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By Antithrombin Interfere with the Inactivation of Thrombin The Identification of Anti-Thrombin Antibodies in the Antiphospholipid Syndrome That Interfere with the Inactivation of Thrombin by Antithrombin This information is current as of September 28, 2021. Kwan-Ki Hwang, Jennifer M. Grossman, Sudha Visvanathan, Reginald U. Chukwuocha, Virgil L. Woods, Jr., Dzung T. Le, Bevra H. Hahn and Pojen P. Chen J Immunol 2001; 167:7192-7198; ; doi: 10.4049/jimmunol.167.12.7192 Downloaded from http://www.jimmunol.org/content/167/12/7192 References This article cites 41 articles, 14 of which you can access for free at: http://www.jimmunol.org/content/167/12/7192.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on September 28, 2021 • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Identification of Anti-Thrombin Antibodies in the Antiphospholipid Syndrome That Interfere with the Inactivation of Thrombin by Antithrombin1 Kwan-Ki Hwang,2* Jennifer M. Grossman,* Sudha Visvanathan,* Reginald U. Chukwuocha,* Virgil L. Woods, Jr.,† Dzung T. Le,‡ Bevra H. Hahn,* and Pojen P. Chen* The combined presence of anti-phospholipid (PL) Ab, including lupus anticoagulants (LAC) and/or anticardiolipin Ab (aCL), and thrombosis is recognized as the antiphospholipid syndrome (APS). LAC are detected as an inhibitory effect on PL-restricted in ␤ vitro blood coagulation tests, and are comprised mainly of Ab against 2 glycoprotein I and prothrombin (PT). Recently, anti-PT Ab (aPT) were found to be associated with thrombosis by some investigators, although this is not confirmed by others. Considering that aPT are heterogeneous in patients and that PT is converted into thrombin, we hypothesize that certain aPT in patients may Downloaded from bind to thrombin, and that some of such anti-thrombin Ab may interfere with thrombin-antithrombin (AT) interaction and thus reduce the AT inactivation of thrombin. To test this hypothesis, we searched for anti-thrombin Ab in APS patients and then studied those found for their effects on the AT inactivation of thrombin. The results revealed that most, but not all, aPT-positive patient plasma samples contained anti-thrombin Ab. To study the functional significance of these Ab, we identified six patient- derived mAb that bound to both PT and thrombin. Of these mAb, three could reduce the AT inactivation of thrombin, whereas others had minimal effect. These findings indicate that some aPT in patients react with thrombin, and that some of such anti- http://www.jimmunol.org/ thrombin Ab could inhibit feedback regulation of thrombin. Because the latter anti-thrombin Ab are likely to promote clotting, it will be important to develop specific assays for such Ab and study their roles in thrombosis in APS patients. The Journal of Immunology, 2001, 167: 7192–7198. ␤ oagulation abnormalities, including thrombosis and re- plexes. The involved plasma proteins include plasma protein 2 gly- ␤ current fetal loss, have emerged as important clinical coprotein-1 ( 2GPI), prothrombin (PT), protein C, and protein S (9– complications in systemic lupus erythematosus (SLE3; 14). To date, the Ab against ␤ GPI and its complexes with cardiolipin C 2 Refs. 1–3). Patients with SLE and acquired coagulation abnormal- probably account for most of the positive findings on tests for aCL in by guest on September 28, 2021 ␤ ities often have anti-phospholipid (PL) Ab (aPL). These include APS (15, 16), whereas Ab against PT and 2GPI are responsible for lupus anticoagulants (LAC), as detected by their abilities to pro- the majority of the LAC activity (11, 17–19). long certain in vitro PL-restricted blood clotting tests, and anticar- Recently, increasing attention has been paid to anti-PT Ab (aPT) diolipin Ab (aCL; Refs. 2 and 4–8). Because LAC is neutralized and their roles in thrombosis in APS patients (18, 20–29). The prev- by addition of excess PL, it was suggested that the LAC Ab might alence of aPT in patients varies among different studies, ranging from interact with PL and thus interfere with blood coagulation on the 30 to 60% in APS patients when tested by ELISA using immobilized limited PL surface in the in vitro test. Therefore, LAC and aCL are human PT on activated polyvinyl chloride plates (20, 23, 25). How- generally referred to as aPL, and the association of thrombosis and ever, aPT were found to be associated with thrombosis (21, 25, 28), fetal loss with LAC and aCL is recognized as antiphospholipid although this is not confirmed by other investigators (23). These con- syndrome (APS; Refs. 2 and 8). flicting data may reflect the heterogeneity of aPT present in