Presence of antithrombin antibody in atherosclerosis patients negative for antiprothrombin antibody, anticardiolipin antibody and lupus anticoagulant

Thrombosis Research (2007) 120, 307–310

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Letter to the Editors-in-Chief Presence of antithrombin antibody in atherosclerosis patients negative for antiprothrombin antibody, anticardiolipin antibody and lupus anticoagulant Arterio/venous thrombosis emerges mostly in association with congenital or acquired coagulation abnormalities, including antiphospholipid antibodies (aPA). Anticardiolipin antibody (aCL) and lupus anticoagulant (LA) are the representative aPA which is the frequently detected autoantibody in systemic lupus erythematosus (SLE), antiphospholipid syndrome (APS), various autoimmune diseases, other diseases, and even in otherwise healthy individuals [1]. Recently, it has been confirmed that aPA consists of a heterogeneous group of antibodies targeting not only phospholipids, but also plasma proteins, such as beta2-glycoprotein I [1], prothrombin (PT) [1,2], protein C (PC), and thrombomodulin (TM). Some researchers have reported that an antibody against thrombin (Thr) existed in patients with SLE and/or APS [3–6], and they have suggested that antithrombin antibody (aThr) could be an additional aPA, which might also cause arterio/venous thrombosis or recurrent fetal loss. Thrombin is a unique enzyme in the coagulation cascade participating in both coagulation and anticoagulant activities/functions. The coagulation properties include the conversion of fibrinogen to fibrin formation and activation of Factors V and VIII, which cause the formation of prothrombinase/factor X complexes, to amplify a conversion of PT to Thr. However, Thr shows anticoagulant properties by binding TM to activate protein C, resulting in inactivation of Factors Va and VIIa to produce thrombin generation (Fig. 1). It is possible that aPA invites not only arterio/ venous thrombosis, but also causes atherosclerosis among patients with aPA, including aThr. Therefore, we conducted an investigation to determine whether aThr existed in patients with atherosclerosis. We studied 35 patients with atherosclerosis, comprised of 23 with essential hypertension (4 of them complicated with hyperlipidemia), 6 with hyperlipidemia, 3 with diabetes mellitus (DM), and 3 with

hyperuricemia (13 males and 22 females from 24 to 62 years old). All patients were allowed to take drugs for the treatment of their respective diseases at the time of the blood sample collection (for the serum and the citrated plasma) but they were prohibited from taking drugs that might have influenced the results of this study for at least 7 days. To evaluate atherosclerosis lesions, we performed ultrasound echotomography of the bilateral carotid arteries employing an echotomographic system (Toshiba Medical, Tokyo). The diagnosis of atherosclerosis was made when the intimal plus medial complex thickness (IMT) of carotid arteries were N 1.2 mm and/or the presence of plaque lesions were confirmed. Twenty-two healthy individuals (aged 22–36 years) served as controls. Written informed consent for this study was obtained from all of the subjects. Measurement of aThr was taken following the procedures established from the results of previously conducted preliminary experiments. One-hundred μl (40 μg/ml) of human thrombin (Enzyme Research Laboratories, USA) solution dissolved in phosphate buffered saline (PBS), or PBS for the background control, were added to a well of an ELISA plate (Maxisorp, Nalge Nunc Internationl, USA) and left at 4 °C overnight. After a vacuum suction removal of the remaining solution, 200 μl of PBS containing 1% bovine serum albumin (BSA)(Calbiochem, Germany)(BSA/PBS) was added and incubated at room temperature for 2 h. Following 3 washings of the plate with PBS containing 0.05% Tween 20 (Tween/PBS), a 100 μl aliquot of the patient's or healthy control's serum diluted to 1:25 in 1%BSA/PBS was added and incubated for 90 min at room temperature. After washing with Tween/PBS 3 times, a 50 μl aliquot of alkaline phosphatase conjugated goat anti-human IgG (Fc) antibody (Bio Source International Inc, USA) diluted in 1%BSA/PBS (1:1,000) was added and incubated at room temperature for 1 h. The plate was washed with Tween/ PBS, then 20 min after an addition of 100 μl of pnitrophenyl phosphate (p-NPP)/diethanolamine (DEA) buffer, 50 μl of 5 M NaOH was added and the developed color was measured at 405 nm. The experiment was conducted in duplicate, and each

