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Anticardiolipin antibodies in heparin-associated thrombocytopenia
THROMBOSIS RESEARCH 67; 601-606,1992 00493848/92 $5.00 + .OO Printed in the USA. Copyright (c) 1992 Pergamon Press Ltd. All rights reserved.
BRIEF ANTICARDIOLIPIN
Yves GRUEL’,
COMMUNICATION
ANTIBODIES IN HEPARIN-ASSOCIATED THROMBOCYTOPENIA
Alain RUPIN112, Herv6 WATIERZ,
Sandrine
VIGIERZ,
Pierre BARDOS2 and Jean LEROY1 1 Laboratoire
(Received
d’Hkmatologie, 2Laboratoire d’lmmunologie, Mbdecine, 37044 Tours Cedex, France.
11.51992;
accepted
in revised form 14.7.1992
Facultk
de
by Editor E. Angles Cano)
INTRODUCTION The antiphospholipid syndrome has been defined by the presence of antiphospholipid antibodies associated with thrombosis and/or thrombocytopenia (1). Heparin-associated thrombocytopenia (HAT) is another immune disorder related to heparin-dependent antibodies but also complicated in about 65 % of cases by arterial and/or venous thrombosis (2). Therefore, it was recently suggested that anticardiolipin antibodies might also be detected in HAT (3). In order to investigate this hypothesis, we looked for anticardiolipin antibodies (ACA) in a group of patients who had developed heparin-associated thrombocytopenia. MATERIAL AND METHODS Twenty-seven patients (13 females and 14 males, age range = 59-90) were All had a normal platelet count before heparin studied. administration for the prevention of postoperative thromboembolic disease (n = 16 with prosthetic surgery in 6 cases) or the treatment of deep vein thrombosis (n = 10) or myocardial infarction (n = 1). After 5 to 20 days of unfractionated heparin therapy, all patients exhibited a platelet In each case, the lower than 100.109/L. consistently count thrombocytopenia resolved within five days following discontinuation of ___-___-_-___-_-___-_~~~_-~~---_ Key Words: Anticardiolipin antibodies, Heparin, Thrombocytopenia 601
602
ANTICARDIOLIPIN
AND HEPARIN
Vol. 67, No. 5
unfractionated heparin and other causes (e.g. infection, other drugs) were excluded. Thrombotic complications were present in 16 cases with either previous venous thromboembolic disease (n = 6) or an aggravation of recent arterial thrombosis (n = 10). In 26 patients, heparin-induced aggregation tests performed as previously described (4), were found to be positive with the platelets from at least one normal donor. Samples for ACA assays in these patients were collected within12 to 72 hours after heparin discontinuation. We also studied 36 hospitalized patients with a normal platelet count (age range = 50-87) and receiving unfractionated heparin for 5 to 15 days either for cardiovascular surgery (coronary artery bypass graft or valvular prosthesis, n = 25) or an ischemic heart disease (n = 11). In this latter had a recent thrombotic event (5 acute myocardial group, 6 cases infarction and 1 deep vein thrombosis). In addition, ACA IgG and IgM were assayed both before and after 5 and10 days of heparin treatment. All samples (serum or plasma) were collected into vacutainer tubes, centrifuged (1,200 g, 15 mins), separated and kept frozen at -30°C until assays. Anticardiolipin antibodies of isotype IgG (ACA IgG) or IgM (ACA IgM) were assayed using a recently reported ELISA method (5). Results were expressed in GPL (ACA IgG) or MPL units (ACA IgM) after establishing standard curves using reference samples containing well-defined amounts of ACA. Finally and in order to seek for a crossreactivity of heparin-dependent antibodies with the cardiolipin molecules, all patients with HAT and found to have significant levels of ACA IgG or IgM were also tested after coincubation of samples with 5 different concentrations of unfractionated heparin, ie 0.25, 0.50, 0.75, 1 or 5 III/ml (6). RESULTS As shown in figure 1, seven patients with HAT exhibited significant levels of ACA IgG (Mean = 19.5, range = 7.5-72 GPL). In addition, two other cases having developed recent arterial thrombosis had high levels of ACA IgM (12.5 and 48.5 MPL). Among the 36 patients receiving heparin but without thrombocytopenia, 2 cases had, after 10 days of anticoagulation for an ischemic heart disease, significant levels of ACA IgG (7 and 30 GPL). Actually, one of these cases (with 30 GPL) had already and before the heparin treatment an ACA level of 45 GPL. On the other hand, 6 additional cases under heparinization and belonging to the group of patients having a cardiac surgery, had also a significant amount of ACA IgM (Mean = 17, range = 8-43 MPL) . For all patients having both heparin-associated thrombocytopenia and ACA, no significant modification of the anticardiolipin reactivity could be
Vol. 67, No. 5
ANTICARDIOLIPIN
AND HEPARIN
IgG
603
IgM
GPILr > 4(1
0 0 0
40
40
30
30
0
20
20
0 0
0
10
8
:
000 0
0
10
0
<5
--~----_----_-_-___
?? ooo.
