Haematologica, Vol 92, Issue 2, e17-e19 doi:10.3324/haematol.10575
Copyright © 2007 by Ferrata Storti Foundation
D. Llobet* ,
M. Borrell*, ,
L. Vila# ,
C. Vallvé* ,
R. Felices* ,
J. Fontcuberta*
An asymptomatic, 29-year-old woman was referred to our hospital before surgery because in the basic study of hemostasis she showed a prolonged thrombin time (TT) and a normal reptilase time (RT). She had not received any anticoagulants so, to account for these abnormal results the presence of an inhibitor or a dysfibrinogenemia was suspected. A 1:1 mixture of the patient’s plasma with control plasma did not correct the TT. Dysfibrinogenemia was excluded because the defibrinated plasma retained the inhibitory activity when mixed with normal plasma. When 0.02 mg/ml of Protamine Sulphate (a concentration that neutralizes 1 U/mL of heparin in normal plasma) was added to the patient’s plasma, the inhibitory activity did not disappear. IgG from the patient and from normal serum was isolated. The patient’s IgG was able to prolong the TT of a normal plasma and of a purified fibrinogen. The patient IgG did not impair the catalytic activity of thrombin, because no difference was observed in the hydrolysis of S-2238 by 1 U NIH human thrombin with normal or patient IgG. The time course of the thrombin–mediated fibrinopeptide-release from normal fibrinogen with the patient’s IgG, showed a delay in the fibrinopeptide B (FPB) release without affecting the fibrinopeptide A (FPA) release. This patient has an IgG antibody that delays fibrinopeptide B release of fibrinogen
An apparently healthy 29 years old woman without a tendency to bleed was referred to our center because of an abnormal coagulation test result that was detected prior to surgery for an ovarian cyst. The cyst was benign and was removed without any bleeding problems during or after the procedure. She had never been exposed to thrombin in fibrin glue. At present, she is healthy and asymptomatic.
In the routine coagulation screening APTT (activated partial thromboplastin time) and PT (prothrombin time) were in the normal range, but TT was prolonged in contrast with RT that was normal. The results of the coagulation test are shown in Table 1. No antiphospholipid antibodies were present. To determine if there were any other immunological abnormality, an immunological profile was performed. The results were normal and no monoclonal paraproteins were found (data not shown). The same results were obtained on 4 separated tests with different samples obtained over 2 years.
The prolonged TT with normal RT might be explained by the presence of heparin, but the patient did not receive heparin or any other anticoagulant. Also the samples had been properly obtained so a dysfibrinogenemia or an inhibitor of fibrin formation was suspected.
The effect of the patient’s IgG on thrombin activity was evaluated using the chromogenic substrate S-2238 for thrombin. No difference was observed in the substrate hydrolysis by 1 U NIH/mlL of human thrombin with normal IgG (residual thrombin obtained: 0.92 U/ml) or with the patient’s IgG (residual thrombin obtained: 1.03 U/ml). This result indicated that the antibody had no effect upon the catalytic activity of thrombin.
Despite the prolonged TT, the normal RT suggested that the antibody might interfere with the release of FPB. To test this possibility, we performed a time-curve of fibrinopeptide release from fibrinogen in the presence of patient and control IgG: normal fibrinogen (0.1 mL at 2 mg/mL) was mixed with 0.1 mL at 4 mg/ml of purified patient or control IgG. The mixtures were incubated 60 minutes at 37°C, then, 20 mL of 10 NIH U/ml of human thrombin was added and the samples incubated at 37°C. The reaction was stopped at different times by incubating the samples at 100°C. The heat-precipitate was removed by centrifugation and the supernatant containing the fibrinopeptides were lyophilized. The concentrations of FPA and FPB released at each incubation time were analized by HPLC 5, the results are showed in Figure 1. When the patient’s IgG was present, fibrinogen released less FPB than with the control IgG. In contrast, the FPA release was similar with patient or control IgG. The differences between FPB released from fibrinogen with control and with the patient’s IgG were maximal at 1 to 2 minutes, and decreased with longer thrombin incubation times. Thus, it can be concluded that the patient’s IgG produced a delay of FPB release from normal fibrinogen without affecting the FPA release. Figure 2 shows the algorithm of the diagnostic procedure.
