(uPA).15 However, the mechanism(s) responsible for the initiation .... Single-chain and two-chain uPA antigen (Sc-uPA and. Tc-uPA) ..... Meyer K, Williams EC.
COAGULATION AND TRANSFUSION MEDICINE Original Article
Excessive Fibrinolysis in Amyloidosis Associated with Elevated Plasma Single-Chain Urokinase HOWARD A. LIEBMAN, M.D.,1 MARY K. CARFAGNO, B.A.,1 ILENE C. WEITZ, M.D.,3 PAUL BERARD, M.D.,1 JERE M. DIIORIO, B.A.,1 EVAN VOSBURGH, M.D.,1 AND ROBERT W. SIMMS, M.D.2
plasminogen activator inhibitor in the patient's plasma were normal. Urokinase-type activator activity and antigen were three to five times elevated in the patient's plasma. Results of immunoprecipitation showed that single-chain urokinase-type activator was the primary urokinase-type activator species in the patient's plasma. Excessive fibrinolysis in patients with amyloidosis results from increased plasma single-chain urokinasetype activator activity. (Key words: Amyloidosis; Fibrinolysis; Single-chain urokinase) Am J Clin Pathol 1992;98:534-541
Hemorrhagic complications frequently occur in patients with amyloidosis.' In most patients, bleeding is believed to result from increased vascular fragility due to amyloid infiltration of blood vessels independent of any abnormal results of hemostatic studies.1,2 In more than 40 patients with amyloidosis, an acquired hemorrhage disorder, resulting from a Factor X deficiency or combined with a Factor IX deficiency, has been reported.3'4 In these patients, Factor X is cleared rapidly from the circulation by binding of the coagulation protein to amyloid deposits.4 A severe bleeding diathesis resulting from excessive fibrinolysis also has been reported in patients with amyloid.5"15 Studies of these patients have found many clinical
and hemostatic similarities. Abnormalities in the fibrinolytic system that have been reported include a decrease in plasma a-2-plasmin inhibitor (a-2-PI)," 1 4 an increase in tissue plasminogen activator (tPA),12 a decrease in plasminogen activator inhibitor activity (PAI-1),' 3 and an increase in plasma urokinase-type activator activity (uPA).15 However, the mechanism(s) responsible for the initiation of fibrinolysis remains obscure. We studied a patient with primary amyloidosis (AL) who had clinically significant bleeding due to excessive fibrinolysis. This patient allows us to demonstrate that the increased fibrinolytic activity was associated with elevated plasma levels of single-chain urokinase-type plasminogen activator (Sc-uPA). This is the first reported acquired bleeding disorder resulting from elevated plasma levels of this fibrinolytic protein. From William B. Castle Hematology Research Laboratory, Divisions 1 opHematology-Oncology and Rheumatology, Department ofMedicine, Boston City Hospital and Boston University School ofMedicine. Boston, MATERIALS AND METHODS Massachusetts, and the1Division ofHematology, Department of Medicine, Cedars Sinai Medical Center, Los Angeles, California. Case History Supported in part by a General Clinical Research Center grant (RR533) from the Division of Research Resources, National Institutes of Health, The patient, a 53-year-old man, was well until February to the Boston University School of Medicine. Dr. Liebman is a recipient 1988, when he developed spontaneous periorbital peteof National Institutes of Health grant HL-39665. chiae without antecedent trauma. During the next year, Received September 4, 1991; revised manuscript accepted for publication October 30, 1991. facial petechiae recurred and became more extensive and Address reprint requests to Dr. Liebman: Division of Hematology, confluent. In May 1989, an abdominal fat pad aspirate University of Southern California, School of Medicine, Raulston 312, demonstrated amyloid deposition, and a diagnosis of pri2025 Zonal Ave., Los Angeles, California 90033. 534
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Severe bleeding resulting from excessive fibrinolysis has been observed in patients with primary amyloidosis. The authors studied a patient with this hemostatic disorder before and during therapy with e-aminocaproic acid. Excessive fibrinolysis was associated with depressed plasma concentrations of coagulation Factors XII, XI, high-molecular-weight kininogen, and Factors VIII and V; and plasminogen and a-2-plasmin inhibitor. These deficiencies were corrected with treatment. The functional and antigenic concentrations of tissue plasminogen activator and
LIEBMAN ET AL. Urokinase-Induced Fi inolysis in Amyloidosis
Protocol The study protocol was approved by the Institutional Review Board at Boston City Hospital. After obtaining informed consent, intravenous epsilon aminocaproic acid (EACA) therapy was started. The patient was treated with intravenous EACA for 3 days and then given oral EACA. Blood samples were collected before therapy and daily
during treatment. Blood samples obtained before therapy (day 0); on days 2, 4, and 7 of therapy, samples were selected for extensive hemostatic evaluation. Tissue plasminogen activator antigen levels were also assayed before and 5 minutes after upper extremity venous occlusion.16 Plasma samples from patients with amyloidosis and no evidence of fibrinolysis were collected for hemostatic studies as part of an ongoing amyloid study. Laboratory Methods Assays were performed using plasma collected in 0.9% sodium citrate. Plasma not used immediately was aliquoted, frozen, and stored at —70 °C. Screening assays, including the prothrombin time, activated partial thromboplastin time, thrombin time, reptilase time, ristocetin cofactor activity, and template bleeding times were performed by standard laboratory methods. Quantitative fibrinogen, fibrin/fibrinogen degradation products (fdp/ FDP), protamine sulfate assay for fibrin monomer, euglobulin clot lysis time, and specific coagulation factor assays were performed in accordance with published techniques used in our laboratory.9 Functional protein C was determined by a coagulation assay based on the prolongation of the activated partial thromboplastin time, with preactivation of plasma protein C by the venom of Agkistrodon Crotalidae contortrix}1 Von Willebrand Factor (vWF) antigen was measured by enzyme-linked immunosorbent assay (ELISA) and vWF multimers were analyzed by the method of Weinstein and colleagues.18 Crosslinked fibrin degradation products (D-dimer) were measured by ELISA (Asserachrom D-Di, American Bioproducts, Parsippony, NJ). 