:
.’.’.‘o
1.ooo”
DDnooo
00000
00000
c4
000000 •l~~~~org: : : : zg ? ooocboocbooo ? ?u ?cIooo
Figure 1 Anticardiolipin antibodies of isotype IgG or IgM assayed in HAT patients without ( 0 ) or with venous/arterial thrombosis ( 0 ) and in patients without thrombocytopenia and receiving unfractionated heparin for cardiovascular surgery ( 0 ) or ischemic heart disease ( El , assay after 10 days of heparin). The dotted line represents the cut-off values (5 GPL and 4 MPL) determined after testing 30 normal controls (5).
recorded after incubation of increasing concentrations
of their samples with cardiolipin in the of heparin (from 0.25 to 5 IU/ml).
presence
DISCUSSION Heparin-associated heparin treatment
thrombocytopenia (HAT) is the presence related to
a severe complication of heparin-dependent
of
604
ANTICARDIOLIPIN
AND HEPARIN
Vol. 67, No. 5
antibodies (2, 6). These antibodies bind onto platelets and lead to cell explain the thrombotic which in part and aggregation activation complications occurring in about 65 % of patients. As antiphospholipid antibodies were also implicated in the pathogenesis of thrombosis and the presence of such antibodies in HAT has been thrombocytopenia, questioned (3). This study shows that about one third (9/27) of patients with HAT may have anticardiolipin antibodies (ACA) mainly of IgG isotype (26 % vs 7.4 % with ACA IgM). Among the patients of similar age and receiving heparin 22 % (8/36) were also for 5 to 15 days, but without thrombocytopenia, ACA positive with a predominance of IgM ACA antibodies (6/36). This apparent high frequency of ACA cannot be attributed to the heparin treatment and might be associated to the existence of an ischemic heart disease or a coronary artery bypass graft as previously shown (7,8,9). In HAT, an association between the presence of ACA IgG and thrombosis cannot be established as we recently found in systemic lupus erythematosus (10) since a) no IgG anticardiolipin activity was detected in 15 of 16 sera from HAT cases with arterial or venous thrombosis, and b) the presence of these antiphospholipid antibodies prior to heparin administration (as clearly shown in one of the controls) cannot be excluded for the 7 patients having both HAT and IgG ACA. In addition, HAT mainly occurs in elderly patients exhibiting more frequently an ACA elevation (11) and undergoing surgery and/or venous thromboembolic disease. Heparins are negatively-charged sulfated molecules and if we do agree with the hypothesis that the specificity of heparin-dependent antibodies is restricted to heparin, it could be argued that, in some cases of HAT, these antibodies may also react with cardiolipin. However, this is unlikely since we did not find any increase in the anticardiolipin activity of ACA positive sera after incubation with heparin concentrations varying between 0.25 and 5 IU/ml. In addition, a lack of cross-reactivity between anticardiolipin antibodies and glycosaminoglycans such as heparin or heparan sulfate has been recently demonstrated (12). In conclusion, this study shows that ACA can be detected in HAT, but the presence of these antibodies must be cautiously interpretated and, could only depend on the indication of heparin treatment (thrombosis, prosthetic devices).