Acquired inhibitors that interfere directly with fibrin formation are rare and most of them occur in patients with autoimmune disease, malignancy or without underlying causes. Most of these inhibitors are related to heparin-like substances that appear from a disturbance of the endothelium. Others are related to a high paraprotein concentration that interferes with the polymerization of the fibrin monomers.6–8 A third group of inhibitors consists of antibodies directed against the fibrinogen molecule6, 9–13 probably at or near the binding sites of the thrombin. These inhibitors delay the fibrinopeptide A or B release. Our patient belongs to this last group. She had an IgG antibody that interfered with the FPB release from normal fibrinogen. This abnormality does not seem to be clinically relevant. Nawarawong6 described a patient with an inhibitor that delayed FPB release and who did not present an abnormal bleeding history. Absence of a bleeding history has also been described in dysfibrinogenemias with delayed FPB release as the only abnormality.14 Unlike the case described by Nawarawong,6 our patient was healthy and did not exhibit any clinical or laboratory abnormalies.
Copyright © 2007 by Ferrata Storti Foundation
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An acquired inhibitor that produced a delay of fibrinopeptide B release in an asymptomatic patient
* Hemostasis Unit, Hematology Department, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain
# Laboratory of Inflammation Mediators, Institute of Research, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain
Correspondence: Montserrat Borrell, Hematology laboratory, Hospital de la Santa Creu i Sant Pau, Avda. St Antoni Ma Claret 167. 08024 Barcelona, Spain, E-mail: mborell{at}santpau.es, Tel: 34932919193 Fax: 34932919192
ABSTRACT
 TOP ABSTRACT Case ReportCoagulation assaysInhibitor detection and...Effect of patient IgG...Effect of patient IgG...DiscussionReferences |
|---|
An asymptomatic, 29-year-old woman was referred to our hospital before surgery because in the basic study of hemostasis she showed a prolonged thrombin time (TT) and a normal reptilase time (RT). She had not received any anticoagulants so, to account for these abnormal results the presence of an inhibitor or a dysfibrinogenemia was suspected. A 1:1 mixture of the patient’s plasma with control plasma did not correct the TT. Dysfibrinogenemia was excluded because the defibrinated plasma retained the inhibitory activity when mixed with normal plasma. When 0.02 mg/ml of Protamine Sulphate (a concentration that neutralizes 1 U/mL of heparin in normal plasma) was added to the patient’s plasma, the inhibitory activity did not disappear. IgG from the patient and from normal serum was isolated. The patient’s IgG was able to prolong the TT of a normal plasma and of a purified fibrinogen. The patient IgG did not impair the catalytic activity of thrombin, because no difference was observed in the hydrolysis of S-2238 by 1 U NIH human thrombin with normal or patient IgG. The time course of the thrombin–mediated fibrinopeptide-release from normal fibrinogen with the patient’s IgG, showed a delay in the fibrinopeptide B (FPB) release without affecting the fibrinopeptide A (FPA) release. This patient has an IgG antibody that delays fibrinopeptide B release of fibrinogen
Key words: Acquired inhibitors, Fibrinopeptide B, Thrombin time, Dysfibrinogenemia.
Case Report
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TOPABSTRACTCase ReportCoagulation assaysInhibitor detection and...Effect of patient IgG...Effect of patient IgG...DiscussionReferences |
|---|
An apparently healthy 29 years old woman without a tendency to bleed was referred to our center because of an abnormal coagulation test result that was detected prior to surgery for an ovarian cyst. The cyst was benign and was removed without any bleeding problems during or after the procedure. She had never been exposed to thrombin in fibrin glue. At present, she is healthy and asymptomatic.
Coagulation assays
|
TOPABSTRACTCase ReportCoagulation assaysInhibitor detection and...Effect of patient IgG...Effect of patient IgG...DiscussionReferences |
|---|
In the routine coagulation screening APTT (activated partial thromboplastin time) and PT (prothrombin time) were in the normal range, but TT was prolonged in contrast with RT that was normal. The results of the coagulation test are shown in Table 1. No antiphospholipid antibodies were present. To determine if there were any other immunological abnormality, an immunological profile was performed. The results were normal and no monoclonal paraproteins were found (data not shown). The same results were obtained on 4 separated tests with different samples obtained over 2 years.
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View this table: [in a new window] [Download PPT slide] |
Table 1. Results of the Coagulation Tests
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Inhibitor detection and characterization
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TOPABSTRACTCase ReportCoagulation assaysInhibitor detection and...Effect of patient IgG...Effect of patient IgG...DiscussionReferences |
|---|
The prolonged TT with normal RT might be explained by the presence of heparin, but the patient did not receive heparin or any other anticoagulant. Also the samples had been properly obtained so a dysfibrinogenemia or an inhibitor of fibrin formation was suspected.
A 1:1 mixture of the patient’s plasma with control plasma was incubated at 37 °C for 1 hour. This mixture exhibited a TT of 66 seconds in contrast to 21 seconds in the TT of control plasma and 122.8 seconds in the patient’s plasma. These results suggested that an inhibitor was present in the patient’s plasma. To confirm this, fibrinogen was precipitated from the patient’s plasma by heat and the defibrinated plasma retained the inhibitory activity. The TT obtained in the patient’s defibrinated plasma mixed with control plasma was 73.9 seconds versus 21.2 in control defibrinated plasma mixed with control plasma. These results indicated that the abnormality was not related to a defect in the fibrinogen molecule.