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Figure 1 Distribution of OD values of antithrombin antibody in patients with atherosclerosis and healthy controls. Antithrombin antibody was judged as positive when the OD value was exceeded the mean OD value + 3SD (= 0.17) of the 22 healthy controls.

aThr was judged as positive when the OD value minus the blank OD value exceeded the mean OD value + 3SD of the 22 healthy controls. To determine whether or not aThr is dependent on phospholipid/Ca2+, we measured phosphatidylserine-dependent aThr in patients sera positive and/or negative for aThr employing a Ca2+/phosphatidylserine-coated ELISA plate. In brief, 30 μl (65 μg/mL) of phosphatidylserin (Sigma, USA) dissolved in ethanol solution were added to the well of an ELISA plate (IWAKI ASSAY PLATE, Iwaki, Japan) and dried with nitrogen gas. After adding an aliquot of 100 μl (40 μg/ml) of human thrombin (Enzyme Research Laboratories) solution dissolved in 5 mM Ca2+-containing PBS or Ca2+-PBS without thrombin for the background control (and also to determine the reactivity to phosphatidylserine alone) to the ELISA well and left at 4 °C overnight. The following procedure was identical to that described above for aThr measurement. To investigate the specificity of aThr measurement, we conducted inhibition experiments by using a method reported by Arvieux et al. [7] with a slight modification. Liposomes containing 40% phosphatidylcholine and 60% phosphatidylserine in PBS were prepared as a stock suspension (540 μg/ml) by vortexing under an ultrasound. Eight hundreds μl of liposomes and an equal volume of thrombin solution diluted in PBS (0, 50, 100 μg/ml) were mixed and incubated at room temperature. After 30 min of incubation, 312 μl of 3 patients sera positive for aThr or 1 healthy control's serum were mixed and diluted in 2288 μl of 1% BSA/PBS (3:25).

Letter to the Editors-in-Chief 800 μl of these diluted patients sera or healthy control's serum were added to the 1600 μl of liposome solution (1:2)(× 25 in final), mixed and incubated for 2 h at room temperature. Then, the serum/liposome solution was ultra-centrifuged at 17,000 rpm (26,000 G) for 30 min at 4 °C. 100 μl of the supernatant, was then subjected to measurement for aThr. The experiment was conducted in duplicate. The following procedure was completely identical to that described in aThr measurement. aPT and beta2-glycoprotein I dependent aCL of the patient's or healthy control's serum were measured employing the ELISA method reported elsewhere [2,8]. LA activity of the patient's or healthy control's plasma was determined using the LA confirmatory test kit (Gradipor, Australia). We also compared the difference of the serum total cholesterol, HDL- and LDL-cholesterol, and triglyceride concentrations between aThr-positive andnegative patients. aThr was positive in 13/35 patients (37.1%), with OD values ranging from 0.24 to 1.41 (the mean OD value + 3SD of 22 healthy controls: 0.17). Conversely, the OD values of both patients group who tested sera positive and/or negative for aThr were less than 0.15 (the mean OD value + 3SD of 22 healthy controls: 0.15) for Thr/phosphatidylserin and/or phosphatidylserine alone (data not shown). Thus, we confirmed that aThr is not dependent on phosphatidylserine. Three patients' sera absorbed with Thr-containing liposomes (50 and 100 μg/ml), showed complete or more than 79% reduction of reactivity to Thr-ELISA (Fig. 2). However, sera from those three patients absorbed with liposomes containing no Thr did not show noticeable change of reactivities to the ThrELISA. None of the 35 patients and 22 healthy controls were positive for aPT. aCL and LA were negative in all 35 patients and 22 healthy controls. No significant statistical difference was observed between the patient groups of those with and those without aThr in the serum concentration of total cholesterol, HDL- and LDL-cholesterol. Recently aThr has been found among patients with aPA, and now aThr has been thought to be a new aPA [3–6]. It is possible that aPA may be a causative factor not only for arterio/venous thrombosis, but also for atherosclerosis by injuring endothelium directly and/ or indirectly [9,10]. Accordingly, we investigated aThr employing ELISA among patients with atherosclerosis, and found that 37% of the 35 patients had aThr. However, none of the aThr-positive patients were additionally positive for aPT, aCL or LA. Our results suggest that aThr, now a possible candidate of aPA, could be present as the sole

