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Elevated plasmin-α2-antiplasmin complex levels in hereditary angioedema: Evidence for the in vivo efficiency of the intrinsic fibrinolytic system
THROMBOSIS RESEARCH 40; 817-821, 1985 0049-3848/85 $3.00 t .OO Printed in the USA. Copyright (c) 1985 Pergamon Press Ltd. All rights reserved. ??
ELEVATED PLASMIN-a2-ANTIPLASMIN COMPLEX LEVELS IN HEREDITARY ANGIOEDEMA: EVIDENCE FOR THE IN VIVO EFFICIENCY OF THE INTRINSIC FIBRINOLYTIC SYSTEM
Torbjarn Nilsson and Ove Back Departments of Clinical Chemistry and Dermatology, University Hospital, S-901 85, UmeZ, Sweden
(Received 15.7.1985; Accepted in revised by Editor H. Suomela)
form 11.9.1985
ABSTRACT The contact activation and fibrinolytic systems were assessed in 5 patients with hereditary angioedema (HAE) Reductioqs in F XII levels and increase in kallikreinlike activity in some patients indicated activation of the contact (intrinsic) system of coagulation. A great increase in plasmin-a2-antiplasmin complex in all subjects indicated that in this disease, there is a constantly ongoing fibrinolysis. Since CT-inhibitor, the deficient protein in HAE, is a poor inhibitor of the well-known extrinsic (tissuetype) plasminogen activator, but the major inhibitor of the contact activation system and a related in vitro phenomenon termed intrinsic fibrinolysis, our data show that this fibrinolytic system is also sometimes operating efficiently in vivo. Furthermore, the known clinical data on HAE are compatible with a role of intrinsic fibrinolysis in the pathophysiology of this disease
INTRODUCTION In hereditary angioedema (HAE) there is a functional deficiency of the Cl-inhibitor (Cl INH), which results in the activation of the classical pathway of the complement system and low plasma levels of C4 and C2. However, this mechanism may not be
Key words: Hereditary angioedema, F XII, Ci-inhibitor, a2-antiplasmin complex, intrinsic fibrinolysis. 817
plasmin-
818
HEREDITARY ANGIOEDEMA
Vol. 40, No. 6
the cause of the symptoms of the disease, since symptoms occur only intermittently whereas the C4 reduction is quite constant, and furthermore it seems doubtful whether any kinin activity can be generated via cleavage of C2, at least not in vitro (1). Cl INH isalso the main inhibitor of factor XII and kallikrein, the contact activation factors of the coagulation system. Therefore it has been speculated that these factors may become activated more easily in HAE patients, thus generating bradykinin which might be responsible for the initiation of the symptoms (1, 2). If so one would expect to find lowered levels of these factors in HAE, but quite high levels of F XII have been reported (3). We have therefore readdressed this question with recently developed assays for the factors of interest.
MATERIALS
AND METHODS
Patients. Citrated plasma was collected in siliconized tubes and stored at -7OOC until analyzed. Five adult patients with the classical variety of HAE were studied. Three patients were untreated while two were on intermittent danazol therapy (4). Factor XII was assayed in a system containing dextran sulphate and a large excess of prekallikrein, and the kallikrein generation was measured with a chromogenic substrate (5). Prekallikrein (PKK) was assayed by measuring the kallikrein generation in a system containing optimized amounts of activator, F XIIf and HMW-kininogen (6). The kit Coatest Prekallikrein (Kabi, Stockholm) was used. Cl-inhibitor was measured by-a chromogenic substrate assay based on inhibition of purified Cls (7). Free kallikrein-like activity (KK-LA) in plasma, probably reflecting activated proteolytic enzymes trapped in a2-macroglobulin (61, was determined with the substrate S-2302 by a slight modification (8) of the method of Friberger (6). Plasminogen-a2-antiplasmin complex (PAP), reflecting ongoing or recent fibrinolysis, was measured by radioimmunoassay (9). a2-Anti lasmin was measured by a chromogenic substrate activity -.+ assay
RESULTS As seen in table 1, the constant findings are the low Cl INH and the markedly increased PAP concentrations. Prekallikrein can be high, normal or low, factor XII is normal or somewhat reduced, and kallikrein-like activity is either normal or very high. The a2-antiplasmin levels are normal. There seems to be a correlation between high PAP levels and low F XII levels (r=0.96), indicating that the PAP generation is linked to consumption of F XII.