ACKNOWLEDGEMENTS We would like to thank Dr Lang and Dr Wrona from the hospital (France) for providing us with samples from heparinized patients.
of Blois
Vol. 67, No. 5
ANTICARDIOLIPIN
AND HEPARIN
605
REFERENCES 1. ASHERSON, R.A. 1742-1746, 1988. 2. WARKENTIN, T.E., 40, 3 l-44, Rev. Med.
A primary
KELTON, 1989.
antiphospholipid
syndrome.
J.G. Heparin-induced
3. COULL, B.M., GOODNIGHT, S.H. prethrombotic states, and stroke. Current Disease and Stroke 25, 13-17, 1990.
J. Rheumatol.
thrombocytopenia.
15,
Ann.
Antiphospholipid antibodies, Concepts of Cerebrovascular
4. LEROY,
J., LECLERC, M.H., DELAHOUSSE, B., GUEROIS G., FOLOPPE, P., GRUEL, Y., TOULEMONDE, F. Treatment of heparin-associated thrombocytopenia and thrombosis with Low Molecular Weight Heparin (CY 216). Sem. Thrombos. 1985. Haemostas. 11, 326-329,
5. RUPIN, A., GRUEL, Y., POUMIER-GASCHARD, P., CHASSAIGNE, M., LEROY, J. and BARDOS, P. Thrombocytopenia in systemic lupus erythematosus : association with antiplatelet and anticardiolipin antibodies. Clin. Zmmunol. Immunopathol. 55, 418-426, 1990. 6. GRUEL, Y., RUPIN, A., DARNIGE, L., MOALIC-REVERDIAU, P., POUMIERGASCHARD, P., BINET, C., BARDOS, P., LEROY. Specific quantification of heparin-dependent antibodies for the diagnosis of heparin-associated thrombocytopenia using an enzyme-linked immunosorbent assay. Thromb. Res. 62, 377-387, 1991. 7. MORTON, K.E., GAVAGHAN, T.P., DAGGARD, G.E., BARON, D.W., HICKIE, J.B., and CHESTERMAN, C.N. Coronary artery bypass graft failure. An autoimmune phenomenom ? Lancet. ii, 1353-1357, 1986. 8. KLEMP, P., COOPER, R.C., STRAUSS, F.J., JORDAAN, E.R., PRZYBOJEWSKI, J.Z., and NEL, N. Anti-cardiolipin antibodies in ischaemic heart disease. Clin. Exp. Immunol. 74, 254-257, 1988. 9. HAMSTEN, A., NORBERG, R., BJORKHOLM, M., DE FAIRE, U., HOLM, G. Antibodies to cardiolipin in young survivors of myocardial infarction: An association with recurrent cardiovascular events. Lancet i, 113-l 16, 1986. 10. RUPIN A., GRUEL, Y., LEROY, J., BARDOS, P. Anticardiolipin antibodies in Association with thrombosis if measured systemic lupus erythematosus. by Elisa with adult bovine serum as buffer rather than fetal calf serum. 60, 415-420, 1990. Thromb. Res.
606
ANTICARDIOLIPIN
AND HEPARIN
Vol. 67, No. 5
11. MANNOUSSAKIS, M.N., TZIOUFAS, A.G., SILIS, M.P., PANGE, P.J.E., GOUDEVENOS, J., and MOUTSAPOULOS, H.M. High prevalence of anticardiolipin and other autoantibodies in a healthy population. Cfin. Exp. Immunol. 69, 557-561, 1987. 12. GALLI, M., CORTELAZZO, S., BARBUI, T. Lack of cross-reactivity between anticardiolipin antibodies and glycosaminoglycans. Thromb. Res. 59, 363367, 1990.
