Acquired Dysfibrinogenemia Caused by Autoantibody Inhibiting Fibrin Polymerization in a Patient with MELAS Syndrome and Bleeding Tendency

Available online at www.annclinlabsci.org 696 Annals of Clinical & Laboratory Science, vol. 46, no. 6, 2016 Acquired Dysfibrinogenemia Caused by Autoantibody Inhibiting Fibrin Polymerization in a Patient with MELAS Syndrome and Bleeding Tendency Nuri Lee1, Ji-Eun Kim1,2, Hyun Ju Yoo1,2, JaYoon Gu1,2, Hyori Kim2,3, Junho Chung2,3, Youngil Koh2,4, and Hyun Kyung Kim1,2 1Department of Laboratory Medicine, 2Cancer Research Institute and 3Department of Biochemistry and Molecular Biology, and 4Department of Internal Medicine, Seoul National University Hospital, Seoul, Korea Abstract. We present a case of acquired dysfibrinogenemia caused by an autoantibody that inhibited fibrin polymerization in a patient previously diagnosed with MELAS (mitochondrial myopathy, encephalopa- thy, lactic acidosis, stroke-like episodes). The patient wesho d prolonged PT, aPTT, and thrombin time. There was no factor deficiency but fibrinogen antigen and activity were decreased. ELISA for detection of fibrinogen antibodies were performed and IgG purified from the patient’s plasma bound to fibrinogen more strongly than did control IgG, indicating the presence of a fibrinogen-specific antibody. Thrombin- mediated fibrin polymerization was severely impaired in the patient, although thrombin-induced fibrinopeptide A release was normal. Scanning electron microscopy was used to investigate the structure of fibrin clots and revealed many pores on the surface of patient’s fibrin clots. Since MELAS is often associated with autoimmune disorders, a work-up for the presence of anti-fibrinogen antibody is necessary when bleeding tendency occurs in MELAS patients along with prolonged thrombin time. Key words: Dysfibrinogenemia, bleeding tendency, MELAS, autoantibody, fibrin polymerization. Introduction patient with acquired dysfibrinogenemia whose antibody specifically inhibited fibrin polymerization Mitochondrial encephalomyopathy with lactic aci- and produced numerous pores on the fibrin clot dosis and stroke-like episodes (MELAS) syndrome surface. has been reported to co-occur with autoimmune disorders such as autoimmune hemolytic anemia, Case Report Graves’ disease, and autoimmune diabetes [1-3]. Acquired dysfibrinogenemia is a hemorrhagic dis- Case presentation and initial work-up. A 62-year-old order caused by the presence of anti-fibrinogen an- female with hematochezia for 5 days was transferred to tibody or abnormal structure of fibrinogen includ- our hospital. Three years ago, she was diagnosed with MELAS; sequencing analysis revealed an A-to-G point ing high sialylation of carbohydrate residue [4]. mutation at position 3243 of the MT-TL1 gene. She had Dysfibrinogenemia has been reported mostly in stroke-like symptoms such as fluctuating mood, dyspha- patients with liver disease, plasma cell disorders, gia, and drowsiness. The underlying diseases were hyper- and inflammatory diseases [4-6]. Although afew tension, heart failure, and hyperlipidemia. She had no patients with MELAS syndrome show hemorrhagic previous bleeding history and did not take any drugs manifestation [7-9], there has been no definite an- that tend to induce bleeding. swer for the mechanism of these bleeding symptoms. Here we describe, for the first time, a MELAS Her platelet count was normal. Coagulation tests showed prolonged prothrombin time (PT) (94.6 sec; normal range, 9.8–12.2) and activated partial thromboplastin Address correspondence to Hyun Kyung Kim, M.D., Ph.D. Department of Laboratory Medicine, Seoul National University time (aPTT) (>400 sec; 26.0–35.3), and decreased fi- College of Medicine, 101 Daehak-ro, Jongno-gu, Seoul 110-744, brinogen activity (<35 mg/dL; 180–380) measured by Korea, phone: 82 2 2072 0853, fax: 82 2 747 0359, e mail: lukekhk@ snu.ac.kr Clauss assay (Table 1). Thrombin time (TT) was also 0091-7370/16/0600-696. © 2016 by the Association of Clinical Scientists, Inc. Dysfibrinogenemia of abnormal fibrin polymerization in MELAS 697 Table 1. Initial laboratory results of the patient. Patient Reference range Hemoglobin (g/dL) 11.4 12–16 Complete blood cells White Blood Cells (/μL) 11,110 4000–10,000 Platelet (× 103/μL) 150 130–400 PT (sec) 94.6 9.8-12.2 aPTT (sec) >400 26.0–35.3 Mixing (incubated) PT (%)* 70.6 (70.5) >75 Mixing (incubated) aPTT (%)* 65.7 (68.4) >75 Fibrinogen activity (mg/dL) <35 180–380 Fibrinogen antigen (ng/mL) 0.6 27.3–50.2 Thrombin time (sec) >240 18.3–25.0 Mixing thrombin time (sec) >240 18.3–25.0 D-dimer (μg/mL) 0.32 0.04–0.49 Coagulation assays Antithrombin (%) 81 80–120 Protein C (%) 77 70–140 Lupus anticoagulant Negative Negative Factor II (%)† 66 70–120 Factor V (%)† 75 70–120 Factor VIII (%)† 123 52–190 Factor IX (%) 74 74–137 Factor X (%)† 44 70–120 Factor VIII antibody