Table 1
Contact activation in 5 HAE patients
Analysis
819
HEREDITARYANGIOEDEMA
Vol. 40, No. 6
system and fibrinolysis
activation
F XII
PKK
KK-LA
CT INH
PAP
ct2AP
%
%
mA/min
umol/l
mg/l
%
Pat No 1x)F
100
75
48
0.17
1.8
87
2X)M
48
117
111
0.33
12.8
91
3M
94
160
3
0.14
2.6
98
4M
80
13
1
0.08
7.5
93
5F
79
67
77
0.21
9.0
111
80
86
48
0.18
6.7
96
Mean value
Normal range 80-120 xl on danazol
80-120
l-5
1.3-1.9
0
80-120
therapy
DISCUSSION
In a recent study, factor XII was said to be very high (174%) in HAE, and fibrinolytic activation was denied since plasminogen and a2-antiplasminwere normal (3). However, these authors used the clotting assay for F XII which may be influenced by the low Cl INH levels in HAE. Since plasmin-a2-antiplasmin complex is not detectable in normal plasma, this assay is more sensitive in detecting fibrinolysis than reductions of plasminogen or a2-antiplasmin levels. Our findings that HAE patients may have reduced F XII and/ or prekallikrein, increased kallikrein-like activity, and increased plasmin-a2-antiplasmin complex levels, demonstrate that in HAE, the low Cl INH renders the contact activation, the complement and the fibrinolytic systems instable and susceptible to activation. Such high PAP levels as in these HAE patients are in fact seldom seen during physiological activation of the extrinsic pathway of fibrinolysis involving the tissue-type plasminogen activator, as for instance in the post-operative period (10) or during the venous occlusion test (11, 12). Furthermore, we have shown that in vitro the Cl INH is a very poor inhibitor of the extrinsic tissue plasminogen activator (13). The presence of an intrinsic fibrinolytic activity has been recognized for a long time, and extensive in vitro studies have been reported (14, 15, 16). To our knowledge, its presence in vivo has never been assessed. Our observation, that the verylow Cl INH activity in HA.H is associated with a marked fibrinolytic
820
HEREDITARY ANGIOEDEMA
Vol. 40, No. 6
activation is compatible with the facts that HAE is not associated with thrombosis, that antifibrinolytic therapy (6_aminohexanoid acid, tranexamic acid) prevents the symptoms in HAE, and that degradation of fibrin, i.e. fibrinolysis, may result in generation of vasoactive peptides17). Whether the intrinsic activators primarily cause fibrinolysis or fibrinogenolysis can however not be decided until these enzymes have been properly characterized. It is interesting here to compare with our findings in patients with cold urticaria. They had slightly reduced Cl INH activity which gave an increased susceptibility to cold-promoted F VII activation, a process ultimately dependent on F XII activation, but no increase in PAP levels (8). We conclude that the exceptionally high degree of fibrinolysis seen in the resting state in HAE patients indicates the in vivo activation of a potent intrinsic fibrinolytic system, as well as its possible involvement in the pathophysiology of hereditary angioedema.
ACKNOWLEDGEMENTS The study was supported by grants from the Edvard Welander Foundation and the Medical Faculty, Umea University. We are grateful to Dr. BjtirnWiman, Karolinska Hospital, Stockholm, for the reagents of the plasmin-a2-antiplasmin complex assay, and Dr. Gerd MichaGlsson and Dr. Lars Hillstrom for supplying plasma from HAE patients.
REFERENCES
1.