individual Accumulated studies show that aPL represent a heterogeneous patient sera and different sets of these autoantibodies in clinically di- group of immunologically and functionally distinct Ab that recognize verse patient populations in different studies. various PL, PL-binding plasma proteins, and/or PL-protein com- To understand the functional and pathogenic property of aPT, Rao and coworkers (11, 18) affinity purified IgG aPT and found *Division of Rheumatology, Department of Medicine, University of California, Los that the purified Ab bound to immobilized phosphatidylserine in † ‡ Angeles, CA 90095; and Departments of Medicine and Pathology, University of 2ϩ California at San Diego, La Jolla, CA 92093 the presence of Ca and PT. These results suggested that IgG aPT cross-linked PT molecules, and thus increased the valence Received for publication April 20, 2001. Accepted for publication October 12, 2001. of interactions between PT and phosphatidylserine. Subse- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance quently, those investigators showed that IgG purified from a with 18 U.S.C. Section 1734 solely to indicate this fact. LAC-positive plasma sample (designated LAC IgG; from a 1 This work was supported by a research grant from the Southern California Chapter patient with hypoprothrombinemia) enhanced the binding of PT of the Arthritis Foundation and by Grants AI32243 and AR42506 from the National Institutes of Health. to HUVEC and increased conversion of PT to thrombin on the surface of HUVEC (22). 2 Address correspondence and reprint requests to Dr. Kwan-Ki Hwang, Medicine/ Rheumatology, 167022, University of California, Los Angeles, CA 90095-1670. E- Thrombin is a key effector enzyme in the coagulation cascade. mail address: [email protected] It converts fibrinogen to fibrin, leading to the formation of fibrin 3 Abbreviations used in this paper: SLE, systemic lupus erythematosus; aCL, anti- clots. It also feedback amplifies the cascade by activating factors V cardiolipin Ab; PL, phospholipid; aPL, anti-PL Ab; APS, antiphospholipid syndrome; ␤ ␤ and VIII, which in turn, enhance conversion of PT to thrombin PT, prothrombin; aPT, anti-PT Ab; AT, antithrombin; AU, abstract unit; 2GPI, 2 glycoprotein-1; EC, endothelial cell; LAC, lupus anticoagulant; PT, prothrombin. (30). Therefore, once thrombin is generated in vivo, it is tightly Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00 The Journal of Immunology 7193 regulated by antithrombin (AT) that binds to thrombin in the pres- min, generation of p-nitroaniline was monitored by measuring OD at 405 ence of heparin-like glycosaminoglycans on the endothelial cell nm. The activity of thrombin was determined as the rate of hydrolysis of (EC) surface and inactivates the enzyme irreversibly (30–32). Con- S-2238 from the linear range of absorbance at 405 nm with time. The effects of monoclonal anti-thrombin Ab on the AT inactivation of sidering that thrombin is derived from the zymogen PT, it is conceiv- thrombin were studied in a functional assay for the thrombin activity in the able that some aPT may bind to thrombin at a site where thrombin presence of AT and heparin, according to Bock et al. (35) with minor interacts with AT, and therefore inhibit AT inactivation of thrombin. modifications. In particular, human AT (Enzyme Research Laboratories) In this study, we report the detection of Ab against thrombin in APS was used at a concentration that was at least 10-fold higher than that of human ␣-thrombin, and experiments were conducted in 50 mM HEPES, patients and the inhibitory effects of three patient-derived IgG mono- 125 mM NaCl, 1 mM EDTA, and 0.1% polyethylene glycol 8000, pH 7.4, clonal anti-thrombin Ab on the AT inactivation of thrombin. These at 25°C in microtiter plates. The assay was initiated by incubating 25 ␮lof findings define a novel anti-thrombin autoantibody in APS and they thrombin (80 nM) separately with 25 ␮l of a test mAb (128 ␮g/ml), normal show that such Ab may interfere with negative feedback regulation of human IgG, or the isotype control monoclonal IgG3 in duplicate for1hat ␮ thrombin in circulation, and thus contribute to thrombosis. room temperature. Then, to each reaction mixture was added 50 lofAT (400 nM or the indicated concentrations) in the buffer containing heparin, resulting in a final heparin concentration of 0.1 USP unit/ml (U/ml) or the Materials and Methods indicated concentrations. Subsequently, 200 ␮l of the chromogenic sub- Patients and healthy controls strate S-2238 was added, and OD at 405 nm was measured at 1 min unless stated otherwise.
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