Negative Negative Factor IX antibody Negative Negative *In mixing (incubated) PT and aPTT tests, percentage correction was calculated as follows: (patient plasma – mixing plasma (1:1)) / (patient plasma – normal control plasma) ×100. If the calculated percentage was below 75%, plasma of the patient was determined as ‘no correction’. †Samples for factor assays were diluted to minimize the effect of inhibitors. Abbreviations: PT, prothrombin time; aPTT, activated partial thromboplastin time. markedly prolonged (>240 sec; 18.3–25.0). Mixing tests IgG immunoglobulin was purified from patient’s plasma for PT, aPTT, and TT showed no correction. The fibrin- and healthy normal plasma by affinity chromatography ogen antigen measured by a commercial ELISA kit using protein A–agarose beads (RepliGen, Waltham, (Zymutest Fibrinogen, Hyphen BioMed, Neuville-sur- MA). Purified IgG was added in triplicate to the wells of Oise, France) was markedly decreased (to 0.6 ng/mL; 96-well microtiter plates precoated with 100 μL human 27.3–50.2). The results of complete blood cells and fibrinogen at various concentrations (Sigma Chemical other coagulation tests are shown in Table 1. Co., St. Louis, MO). Horseradish peroxidase–conjugat- ed rabbit antibody to human IgG was used as secondary Blood sampling. Blood was collected into 0.109 M tri- antibody. Binding of IgG from patient’s plasma to im- sodium citrate tubes by venipuncture. Platelet-poor mobilized fibrinogen was significantly stronger than that plasma was prepared by centrifugation for 15 min at of IgG from normal plasma at all concentrations of fi- 2500×g and stored at −80°C. Normal plasma and IgG brinogen tested, confirming that IgG from patient’s were collected from healthy adult volunteers (n=10). plasma specifically targeted fibrinogen (Figure 1). The study was ovappr ed by the Institutional Review Board of Seoul National University Hospital and all Interference of the anti-fibrinogen antibody with fi- blood samples were collected after written informed brin polymerization but not with fibrinopeptide re- consent was obtained. lease. Dysfibrinogenemia caused by anti-fibrinogen antibodies is discriminated depending on the stage of fibrin Detection of anti-fibrinogen antibody. As the patient clot formation. For fibrin monomer, fibrinopeptide A showed decreased fibrinogen activity and fibrinogen an- release assay is applied to detect antibodies inhibit the tigen along with uncorrected mixing tests for PT, aPTT, proteolysis of fibrinopeptide A from fibrinogen and fi- and TT, she was suspected of having anti-fibrinogen an- brin polymerization assay is conducted for antibodies of tibody. To confirm the presence of such antibody, the fibrin polymer [10-12]. 698 Annals of Clinical & Laboratory Science, vol. 46, no. 6, 2016 critical point drying, and coating as described previously [14]. At least 3 different areas per clot were observed and photographed digitally using a field emission scanning electron microscope (JSM-6700F; JEOL Ltd., Tokyo, Japan). The patient’s fibrin clots wesho d more pores on the surface (although fibrin strands were intact) than normal fibrin clots (Figure 3). Clinical course. After all the tests, the patient was diagnosed with acquired dysfibrinogenemia caused by anti- fibrinogen antibody that targeted fibrin polymerization. Although she was treated with plasmapheresis, steroid impulse, and transfusion, she died on the 20th hospital day after renal, pulmonary, and cardiovascular failure. Figure 1. Binding of IgG purified from the patient’s plasma to fibrinogen. IgG purified from patient’s plasma bound to fibrinogen more strongly than did IgG from Discussion normal plasma, indicating the presence of fibrinogen-specific antibody. Data are expressed as mean ± SEM. The reported case was a MELAS patient whose *P<0.05, ** P<0.01 vs. normal plasma. bleeding was due to the development of anti-fibrinogen antibody that inhibited fibrin polymerization. We further investigated whether the anti-fibrinogen an- MELAS is a clinically heterogeneous but distinct tibody inhibits thrombin-mediated release of fibrino- syndrome caused by defects in mitochondrial func- peptide A from fibrinogen or interferes with subsequent tion [7]. Considering that MELAS syndrome is of- fibrin polymerization. For fibrinopeptide A release as- ten associated with autoimmune disorders [1-3], it say [10,11], patient’s or normal IgG (45 uL at 0.8 mg/ is plausible that the mitochondrial mutation identi- mL) was premixed with human fibrinogen (90 μL at 0.2 mg/mL; Hyphen BioMed). Human thrombin (0.02 fied in the patient may have driven the develop- NIH unit/mL) was added and fibrinopeptide A levels in ment of anti-fibrinogen antibody. To our knowl- the supernatants were measured at different time points edge, this is the first report

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