FIELDS, T., GHEBREHIWET, B. and KAPLAN, A.P. Kinin formation in hereditary angioedema plasma: evidence against kinin derivation from C2 and in support of 'spontaneous' formation of bradykinin. J.Allerg.Clin.Immunol. 2, 54-60, 1983.
2.
CURD, J.G., YELVINGTON, M. and BURRIDGE, N. Generation of bradykinin during incubation of hereditary angioedema plasma. Mol.Immunol. 19, 1365, 1983.
3.
N. and DICK, W. CompleCULLMAN, W., KUVARY, P.M., MijTLLER, ment, coagulation and fibrinolytic parameters in hereditary angioedema (HAE). Clin.Exp.Immunol. 49, 618-622, 1982.
4.
GELFAND, J.A., SHERINS, R.J., ALLING, D.W. and FRANK, M.M. Treatment of hereditary angioedema with danazol. Reversal of clinical and biochemical abnormalities. N.Engl.J.Med. 295, 1444-1448, 1976.
Vol. 40, No. 6
HEREDITARYANGIOEDEMA
821
5.
TANKERSLEY, D.L., ALVING, B.M. and FINLAYSON, J.S. Activation of factor XII by dextran sulphate: the basis for an assay of factor XII. -Blood 62, 448-456, 1983.
6.
FRIBERGER, P. Chromogenic peptide substrates. Their use for the assay of factors in the fibrinolytic and the plasma kallikrein-kinin systems. Scand.J.Clin.Lab.Invest. suppl. 162, 1982.
7.
WIMAN, B. and NILSSON, T. A new simple method for determination of CI-esterase inhibitor activity in plasma. Clin.Chim. Acta 128, 359-366, 1983. --
8.
NILSSON, T. and BACK, 0. On the role of the Ci-esterase inhibitor in cold urticaria. Acta Derm.Venereol. (Stockh) 64, 197-202, 1984.
9.
WIMAN, B., JACOBSSON, L., ANDERSSON, M. and MELLBRING, G. Determination of plasmin-a2-antiplasmin complex in plasma samples by means of a radioimmunoassay. Scand.J.Clin.Lab. Invest. 43, 27-33, 1983.
10. WIMAN, B., MELLBRING, G. and tiNBY, M. Plasminogen activator release during venous stasis and exercise as determined by a new specific assay. Clin.Chim.Acta 127, 279-288, 1982. 11. BERGSDORF, N., NILSSON, T. and WALLEN, P. An enzyme linked immunosorbent assay for determination of tissue plasminogen activator applied to patients with thromboembolic disease. Thromb.Haemost. 50, 740-744, 1983. 12. MELLBRING, G., DAHLGREN, S., REIZ, S. and WIMAN, B. Fibrinolytic activity in plasma and deep vein thrombosis after major abdominal surgery. Thromb.Res. 32, 575-584, 1983. 13. RANBY, M., BERGSDORF, N. and NILSSON, T. Enzymatic properties of one- and two-chain form of tissue plasminogen activator. Thromb.Res. 27, 175-183, 1982. 14. NIEWIAROWSKI, S. and PROU-WARTELLE, 0. Rale du facteur contact (facteur Hagernan) dans la fibrinolyse. Thromb.Diath. Haemorrh. 3, 593-603, 1959. 15. OGSTON, D., OGSTON, C.M., RATNOFF, O.D. and FORBES, C.D. Studies on a complex mechanism for the activation of plasminogen by kaolin and chloroform: the participation of Haqeman factor and additional cofactors. J,Clin.Invest. 48, 17861801, 1969. 16. KLUFT, C., TRUMPI-KALSHOVEN, M.M., JIE, A.F.H. and VELDHUYZENSTOLK, E.C. Factor XII-dependent fibrinolysis: A double function of plasma kallikrein and the occurrence of a previously undescribed factor XII and kallikrein-dependent plasminogen proactivator. Thromb.Haemost. 41, 756-773, 1979. 17. SALDEEN, T. Vasoactive peptides derived from degradation of fibrinogen and fibrin. Ann.N.Y.Acad.Sci. 408, 424-437, 1983.