BRIEF ANTICARDIOLIPIN
Yves GRUEL’,
COMMUNICATION
ANTIBODIES IN HEPARIN-ASSOCIATED THROMBOCYTOPENIA
Alain RUPIN112, Herv6 WATIERZ,
Sandrine
VIGIERZ,
Pierre BARDOS2 and Jean LEROY1 1 Laboratoire
(Received
d’Hkmatologie, 2Laboratoire d’lmmunologie, Mbdecine, 37044 Tours Cedex, France.
11.51992;
accepted
in revised form 14.7.1992
Facultk
de
by Editor E. Angles Cano)
INTRODUCTION The antiphospholipid syndrome has been defined by the presence of antiphospholipid antibodies associated with thrombosis and/or thrombocytopenia (1). Heparin-associated thrombocytopenia (HAT) is another immune disorder related to heparin-dependent antibodies but also complicated in about 65 % of cases by arterial and/or venous thrombosis (2). Therefore, it was recently suggested that anticardiolipin antibodies might also be detected in HAT (3). In order to investigate this hypothesis, we looked for anticardiolipin antibodies (ACA) in a group of patients who had developed heparin-associated thrombocytopenia. MATERIAL AND METHODS Twenty-seven patients (13 females and 14 males, age range = 59-90) were All had a normal platelet count before heparin studied. administration for the prevention of postoperative thromboembolic disease (n = 16 with prosthetic surgery in 6 cases) or the treatment of deep vein thrombosis (n = 10) or myocardial infarction (n = 1). After 5 to 20 days of unfractionated heparin therapy, all patients exhibited a platelet In each case, the lower than 100.109/L. consistently count thrombocytopenia resolved within five days following discontinuation of ___-___-_-___-_-___-_~~~_-~~---_ Key Words: Anticardiolipin antibodies, Heparin, Thrombocytopenia 601
602
ANTICARDIOLIPIN
AND HEPARIN
Vol. 67, No. 5
unfractionated heparin and other causes (e.g. infection, other drugs) were excluded. Thrombotic complications were present in 16 cases with either previous venous thromboembolic disease (n = 6) or an aggravation of recent arterial thrombosis (n = 10). In 26 patients, heparin-induced aggregation tests performed as previously described (4), were found to be positive with the platelets from at least one normal donor. Samples for ACA assays in these patients were collected within12 to 72 hours after heparin discontinuation. We also studied 36 hospitalized patients with a normal platelet count (age range = 50-87) and receiving unfractionated heparin for 5 to 15 days either for cardiovascular surgery (coronary artery bypass graft or valvular prosthesis, n = 25) or an ischemic heart disease (n = 11). In this latter had a recent thrombotic event (5 acute myocardial group, 6 cases infarction and 1 deep vein thrombosis). In addition, ACA IgG and IgM were assayed both before and after 5 and10 days of heparin treatment. All samples (serum or plasma) were collected into vacutainer tubes, centrifuged (1,200 g, 15 mins), separated and kept frozen at -30°C until assays. Anticardiolipin antibodies of isotype IgG (ACA IgG) or IgM (ACA IgM) were assayed using a recently reported ELISA method (5). Results were expressed in GPL (ACA IgG) or MPL units (ACA IgM) after establishing standard curves using reference samples containing well-defined amounts of ACA. Finally and in order to seek for a crossreactivity of heparin-dependent antibodies with the cardiolipin molecules, all patients with HAT and found to have significant levels of ACA IgG or IgM were also tested after coincubation of samples with 5 different concentrations of unfractionated heparin, ie 0.25, 0.50, 0.75, 1 or 5 III/ml (6). RESULTS As shown in figure 1, seven patients with HAT exhibited significant levels of ACA IgG (Mean = 19.5, range = 7.5-72 GPL). In addition, two other cases having developed recent arterial thrombosis had high levels of ACA IgM (12.5 and 48.5 MPL). Among the 36 patients receiving heparin but without thrombocytopenia, 2 cases had, after 10 days of anticoagulation for an ischemic heart disease, significant levels of ACA IgG (7 and 30 GPL). Actually, one of these cases (with 30 GPL) had already and before the heparin treatment an ACA level of 45 GPL. On the other hand, 6 additional cases under heparinization and belonging to the group of patients having a cardiac surgery, had also a significant amount of ACA IgM (Mean = 17, range = 8-43 MPL) . For all patients having both heparin-associated thrombocytopenia and ACA, no significant modification of the anticardiolipin reactivity could be
Vol. 67, No. 5
ANTICARDIOLIPIN
AND HEPARIN
IgG
603
IgM
GPILr > 4(1
0 0 0
40
40
30
30
0
20
20
0 0
0
10
8
:
000 0
0
10
0
<5
--~----_----_-_-___
?? ooo.
