteolytic activation on cell surfaces still may be disturbed.
References Aasen AO, Smith-Erichsen N, Gallimore MJ, Amundsen E. Studies on components of the plasma kallikrein-kinin system in plasma samples from normal individuals and patients with septic shock. In: Schumer W, Spitzer JJ, Marshall BE, Eds.
368
T.S. Karlsrud et al. / lmmunopharmacology 33 (1996) 365-368
Avd. in Shock Res. Vol 4. New York: A.R. Liss. Inc., 1980; 1-10. Aasen AO, Smith-Eriksen N, Amundsen E. Plasma kallikrein-kinin system in septicemia. Arch Surg 1983; 118: 343-345. Assfalg-Machleidt I, Jochum M, Klaubert W, Inthorn D, Machleidt W. Enzymatically active cathepsin B dissociating from its inhibitor complexes is elevated in blood plasma of patients with septic shock and some malignant tumors. Biol Chem Hoppe-Seyler 1988; 369(suppl): 263-269. Cugno M, Hack CE, de Boer JP, Eerenberg AJM, Agostoni A,
Cicardi M. Generation of plasmin during attacks of hereditary angioedema. J Lab Clin Med 1993; 121: 38-43. Kalter ES, Daha MR, ten Cate JW, Verhoef J, Bouma BN. Activation and inhibition of Hageman factor-dependent pathways and the complement system in uncomplicated bacteremia or bacterial shock. J Infect Dis 1985; 151: 1019-1027. Vogel R, Assfalg-Machleidt I, Esterl A, Machleidt W, MtillerEsterl W. Proteinase-sensitive regions in the heavy chain of low molecular weight kininogen map to the inter-domain junctions. J Biol Chem 1988; 263: 12661-12668.