ELEVATED PLASMIN-a2-ANTIPLASMIN COMPLEX LEVELS IN HEREDITARY ANGIOEDEMA: EVIDENCE FOR THE IN VIVO EFFICIENCY OF THE INTRINSIC FIBRINOLYTIC SYSTEM
Torbjarn Nilsson and Ove Back Departments of Clinical Chemistry and Dermatology, University Hospital, S-901 85, UmeZ, Sweden
(Received 15.7.1985; Accepted in revised by Editor H. Suomela)
form 11.9.1985
ABSTRACT The contact activation and fibrinolytic systems were assessed in 5 patients with hereditary angioedema (HAE) Reductioqs in F XII levels and increase in kallikreinlike activity in some patients indicated activation of the contact (intrinsic) system of coagulation. A great increase in plasmin-a2-antiplasmin complex in all subjects indicated that in this disease, there is a constantly ongoing fibrinolysis. Since CT-inhibitor, the deficient protein in HAE, is a poor inhibitor of the well-known extrinsic (tissuetype) plasminogen activator, but the major inhibitor of the contact activation system and a related in vitro phenomenon termed intrinsic fibrinolysis, our data show that this fibrinolytic system is also sometimes operating efficiently in vivo. Furthermore, the known clinical data on HAE are compatible with a role of intrinsic fibrinolysis in the pathophysiology of this disease
INTRODUCTION In hereditary angioedema (HAE) there is a functional deficiency of the Cl-inhibitor (Cl INH), which results in the activation of the classical pathway of the complement system and low plasma levels of C4 and C2. However, this mechanism may not be
Key words: Hereditary angioedema, F XII, Ci-inhibitor, a2-antiplasmin complex, intrinsic fibrinolysis. 817
plasmin-
818
HEREDITARY ANGIOEDEMA
Vol. 40, No. 6
the cause of the symptoms of the disease, since symptoms occur only intermittently whereas the C4 reduction is quite constant, and furthermore it seems doubtful whether any kinin activity can be generated via cleavage of C2, at least not in vitro (1). Cl INH isalso the main inhibitor of factor XII and kallikrein, the contact activation factors of the coagulation system. Therefore it has been speculated that these factors may become activated more easily in HAE patients, thus generating bradykinin which might be responsible for the initiation of the symptoms (1, 2). If so one would expect to find lowered levels of these factors in HAE, but quite high levels of F XII have been reported (3). We have therefore readdressed this question with recently developed assays for the factors of interest.
MATERIALS
AND METHODS
Patients. Citrated plasma was collected in siliconized tubes and stored at -7OOC until analyzed. Five adult patients with the classical variety of HAE were studied. Three patients were untreated while two were on intermittent danazol therapy (4). Factor XII was assayed in a system containing dextran sulphate and a large excess of prekallikrein, and the kallikrein generation was measured with a chromogenic substrate (5). Prekallikrein (PKK) was assayed by measuring the kallikrein generation in a system containing optimized amounts of activator, F XIIf and HMW-kininogen (6). The kit Coatest Prekallikrein (Kabi, Stockholm) was used. Cl-inhibitor was measured by-a chromogenic substrate assay based on inhibition of purified Cls (7). Free kallikrein-like activity (KK-LA) in plasma, probably reflecting activated proteolytic enzymes trapped in a2-macroglobulin (61, was determined with the substrate S-2302 by a slight modification (8) of the method of Friberger (6). Plasminogen-a2-antiplasmin complex (PAP), reflecting ongoing or recent fibrinolysis, was measured by radioimmunoassay (9). a2-Anti lasmin was measured by a chromogenic substrate activity -.+ assay
RESULTS As seen in table 1, the constant findings are the low Cl INH and the markedly increased PAP concentrations. Prekallikrein can be high, normal or low, factor XII is normal or somewhat reduced, and kallikrein-like activity is either normal or very high. The a2-antiplasmin levels are normal. There seems to be a correlation between high PAP levels and low F XII levels (r=0.96), indicating that the PAP generation is linked to consumption of F XII.