19 Plasminogen concentration in plasma was determined by a chromogenic assay (actichrome PLG, American Diagnostica, Greenwich, CT) using a normal plasma pool standard. Alpha-2-plasmin inhibitor concentration in plasma was measured by electroimmunoassay using goat antihuman a-2-PI.20 A comparison of free 2-PI and plasmin/a-2-PI complex in patient and control plasma was performed using the western blot method.21 After transfer of plasma proteins to nitrocellulose paper, a-2-PI was detected with a murine monoclonal antibody (MoAb 3612, American Diagnostica) followed by goat anti-murine antibody conjugated to horseradish peroxidase. Blots were developed using 4-chloro-l naphthol. Tissue plasminogen activator antigen and PAI-1 antigen were measured in plasma by ELISA (Tint Elize tPA and PAI-1, American Diagnostics). Tissue plasminogen activator and PAI-1 activities were measured in freshly collected plasma by a chromogenic assay (Stachrom tPA, Diagnostica Stago, Asnieres-Sur-Seine, France and Spectrolyse/PL, American Diagnostica).22-23
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mary amyloidosis was made. Therapy with colchicine was started, but the patient continued to complain of postural dizziness, fatigue, weight loss, intermittent stabbing pain in his legs and hands, and recurrent epistaxis. Petechiae and ecchymosis frequently occurred in areas of minor trauma or pressure, including buccal hematomas. Physical examination showed normal vital signs without orthostatic hypotension. The patient had multiple buccal hematomas measuring as large as 2 cm in diameter. Petechiae and ecchymoses in the periorbital region, the nasal bridge, neck, upper torso, and lower legs were found. The liver and spleen were not enlarged. Stool tested negative with guaiac reagent. Results of neurologic examination were normal, except for decreased pin prick and touch in the extremities, which is consistent with early polyneuropathy. Results of laboratory studies were unremarkable, except for hemostatic assays. He had normal results for hemoglobin, hematocrit, leukocyte count, and platelet count. His serum electrolytes, renal function, and liver function tests also were normal. Activated partial thromboplastin time was 36 seconds compared to a control of 26 seconds; prothrombin time was 29 seconds with a control of 12 seconds, and thrombin time was 18 seconds with a control of 14 seconds. Serum protein and immunoelectrophoresis showed a monoclonal immunoglobulin (Ig) G lambda spike of approximately 600 mg/dL. Urine electrophoresis detected free lambda light chains. Quantitative immunoglobulin were as follows: IgG, 1,830 mg/dL (639 to 1,349 mg/dL); IgA, 87 mg/dL (70 to 312 mg/dL); and IgM, 43 mg/dL (56 to 352 mg/dL). A bone marrow aspirate and biopsy found moderate plasmacytosis (15%) with mature-appearing plasma cells and normal bone marrow maturation. Skeletal survey found no bone lesions. Results of abdominal computed tomographic and liver spleen scans were normal. Echocardiogram showed normal left ventricular wall motion and function. Skin biopsy showed potassium permanganate-resistant amyloid deposition. Evaluation of the patient's coagulopathy included a euglobulin clot lysis time of less than 60 minutes, which was diagnostic of accelerated fibrinolysis. The patient was admitted to the Boston University Clinical Research Center at Boston City Hospital for evaluation and treatment of accelerated fibrinolysis.
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COAGULATION AND TRANSFUSION MEDICINE Original Article Single-chain urokinase-type plasminogen activator and Tc-uPA from human urine were used as controls. The uPA antigen was dissociated from the antibody-Sepharose by heating at 56 °C for 10 minutes in polyacrylamide gel electrophoresis-sodium dodecyl sulfate sample buffer containing 2-mercaptoethanol and run on 10% polyacrylamide gels. After electrophoresis, the proteins were transferred to nitrocellulose paper and uPA antigen was detected with affinity-purified rabbit anti-uPA antibodies. RESULTS Screening Studies The hemostatic studies performed on this patient are listed in Table 1. Screening studies performed before therapy with EACA showed prolongation of the prothrombin time, activated partial thromboplastin time, and thrombin time assays. The addition of normal plasma in a 1:1 mixture resulted in only a partial correction of the prolonged assays, without further prolongation after incubation at 37 °C for 1 hour. Specific factor assays demonstrated multiple deficiencies, most pronounced for Factor XII, Factor XI, and high-molecular-weight kininogen. Factor V and Factor VIII also were depressed, a finding reported in other patients with this hemostatic
TABLE 1. HEMOSTATIC STUDIES Patient Day
0
2
4
7
EACA dose (g/day) Screening assays Euglobulin lysis (min) PT (sec) aPTT (sec) TT (sec) Reptilase (sec) Fibrinogen (mg/dL) Fibrin monomer Fdp/FDP (^g/mL) D-dimer (ng/mL) Bleeding time (min) Factor assays Factor II (%) Factor X (%) Factor IX (%) Factor VII (%) Protein C (%) Factor V (%) Factor VIII (%) HMWK (%) Factor XI (%) Factor XII (%) vWF antigen (%) R CoF (%)
0
36 (IV)
12 (oral)
18 (oral)
10 120 15.3 39.3 19.6 16.3 172 Negative 120 12.0-12.3 26.2-26.7 14.4-14.9 12.2-12.9 238-242 Negative
