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Acquired inhibitors of certain coagulation factors are relatively common, but autoantibodies against thrombin occur infrequently.4 Most acquired inhibitors of thrombin appear in patients exposed to topical preparations of bovine thrombin or fibrin "glue" during major surgery; these antibodies react mainly with bovine coagulation proteins.5,6,7,8 Acquired inhibitors directed primarily against human thrombin are usually associated with predisposing conditions7 and are extremely rare in patients without an underlying disease.
In this report, we describe a patient with a fatal bleeding diathesis caused by a spontaneously acquired inhibitor of thrombin. Clinical evaluation, laboratory investigation, and a full autopsy failed to reveal an underlying disease. Certain features of the clinical presentation and laboratory results should permit the early recognition of antibody inhibitors of thrombin and allow the institution of therapy.
Case Report
The patient, a 72-year-old woman, presented with rectal bleeding, ecchymoses on the chest and back, and a hematoma of four days' duration on the upper right arm, measuring 6 by 10 cm. The hemoglobin level was 6.8 g per deciliter (4.22 mmol per liter), and the hematocrit was 20 percent, with normal red-cell indexes. The platelet count and the white-cell count were also normal. Results of coagulation tests were as follows: prothrombin time, 17.1 seconds (international normalized ratio, 2.1), activated partial-thromboplastin time, 60 seconds (ratio, 2.0), and thrombin time, 118 seconds (normal range, 16 to 27 seconds). The fibrinogen level was 3.81 g per liter (11.2 µmol per liter) (normal range, 1.5 to 4.0 g per liter [4.4 to 11.8 µmol per liter]), and the d-dimer assay for detection of fibrin-degradation products was not elevated. Levels of serum iron, folate, and B12 were normal, and the reticulocyte index was 4.6 percent (normal range, 0.5 to 1.7 percent).
The patient was not taking any medications, including nonsteroidal antiinflammatory agents, and denied any history of bleeding or alcohol abuse. Except for an appendectomy at the age of 10, she had never undergone a major surgical procedure.
The patient received two units of packed red cells, which increased her hemoglobin level to 10.4 g per deciliter (6.45 mmol per liter). On the fourth day after admission, flexible sigmoidoscopy established that the source of the patient's rectal bleeding was an anal hemorrhoid. Further studies of her coagulopathy demonstrated an IgG antibody directed against thrombin. Her hemoglobin levels remained stable while additional studies, including chest radiography, mammography, serum and urine protein electrophoresis with immunofixation, antinuclear-antibody screening with an autoantibody panel, and a bone marrow biopsy, were performed to rule out an occult cancer or autoimmune disorder. The results of all these studies were within normal limits. Treatment with 100 mg of prednisone per day was begun. On the 12th day after admission, she received another two units of packed red cells and was discharged, only to be readmitted later that same evening because of pain in the right hip.
On her readmission, physical examination was unremarkable and the hemoglobin level was 10.2 g per deciliter (6.33 mmol per liter). The patient's course was uneventful until the fourth day after readmission, when she was found to have increasing abdominal distention, large bilateral flank hematomas, and a hemoglobin level of 6.1 g per deciliter (3.79 mmol per liter). Despite plasma exchange and immunosuppressive therapy, she continued to hemorrhage and required intubation for ventilatory support. She had no response to further therapy and died 22 days after her original admission.
A postmortem examination revealed a large left retroperitoneal hematoma containing 3.5 liters of blood and a massive left hemothorax containing 4 liters of blood. It was concluded that the cause of death was respiratory failure, resulting from the hemothorax.
Methods
Enzyme Immunoassays
Human prothrombin (Enzyme Research Laboratories), human thrombin (Sigma), bovine thrombin (Sigma), and bovine factor Va (Enzyme Research Laboratories) were diluted to 1 µg per milliliter in phosphate-buffered saline and dialyzed against 0.05 M sodium carbonate (pH 9.6) at 4° C. Ninety-six–well plates were coated with 0.2 µg of protein per well and incubated overnight at 4° C. Serial plasma dilutions (1:10 to 1:1,000,000) in phosphate-buffered saline were incubated for two hours at room temperature. After these samples were washed four times with Stago Wash (Stago Diagnostics), peroxidase-conjugated goat antihuman IgG (Sigma) was added at a dilution of 1:1000 in Stago dilution buffer (Stago Diagnostics) and incubated for two hours at room temperature. We added 200 µl of peroxidase substrate (Sigma) and quantitated the development of color by measuring absorbance at 490 nm (Vmax Kinetic Microplate Reader, Molecular Devices).
Immunoblotting
Human prothrombin, human thrombin, bovine thrombin, and bovine factor Va were diluted to 1 µg per microliter in phosphate-buffered saline. From each sample, 2.5 µg was electrophoresed in 12 percent polyacrylamide gel and then transferred to nitrocellulose membranes (Schleicher and Schuell) on a miniapparatus (BioRad). The filters were pretreated overnight at room temperature in blocking buffer (5 percent bovine serum albumin, 0.2 percent polysorbate 20 [Tween 20], 0.01 percent sodium azide) and then incubated with plasma from the patient diluted to 1:500 in blocking buffer for three hours at 25°C. After the filters were washed four times with 0.1 percent Tween 20 in 1x phosphate-buffered saline, peroxidase-conjugated goat antihuman IgG (Boehringer–Mannheim) at a dilution of 1:50,000 was added for one hour at 25°C. After the filters were washed six times as described above, enhanced chemiluminescence (ECL, Amersham) was performed and all filters were exposed to Hyperfilm-ECL (Amersham) for two minutes.