Table 1
Contact activation in 5 HAE patients
Analysis
819
HEREDITARYANGIOEDEMA
Vol. 40, No. 6
system and fibrinolysis
activation
F XII
PKK
KK-LA
CT INH
PAP
ct2AP
%
%
mA/min
umol/l
mg/l
%
Pat No 1x)F
100
75
48
0.17
1.8
87
2X)M
48
117
111
0.33
12.8
91
3M
94
160
3
0.14
2.6
98
4M
80
13
1
0.08
7.5
93
5F
79
67
77
0.21
9.0
111
80
86
48
0.18
6.7
96
Mean value
Normal range 80-120 xl on danazol
80-120
l-5
1.3-1.9
0
80-120
therapy
DISCUSSION
In a recent study, factor XII was said to be very high (174%) in HAE, and fibrinolytic activation was denied since plasminogen and a2-antiplasminwere normal (3). However, these authors used the clotting assay for F XII which may be influenced by the low Cl INH levels in HAE. Since plasmin-a2-antiplasmin complex is not detectable in normal plasma, this assay is more sensitive in detecting fibrinolysis than reductions of plasminogen or a2-antiplasmin levels. Our findings that HAE patients may have reduced F XII and/ or prekallikrein, increased kallikrein-like activity, and increased plasmin-a2-antiplasmin complex levels, demonstrate that in HAE, the low Cl INH renders the contact activation, the complement and the fibrinolytic systems instable and susceptible to activation. Such high PAP levels as in these HAE patients are in fact seldom seen during physiological activation of the extrinsic pathway of fibrinolysis involving the tissue-type plasminogen activator, as for instance in the post-operative period (10) or during the venous occlusion test (11, 12). Furthermore, we have shown that in vitro the Cl INH is a very poor inhibitor of the extrinsic tissue plasminogen activator (13). The presence of an intrinsic fibrinolytic activity has been recognized for a long time, and extensive in vitro studies have been reported (14, 15, 16). To our knowledge, its presence in vivo has never been assessed. Our observation, that the verylow Cl INH activity in HA.H is associated with a marked fibrinolytic
820
HEREDITARY ANGIOEDEMA
Vol. 40, No. 6
activation is compatible with the facts that HAE is not associated with thrombosis, that antifibrinolytic therapy (6_aminohexanoid acid, tranexamic acid) prevents the symptoms in HAE, and that degradation of fibrin, i.e. fibrinolysis, may result in generation of vasoactive peptides17). Whether the intrinsic activators primarily cause fibrinolysis or fibrinogenolysis can however not be decided until these enzymes have been properly characterized. It is interesting here to compare with our findings in patients with cold urticaria. They had slightly reduced Cl INH activity which gave an increased susceptibility to cold-promoted F VII activation, a process ultimately dependent on F XII activation, but no increase in PAP levels (8). We conclude that the exceptionally high degree of fibrinolysis seen in the resting state in HAE patients indicates the in vivo activation of a potent intrinsic fibrinolytic system, as well as its possible involvement in the pathophysiology of hereditary angioedema.
ACKNOWLEDGEMENTS The study was supported by grants from the Edvard Welander Foundation and the Medical Faculty, Umea University. We are grateful to Dr. BjtirnWiman, Karolinska Hospital, Stockholm, for the reagents of the plasmin-a2-antiplasmin complex assay, and Dr. Gerd MichaGlsson and Dr. Lars Hillstrom for supplying plasma from HAE patients.
REFERENCES
1.
FIELDS, T., GHEBREHIWET, B. and KAPLAN, A.P. Kinin formation in hereditary angioedema plasma: evidence against kinin derivation from C2 and in support of 'spontaneous' formation of bradykinin. J.Allerg.Clin.Immunol. 2, 54-60, 1983.