There are reports1–4 of patients who presented a heparin-like anticoagulant that prolonged the TT and could be neutralized by Protamine Sulphate (PS). To determine if this anticoagulant was heparin-like, we treated the patient’s plasma with 0.02 mg/mL of PS that is the minimum concentration of PS that neutralized 1 U/mL of heparin in a control plasma. Control plasma with 1 U/mL of heparin and 0.02 mg/mL of PS gave a TT of 19.2 seconds. After addition of 0.02 mg/mL of PS to the patient and control plasma, the TT were 170 seconds and 19.2 seconds respectively. This indicated that the anticoagulant was not neutralized by PS and that it was not a heparin-like substance.
We proceeded to purified IgG from patient and control serum using a column of protein-G-sepharose. The patient’s IgG when mixed with control plasma and compared with control IgG prolonged the TT from 29.3 to 66.1 seconds. The same results were obtained when IgG was mixed with purified fibrinogen. Patient and control IgG mixed with purified fibrinogen showed a TT of 95,6 and 62,5 seconds respectively. When IgG was removed from the patient’s serum (serum without IgG) the inhibitory activity was lost (serum without IgG plus control plasma show a TT: 20.5 seconds). These results indicated that the inhibitor was an IgG antibody.
Effect of patient IgG on thrombin activity
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TOPABSTRACTCase ReportCoagulation assaysInhibitor detection and...Effect of patient IgG...Effect of patient IgG...DiscussionReferences |
|---|
The effect of the patient’s IgG on thrombin activity was evaluated using the chromogenic substrate S-2238 for thrombin. No difference was observed in the substrate hydrolysis by 1 U NIH/mlL of human thrombin with normal IgG (residual thrombin obtained: 0.92 U/ml) or with the patient’s IgG (residual thrombin obtained: 1.03 U/ml). This result indicated that the antibody had no effect upon the catalytic activity of thrombin.
Effect of patient IgG on fibrinopeptide release from fibrinogen
|
TOPABSTRACTCase ReportCoagulation assaysInhibitor detection and...Effect of patient IgG...Effect of patient IgG...DiscussionReferences |
|---|
Despite the prolonged TT, the normal RT suggested that the antibody might interfere with the release of FPB. To test this possibility, we performed a time-curve of fibrinopeptide release from fibrinogen in the presence of patient and control IgG: normal fibrinogen (0.1 mL at 2 mg/mL) was mixed with 0.1 mL at 4 mg/ml of purified patient or control IgG. The mixtures were incubated 60 minutes at 37°C, then, 20 mL of 10 NIH U/ml of human thrombin was added and the samples incubated at 37°C. The reaction was stopped at different times by incubating the samples at 100°C. The heat-precipitate was removed by centrifugation and the supernatant containing the fibrinopeptides were lyophilized. The concentrations of FPA and FPB released at each incubation time were analized by HPLC 5, the results are showed in Figure 1. When the patient’s IgG was present, fibrinogen released less FPB than with the control IgG. In contrast, the FPA release was similar with patient or control IgG. The differences between FPB released from fibrinogen with control and with the patient’s IgG were maximal at 1 to 2 minutes, and decreased with longer thrombin incubation times. Thus, it can be concluded that the patient’s IgG produced a delay of FPB release from normal fibrinogen without affecting the FPA release. Figure 2 shows the algorithm of the diagnostic procedure.
 View larger version (8K): [in a new window] [Download PPT slide] |
Figure 1. Release of FPA and FPB of Patient and Control. 0.1 ml of patient or control IgG (4mg/ml) were mixed with 0.1 ml of fibrinogen at 2 mg/ml, incubated 1 hour at 37°C and treated with 20 µl of 10 NIH U/ml of thrombin. The reaction was stopped at various times by heating at 100°C. The precipitate was removed by centrifugation and the supernatant lyophilized. These samples were dissolved in 100 µl of acetic acid, centrifuged for 4 minutes at 15000 g and finally 20µl from the supernatant were injected into the HPLC. Closed triangles show FPA released from samples with control IgG; open triangles show FPA released from samples with patient IgG; closed stars show FPB released from samples with control IgG and open stars show FPB released from samples with patient IgG
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 View larger version (14K): [in a new window] [Download PPT slide] |
Figure 2. Algorithm chart of the diagnostic procure: Pp: patient plasma, Cp: control plasma, PS: protamine sulphate, Fg: fibrinogen, IgG: patient IgG, Thr: Thrombin, FP: Fibrinopeptide, FPB: Fibrinopeptide B.