Letter to the Editors-in-Chief

Figure 2 Inhibition percentage of antithrombin antibody binding to immobilized thrombin on ELISA plate by thrombin/ liposome. Eight hundreds μl of liposome and equal volume of thrombin solution (0, 50, 100 μg/ml) diluted in PBS were mixed and incubated at room temperature. After a 30 min incubation, antithrombin antibody positive 3 patient's serum (▴–▴, ■–■, ♦–♦) and 1 healthy control serum (●- -●) were added, mixed and incubated for 2 h at room temperature. One hundred μl of the supernatant obtained by ultracentrifugation was then subjected to measure antithrombin antibody.

antibody in some patients' group without APS, including those with atherosclerosis. Accordingly, it is possible that aThr along with other aPA could be a new diagnostic marker and/or predictive risk factor for atherosclerosis/thrombosis. Nevertheless, it is still unclear how aThr antibody invites atherosclerosis/thrombosis in a patient with aThr. It is possible that the emergence of aThr in patients with atherosclerosis is the consequence of the presence of atherosclerotic lesions which have latent antigenic properties. However, we speculate that aThr, possibly an autoantibody like other antiphospholipid antibodies existent in nature, could have caused atherosclerosis in these patient groups. aThr may compete with ATIII to interrupt binding of ATIII to thrombin, thus failing to inactivate thrombin. Residual active type thrombin activates protease activated receptor-1 (PAR-1) on platelets to intensify an induction of cellular adhesion molecules, such as VCAM-1, ICAM-1, and E-selectin, on endothelial cells. Active thrombin also activates PAR-1 on endothelium to increase production of thrombospondin-1, E-selectin, plasminogen activator inhibitor-1 (PAI-1), tissue factor, and NF-κB. These factors may cause accumulation on and invasion into the endothelium of monocytes, initiating a formation of atherosclerosis lesion. However, any explanation remains speculative. Hwang et al. [3] demonstrated that patientderived, aCL-positive 6 monoclonal IgG antibodies were positive for both aPT and aThr by ELISA. Also, the binding of three of the 6 monoclonal antibodies

309 to PT and Thr was inhibited more effectively with soluble Thr than with PT. In addition, those 3 monoclonal antibodies reduced the ATIII inactivation of thrombin in vivo. From these results, they concluded that some aThr in patients could interfere with the ATIII inactivation of thrombin, thus allowing the promotion and/or sustainability of thrombosis in patients with aThr/aPT. Furthermore, Hwang et al. recently reported [4] that 6 of 6 monoclonal aCL, which were the same antibodies they employed in the previous report (3), had bound to PC and activated PC (APC). One of the 6 APC-reactive monoclonal antibodies inhibited the anticoagulant function of APC. From these findings, they speculated that some aThr may play a role as prothrombotic factors by inhibiting APC function. Kolev et al. [5] reported the existence of aThrpositive IgG and isolated this IgG from the plasma of a patient with secondary APS (positive for aCL and LA). This IgG expressed fibrinogen-clotting and amidolytic activity of thrombin, and activated factor XIII. Also, they have confirmed that IgG hindered an inhibitory function of hirudin on the intrinsic thrombin activity. From these results, they concluded that the identification of an aThr could be a clinically as well as a pathologically valuable tool in finding patients with a high-risk of thrombosis. It is likely that our aThr found in patients with atherosclerosis is a different antibody from those of Hwang et al. [3,4] and Kolev et al. [5] because our patients were exclusively positive for aThr with no aPT, including aCL and LA. It is also possible that aThr found by Hwang et al. [3,4] was different from the one identified by Kolev et al. [5], because the latter did not react to PC. Furthermore, there still remains a possibility that prothrombotic properties of aThr confirmed by Hwang, et al. [3,4] and by Kolev et al. [5] could be attributable to co-existing aCL, LA and/or aPT in monoclonal antibodies and serum IgG from patient(s). However, these results suggest an existence of heterogeneous aThr, and that pathogenesis of thrombosis in patients with aThr may be multifactorial. To conclude, we confirmed the sole existence of aThr in atherosclerosis patients. However, further examination using more samples as well as a longterm follow-up study of the aThr-positive patients are needed to verify the clinical significance of aThr. Also, the clinicopathological mechanisms of aThr in atherosclerosis and/or thrombosis remain to be clarified in the future.