Results
The dilute-Russell's-viper-venom assay for the lupus inhibitor and an anticardiolipin-antibody assay were negative. Fibrinogen levels were normal when measured with kinetic and nephelometric methods, thereby ruling out a dysfibrinogenemia. The thrombin time was remeasured with human thrombin in place of bovine thrombin and continued to be markedly prolonged (111 seconds). Incubation of the patient's plasma with normal plasma at a 1:1 ratio for 1 hour did not affect the thrombin time, which remained elevated at 99 seconds. These results suggested the presence of an inhibitor of human thrombin.
Assays for factors II, V, VIII, IX, and X (Table 1) showed decreased activity for all factors except factor V. In each case, a 1:1 mixture of the patient's plasma with normal plasma did not appreciably change the factor-activity level, whereas increasing dilution of the patient's plasma resulted in increased factor activities. Since the final step of each factor-activity assay requires that thrombin cleave fibrinogen to initiate clot formation, these activity assays also suggested the presence of an inhibitor of thrombin. After passing the patient's plasma over a protein A–Sepharose column (Sigma) to remove the IgG fraction, we obtained normal factor-activity levels. The effluent from the column gave normal prothrombin, activated-partial-thromboplastin, and thrombin times. We therefore concluded that the inhibitor was an IgG antibody.
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Our patient, a previously healthy elderly woman without any personal or family history of bleeding, presented to the emergency room with an acute hemorrhage. Coagulation tests revealed a prolonged prothrombin time, a prolonged activated-partial-thromboplastin time, and a markedly prolonged thrombin time. Factor-activity levels were markedly decreased, and mixing studies suggested the presence of an inhibitor of thrombin. We found that the inhibitor was an IgG antibody against a site on thrombin exposed or created after its conversion from prothrombin. The inhibitor's ability to interfere with cleavage of fibrinogen by both human and bovine thrombin suggests that it is directed against one or more conserved epitopes at the active site of thrombin, the fibrinogen-binding site, or both. Such binding characteristics imply a similarity to hirudin, a potent thrombin inhibitor known to occupy various functional sites on the thrombin molecule.2 Further studies of our patient's inhibitor may determine its exact binding specificity.
Most examples of acquired thrombin inhibitors occur in patients exposed to bovine topical thrombin or fibrin-glue preparations during major surgery.7 Prospective studies have shown that one third to one half of such patients become immunized to bovine thrombin and bovine factor Va and have abnormal clotting-based coagulation.8,11 Most of these patients are asymptomatic, but some with thrombin inhibitors that cross-react strongly with human thrombin have severe hemostatic complications and may even bleed to death.11
Acquired inhibitors directed specifically against human thrombin are rare, and they almost always occur in patients with an underlying disease, especially of the autoimmune type.7 Indeed, the first characterized inhibitor of thrombin was found in a patient with rheumatoid arthritis in 1957.12 In 1964, Hawiger et al.13 isolated an inhibitor of thrombin from a patient with systemic lupus erythematosus. An acquired inhibitor of thrombin has also been described in a patient in whom a lupus-like syndrome developed during procainamide therapy.14 Antibody inhibitors of thrombin differ from the more common antiprothrombin antibodies found in some patients with lupus inhibitors.15 These antiprothrombin antibodies are not neutralizing inhibitors, but by binding prothrombin they accelerate its clearance from the plasma.16 In some cases (about 10 percent),17 the rapid elimination of prothrombin can cause profound hypoprothrombinemia and abnormal bleeding. In patients with acquired thrombin inhibitors, by contrast, the thrombin time is more prolonged than the prothrombin time, and a bleeding diathesis, which may be severe, is typical. In certain cases the acquired thrombin inhibitor binds to a site involved in the molecule's anticoagulant function and produces thrombotic disease.18 Some patients with either monoclonal or polyclonal gammopathies occasionally have inhibitors of thrombin.19,20,21,22
Four reports23,24,25,26 have described thrombin inhibitors in patients without a diagnosed predisposing illness. One patient had evidence of underlying autoimmune disease23; one probably had a lupus inhibitor24; a third had recently undergone major surgery,25 raising the possibility of exposure to bovine thrombin; and the fourth had a history of recurrent thrombosis.26 Despite an extensive workup for occult cancer and a complete autopsy, no evidence of systemic disease was uncovered in our patient. We therefore conclude that the patient's inhibitor occurred spontaneously. Despite therapy begun only one week after presentation, the patient had a massive hemorrhage and bled to death. Given her outcome and that of others like her,11,23 we recommend searching for an inhibitor of thrombin in patients with bleeding or thrombosis who present with prolongations in prothrombin time, activated-partial-thromboplastin time, and thrombin time but who have normal platelet counts and fibrinogen levels. Immediate and aggressive treatment of such thrombin inhibitors may reduce morbidity and mortality in these patients.
Dr. Skålhegg is supported by a grant from the Norwegian Research Council.
Source Information
From the Departments of Laboratory Medicine (A.R.L.S., R.H., G.S., W.C.), Pharmacology (B.S.S.), and Pathology (R.P.), University of Washington Medical Center, Seattle.
Address reprint requests to Dr. La Spada at the Department of Laboratory Medicine, Box 357110, University of Washington Medical Center, 1959 N.E. Pacific St., Seattle, WA 98195.
References
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-thrombin (molecular weight, 38,000) and arrow b points to a form of bovine 
-thrombin generated by additional cleavages in this species.