2.
CURD, J.G., YELVINGTON, M. and BURRIDGE, N. Generation of bradykinin during incubation of hereditary angioedema plasma. Mol.Immunol. 19, 1365, 1983.
3.
N. and DICK, W. CompleCULLMAN, W., KUVARY, P.M., MijTLLER, ment, coagulation and fibrinolytic parameters in hereditary angioedema (HAE). Clin.Exp.Immunol. 49, 618-622, 1982.
4.
GELFAND, J.A., SHERINS, R.J., ALLING, D.W. and FRANK, M.M. Treatment of hereditary angioedema with danazol. Reversal of clinical and biochemical abnormalities. N.Engl.J.Med. 295, 1444-1448, 1976.
Vol. 40, No. 6
HEREDITARYANGIOEDEMA
821
5.
TANKERSLEY, D.L., ALVING, B.M. and FINLAYSON, J.S. Activation of factor XII by dextran sulphate: the basis for an assay of factor XII. -Blood 62, 448-456, 1983.
6.
FRIBERGER, P. Chromogenic peptide substrates. Their use for the assay of factors in the fibrinolytic and the plasma kallikrein-kinin systems. Scand.J.Clin.Lab.Invest. suppl. 162, 1982.
7.
WIMAN, B. and NILSSON, T. A new simple method for determination of CI-esterase inhibitor activity in plasma. Clin.Chim. Acta 128, 359-366, 1983. --
8.
NILSSON, T. and BACK, 0. On the role of the Ci-esterase inhibitor in cold urticaria. Acta Derm.Venereol. (Stockh) 64, 197-202, 1984.
9.
WIMAN, B., JACOBSSON, L., ANDERSSON, M. and MELLBRING, G. Determination of plasmin-a2-antiplasmin complex in plasma samples by means of a radioimmunoassay. Scand.J.Clin.Lab. Invest. 43, 27-33, 1983.
10. WIMAN, B., MELLBRING, G. and tiNBY, M. Plasminogen activator release during venous stasis and exercise as determined by a new specific assay. Clin.Chim.Acta 127, 279-288, 1982. 11. BERGSDORF, N., NILSSON, T. and WALLEN, P. An enzyme linked immunosorbent assay for determination of tissue plasminogen activator applied to patients with thromboembolic disease. Thromb.Haemost. 50, 740-744, 1983. 12. MELLBRING, G., DAHLGREN, S., REIZ, S. and WIMAN, B. Fibrinolytic activity in plasma and deep vein thrombosis after major abdominal surgery. Thromb.Res. 32, 575-584, 1983. 13. RANBY, M., BERGSDORF, N. and NILSSON, T. Enzymatic properties of one- and two-chain form of tissue plasminogen activator. Thromb.Res. 27, 175-183, 1982. 14. NIEWIAROWSKI, S. and PROU-WARTELLE, 0. Rale du facteur contact (facteur Hagernan) dans la fibrinolyse. Thromb.Diath. Haemorrh. 3, 593-603, 1959. 15. OGSTON, D., OGSTON, C.M., RATNOFF, O.D. and FORBES, C.D. Studies on a complex mechanism for the activation of plasminogen by kaolin and chloroform: the participation of Haqeman factor and additional cofactors. J,Clin.Invest. 48, 17861801, 1969. 16. KLUFT, C., TRUMPI-KALSHOVEN, M.M., JIE, A.F.H. and VELDHUYZENSTOLK, E.C. Factor XII-dependent fibrinolysis: A double function of plasma kallikrein and the occurrence of a previously undescribed factor XII and kallikrein-dependent plasminogen proactivator. Thromb.Haemost. 41, 756-773, 1979. 17. SALDEEN, T. Vasoactive peptides derived from degradation of fibrinogen and fibrin. Ann.N.Y.Acad.Sci. 408, 424-437, 1983.