References [1] deGroot PG, Derksen RH. Pathophysiology of the antiphospholipid syndrome. J Thromb Haemost 2005;3:1854—60.

310 [2] Sanaka T, Matsuda J, Teramoto T. Detection and clinical significance of Ba2+ and Sr2+-dependent antiprothrombin antibodies in patients with systemic lupus erythematosus and antiphospholipid syndrome. Lupus 2003;12:117—23. [3] Hwang K-K, Grossman J, Visvanathan S, Chukwuocha RR, Woods VL, Le DT, et al. Identification of anti-thrombin antibodies in the antiphospholipid syndrome that interfere with the inactivation of thrombin by antithrombin. J Immunol 2001;167:7192—8. [4] Hwang KK, Yang CD, Yan W, Grossman JM, Hahn BH, Chen PP. A thrombin-cross-reactive anticardiolipin antibody binds to and inhibits the anticoagulant function of activated protein C. Arthritis Rheum 2003;48:1622—30. [5] Kolev K, Lerant I, Skopal J, Kelemen A, Nagy Z, Machovich R. Impaired inactivation by antithrombin and hirudin and preserved fibrinogen-clotting activity of thrombin in complex with antithrombin antibody from a patient with antiphospholipid syndrome. Thromb Haemost 2005;94: 82—7. [6] Miesbach W, Mathihias T, Scharrer I. Identification of thrombin antibodies in patients with antiphospholipid antibodies in patients with antiphospholipid syndrome. Ann NY Acad Sci 2005;1050:250—6. [7] Arvieux J, Darminge L, Caron C, Reber G, Bensa JC. Development of an ELISA for autoantibodies to prothrombin showing their prevalence in patients with lupus anticoagulants. Thromb Haemost 1995;74:1120—5.

Letter to the Editors-in-Chief [8] Matsuda J, Saitoh N, Gohchi K, Gotoh M, Tsukamoto M. Distinguishing beta2-glycoporotein I dependent (systemic lupus erythematosus type) and independent (syphilis type) anti-cardiolipin antibody with Tween 20. Br J Haematol 1993;85:799—802. [9] Nicolo D, Monestier M. Antiphospholipid antibodies and atherosclerosis. Clin Immunol 2004;112:183—9. [10] Matsuura E, Kobayashi K, Inoue K, Lopez LR, Shoenfeld Y. Oxidized LDL/beta2-glycoprotein I complexes: new aspects in atherosclerosis. Lupus 2005;14:736—41.

Juzo Matsuda* Takeo Miebori Atsushi Matsuyama Department of Medicine, Teikyo University School of Medicine, Kaga 2-11-1, Itabashi-Ku, Tokyo 173-8605, Japan E-mail address: [email protected]. ⁎Corresponding author. Tel.: +81 3 3964 4146; fax: +81 3 5375 1308.

31 March 2006