:
.’.’.‘o
1.ooo”
DDnooo
00000
00000
c4
000000 •l~~~~org: : : : zg ? ooocboocbooo ? ?u ?cIooo
Figure 1 Anticardiolipin antibodies of isotype IgG or IgM assayed in HAT patients without ( 0 ) or with venous/arterial thrombosis ( 0 ) and in patients without thrombocytopenia and receiving unfractionated heparin for cardiovascular surgery ( 0 ) or ischemic heart disease ( El , assay after 10 days of heparin). The dotted line represents the cut-off values (5 GPL and 4 MPL) determined after testing 30 normal controls (5).
recorded after incubation of increasing concentrations
of their samples with cardiolipin in the of heparin (from 0.25 to 5 IU/ml).
presence
DISCUSSION Heparin-associated heparin treatment
thrombocytopenia (HAT) is the presence related to
a severe complication of heparin-dependent
of
604
ANTICARDIOLIPIN
AND HEPARIN
Vol. 67, No. 5
antibodies (2, 6). These antibodies bind onto platelets and lead to cell explain the thrombotic which in part and aggregation activation complications occurring in about 65 % of patients. As antiphospholipid antibodies were also implicated in the pathogenesis of thrombosis and the presence of such antibodies in HAT has been thrombocytopenia, questioned (3). This study shows that about one third (9/27) of patients with HAT may have anticardiolipin antibodies (ACA) mainly of IgG isotype (26 % vs 7.4 % with ACA IgM). Among the patients of similar age and receiving heparin 22 % (8/36) were also for 5 to 15 days, but without thrombocytopenia, ACA positive with a predominance of IgM ACA antibodies (6/36). This apparent high frequency of ACA cannot be attributed to the heparin treatment and might be associated to the existence of an ischemic heart disease or a coronary artery bypass graft as previously shown (7,8,9). In HAT, an association between the presence of ACA IgG and thrombosis cannot be established as we recently found in systemic lupus erythematosus (10) since a) no IgG anticardiolipin activity was detected in 15 of 16 sera from HAT cases with arterial or venous thrombosis, and b) the presence of these antiphospholipid antibodies prior to heparin administration (as clearly shown in one of the controls) cannot be excluded for the 7 patients having both HAT and IgG ACA. In addition, HAT mainly occurs in elderly patients exhibiting more frequently an ACA elevation (11) and undergoing surgery and/or venous thromboembolic disease. Heparins are negatively-charged sulfated molecules and if we do agree with the hypothesis that the specificity of heparin-dependent antibodies is restricted to heparin, it could be argued that, in some cases of HAT, these antibodies may also react with cardiolipin. However, this is unlikely since we did not find any increase in the anticardiolipin activity of ACA positive sera after incubation with heparin concentrations varying between 0.25 and 5 IU/ml. In addition, a lack of cross-reactivity between anticardiolipin antibodies and glycosaminoglycans such as heparin or heparan sulfate has been recently demonstrated (12). In conclusion, this study shows that ACA can be detected in HAT, but the presence of these antibodies must be cautiously interpretated and, could only depend on the indication of heparin treatment (thrombosis, prosthetic devices).