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Discussion
|
TOPABSTRACTCase ReportCoagulation assaysInhibitor detection and...Effect of patient IgG...Effect of patient IgG...DiscussionReferences |
|---|
Acquired inhibitors that interfere directly with fibrin formation are rare and most of them occur in patients with autoimmune disease, malignancy or without underlying causes. Most of these inhibitors are related to heparin-like substances that appear from a disturbance of the endothelium. Others are related to a high paraprotein concentration that interferes with the polymerization of the fibrin monomers.6–8 A third group of inhibitors consists of antibodies directed against the fibrinogen molecule6, 9–13 probably at or near the binding sites of the thrombin. These inhibitors delay the fibrinopeptide A or B release. Our patient belongs to this last group. She had an IgG antibody that interfered with the FPB release from normal fibrinogen. This abnormality does not seem to be clinically relevant. Nawarawong6 described a patient with an inhibitor that delayed FPB release and who did not present an abnormal bleeding history. Absence of a bleeding history has also been described in dysfibrinogenemias with delayed FPB release as the only abnormality.14 Unlike the case described by Nawarawong,6 our patient was healthy and did not exhibit any clinical or laboratory abnormalies.
References
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TOPABSTRACTCase ReportCoagulation assaysInhibitor detection and...Effect of patient IgG...Effect of patient IgG...DiscussionReferences |
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- Chapman GS, George CB, Danley DL. Heparin-like anticoagulant associated with plasma cell myeloma. Am J Clin Pathol 1985;83:764-6.[Web of Science][Medline]
- Tefferi A, Owen BA, Nichols WL, Witzig TE, Owen WG. Isolation of a heparin-like anticoagulant from the plasma of a patient with metastatic bladder carcinoma. Blood 1989;74:252-4.
[Abstract/Free Full Text] - Khoory MS, Nesheim ME, Bowie EJ, Mann KG. Circulating heparin sulphate proteoglycan anticoagulant from a patient with a plasma cell disorder. J Clin Invest 1980;65:666-74.[Web of Science][Medline]
- Bussel JB, Steinherz PG, Miller DR, Hilgartner MW. A heparin-like anticoagulant in an 8-month-old boy with acute monoblastic leukaemia. Am J Hematol 1984;16:83-90.[Web of Science][Medline]
- Kehl M, Lottspeich F, Henschen A. Analysis of human fibrinopeptides by high-performance liquid chromatography. Hoppe-Seyler’s Z Physiol Chem 1981;362:1661-4.[Web of Science][Medline]
- Nawarawong W, Wyshock E, Meloni FJ, Weitz J, Schmaier AH. The rate of fibrinopeptide B release modulates the rate of clot formation: a study with an acquired inhibitor to fibrinopeptide B release. Br J Haematol 1991;79:296-301.[Web of Science][Medline]
- Davey FR, Gordon GB, Boral LI, Gottlieb AJ. Gamma globulin inhibition of fibrin clot formation. Ann Clin Lab Sci 1976;6:72-7.[Abstract]
- ohler A, Redondo M, Lammle B. Increased thrombin time in a patient wih multiple myeloma. Ther Umsch 1999;56:491-4.[CrossRef][Medline]
- Gris JC, Schved JF, Branger B, Aguilar-Martinez P, Vécina F, Oulès R, et al. Autoantibody to plasma fibrinopeptide A in a patient with a severe acquired haemorragic syndrome. Blood Coagul Fibrinolysis 1992;3:519-29.[CrossRef][Web of Science][Medline]
- Hoots WK, Carrell NA, Wagner RH, Cooper HA, McDonagh J. A naturally occurring antibody that inhibits fibrin monomer polymerization. N Engl J Med 1981;304:857-61.[Abstract]
- Ghosh S, McEvoy P, McVerry BA. Idiopathic autoantibody that inhibits fibrin monomer polymerization. Br J Haematol 1983;53:65-72.[Web of Science][Medline]
- Ruiz-Arguelles A. Spontaneous reversal of acquired auto immune dysfibrinogenemia probably due to an antiidiotypic autoantibody directed to an interspecies cross-reactive idio-type expressed on fibrinogen antibodies. J Clin Invest 1988;82:958-63.[Web of Science][Medline]
- Marciniak E, Greenwood MF. Acquired coagulation inhibitor delaying fibrinopeptide release. Blood 1979;53:81-92.
[Abstract/Free Full Text] - Hanss M, Biot F. A Database For Human Fibrinogen Variants. Ann N 2001;936:89-90. http//www.geht.org/databaseang/fibrinogen.
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