ACKNOWLEDGEMENTS We would like to thank Dr Lang and Dr Wrona from the hospital (France) for providing us with samples from heparinized patients.
of Blois
Vol. 67, No. 5
ANTICARDIOLIPIN
AND HEPARIN
605
REFERENCES 1. ASHERSON, R.A. 1742-1746, 1988. 2. WARKENTIN, T.E., 40, 3 l-44, Rev. Med.
A primary
KELTON, 1989.
antiphospholipid
syndrome.
J.G. Heparin-induced
3. COULL, B.M., GOODNIGHT, S.H. prethrombotic states, and stroke. Current Disease and Stroke 25, 13-17, 1990.
J. Rheumatol.
thrombocytopenia.
15,
Ann.
Antiphospholipid antibodies, Concepts of Cerebrovascular
4. LEROY,
J., LECLERC, M.H., DELAHOUSSE, B., GUEROIS G., FOLOPPE, P., GRUEL, Y., TOULEMONDE, F. Treatment of heparin-associated thrombocytopenia and thrombosis with Low Molecular Weight Heparin (CY 216). Sem. Thrombos. 1985. Haemostas. 11, 326-329,
5. RUPIN, A., GRUEL, Y., POUMIER-GASCHARD, P., CHASSAIGNE, M., LEROY, J. and BARDOS, P. Thrombocytopenia in systemic lupus erythematosus : association with antiplatelet and anticardiolipin antibodies. Clin. Zmmunol. Immunopathol. 55, 418-426, 1990. 6. GRUEL, Y., RUPIN, A., DARNIGE, L., MOALIC-REVERDIAU, P., POUMIERGASCHARD, P., BINET, C., BARDOS, P., LEROY. Specific quantification of heparin-dependent antibodies for the diagnosis of heparin-associated thrombocytopenia using an enzyme-linked immunosorbent assay. Thromb. Res. 62, 377-387, 1991. 7. MORTON, K.E., GAVAGHAN, T.P., DAGGARD, G.E., BARON, D.W., HICKIE, J.B., and CHESTERMAN, C.N. Coronary artery bypass graft failure. An autoimmune phenomenom ? Lancet. ii, 1353-1357, 1986. 8. KLEMP, P., COOPER, R.C., STRAUSS, F.J., JORDAAN, E.R., PRZYBOJEWSKI, J.Z., and NEL, N. Anti-cardiolipin antibodies in ischaemic heart disease. Clin. Exp. Immunol. 74, 254-257, 1988. 9. HAMSTEN, A., NORBERG, R., BJORKHOLM, M., DE FAIRE, U., HOLM, G. Antibodies to cardiolipin in young survivors of myocardial infarction: An association with recurrent cardiovascular events. Lancet i, 113-l 16, 1986. 10. RUPIN A., GRUEL, Y., LEROY, J., BARDOS, P. Anticardiolipin antibodies in Association with thrombosis if measured systemic lupus erythematosus. by Elisa with adult bovine serum as buffer rather than fetal calf serum. 60, 415-420, 1990. Thromb. Res.
606
ANTICARDIOLIPIN
AND HEPARIN
Vol. 67, No. 5
11. MANNOUSSAKIS, M.N., TZIOUFAS, A.G., SILIS, M.P., PANGE, P.J.E., GOUDEVENOS, J., and MOUTSAPOULOS, H.M. High prevalence of anticardiolipin and other autoantibodies in a healthy population. Cfin. Exp. Immunol. 69, 557-561, 1987. 12. GALLI, M., CORTELAZZO, S., BARBUI, T. Lack of cross-reactivity between anticardiolipin antibodies and glycosaminoglycans. Thromb. Res. 59, 363367, 1990.