ORIGINAL ARTICLE Prothrombotic Effect of Anti-beta-2 Glycoprotein-1 Antibodies on the Expression of Tissue Factor, Thrombomodulin, and Plasminogen Activator Inhibitor-1 in Endothelial Cells Rikarni1,2, Rahajuningsih Dharma2, Karmel L. Tambunan3, Harry Isbagyo3, Beti E. Dewi4, Nuzirwan Acang5, Rianto Setiabudy6, Adi K. Aman7 Department of Clinical Pathology, Faculty of Medicine, University of Andalas - M. Djamil Hospital, Padang, Indonesia. Department of Clinical Pathology, Faculty of Medicine Universitas Indonesia - Cipto Mangunkusumo Hospital, Jakarta, Indonesia 3 Department of Internal Medicine, Faculty of Medicine Universitas Indonesia - Cipto Mangunkusumo Hospital, Jakarta, Indonesia 4 Department of Microbiology, Faculty of Medicine Universitas Indonesia - Cipto Mangunkusumo Hospital, Jakarta, Indonesia. 5 Department of Pharmacology, Faculty of Medicine Universitas Indonesia - Cipto Mangunkusumo Hospital, Jakarta, Indonesia. 6 Department of Internal Medicine, Faculty of Medicine University of Andalas - M. Djamil Hospital, Padang, Indonesia 7 Department of Clinical Pathology, Faculty of Medicine University of North Sumatera – Adam Malik Hospital, Medan, Indonesia. 1 2 Correspondence mail: Department of Clinical Pathology, Faculty of Medicine, University of Andalas - M Djamil Hospital. Jl. Perintis Kemerdekan Padang, Sumatera Barat, Indonesia. email: rikarni_dr@yahoo.com. ABSTRAK Tujuan: untuk menganalisis efek imunoglobulin (Ig)G dan IgM anti-beta2glikoprotein-1(β2GP1) terhadap ekspresi messenger RNA (mRNA) tissue factor (TF), mRNA trombomodulin (TM), dan mRNA plasminogen activator inhibitor-1(PAI-1) pada endotel. Metode: studi eksperimental laboratorium dengan memajankan antibodi antiβ2GP1 pada human umbilical vein endothelial cells (HUVEC). Sampel adalah IgG anti-β2GP1 dipurifikasi dari 6 pasien sindrom antifosfolipid (APS) dan IgM anti-β2GP1 dipurifikasi dari 6 pasien APS. Kontrol adalah IgG dipurifikasi dari 6 orang sehat (IgG-NHS) dan IgM dipurifikasi dari 6 orang sehat (IgM-NHS). HUVEC dipajan dengan IgG anti-β2GP1, IgM anti-β2GP1, IgG-NHS, IgM-NHS selama 4 jam. Pengukuran ekspresi relatif mRNA TF, mRNA TM, dan mRNA PAI-1 dilakukan sebelum dan sesudah pemajanan dengan metode real time reverse transcription polymerase chain reaction. Penelitian dilakukan di Rumah Sakit Cipto Mangunkusumo/Fakultas Kedokteran Universitas Indonesia. Hasil: ekspresi relatif mRNA TF, mRNA TM, dan mRNA PAI-1 pada HUVEC yang dipajan dengan IgG anti-β2GP1 adalah 3,14(0,93)-, 0,31(0,13)-, 5,33(2,75)-kali dibandingkan pada HUVEC yang dipajan dengan IgG-NHS. Ekspresi relatif mRNA TF, mRNA TM, dan mRNA PAI-1 pada HUVEC yang dipajan IgM antiβ2GP1 adalah 4,33(1,98)-, 0,33(0,22)-, 5,47(2.64)-kali dibandingkan pada HUVEC yang dipajan IgMNHS. Hasil analisis statistik, sebelum dan sesudah pemajanan HUVEC dengan IgG anti-β2GP1, memperlihatkan perbedaan bermakna ekspresi relatif mRNA TF {1,09(0,76) berbanding 3,14(0,93), p=0,003}, mRNA TM {0,91(0,11) berbanding 0,31±0,13, p=0,001}, dan mRNA PAI-1 {0,93(0,13) berbanding 5,33(2,75, p=0,013}, Hasil analisis statistik, sebelum dan sesudah pemajanan HUVEC dengan IgM anti-β2GP1 memperlihatkan perbedaan bermakna ekspresi relatif mRNA TF {1,03(0,11) berbanding 4,33(1,98), p=0,008}, mRNA TM {0,93(0,08) berbanding 0,33(0,22), p=0,003}, dan mRNA PAI-1 {1,02(0,10) berbanding 5,47(2,64), p=0,01}. Kesimpulan: IgG anti-β2GP1 dan IgM anti-β2GP1 dari pasien APS meningkatkan ekspresi relatif mRNA TF, mRNA PAI-1, dan menurunkan Acta Medica Indonesiana - The Indonesian Journal of Internal Medicine 31 Rikarni Acta Med Indones-Indones J Intern Med mRNA TM pada endotel. Hal itu membuktikan bahwa mekanisme trombosis pada APS dapat terjadi melalui jalur aktivasi koagulasi, hambatan fibrinolisis dan penurunan antikoagulan. Kata kunci: APS, IgG/IgM, anti-β2GP1, mRNA TF, mRNA TM, m RNA PAI-1. ABSTRACT Aim: to analyse the effects of immunoglobulin (Ig)G and IgM anti-beta-2 glycoprotein-1 (anti-β2GP1) on the expression of tissue factor (TF), thrombomodulin (TM), and plasminogen activator inhibitor-1(PAI-1) of endothelial cells in the messenger RNA level. Methods: laboratory experimental study in human umbilical vein endothelial cells (HUVEC) was done at Cipto Mangunkusumo Hospital/Faculty of Medicine, Universitas Indonesia. Samples are purified IgG anti-β2GP1 from six antiphospholipid syndrome (APS) patients serum and IgM anti-β2GP1 from six APS patients serum. For controls, purified IgG from six normal human serum (IgM-NHS) and purified IgM from six normal human serum (IgM-NHS) were used. HUVEC were treated with purified IgG anti-β2GP1, IgM anti-β2GP1, IgG-NHS, IgM-NHS for four hours of incubation. We measured TF, TM, and PAI-1 of HUVEC in mRNA relative expression levels (before and after treatment) by real time reverse transcription polymerase chain reaction. Results: the mean value of TF, TM, and PAI-1 mRNA levels in HUVEC after treated with IgG anti-β2GP1 compared to Ig-NHS were 3.14 (0.93)-, 0.31 (0.13)-, 5.33 (2.75)-fold respectively. In other hand, after treated with IgM anti-β2GP1 compared to IgM-NHS, mRNA levels of TF, TM, and PAI-1 were 4.33 (1.98)-, 0.33 (0.22)-, 5.47 (2.64)-fold respectively. Before and after treatment with IgG anti-β2GP1 showed significant differences of TF mRNA levels {1.09 (0.76) versus 3.14 (0.93), p=0.003}, TM mRNA levels {0.91 (0.11) versus 0.31(0.13), p=0.001}, and PAI-1 mRNA levels 0.93 (0.13) versus 5.33 (2.75), p=0.013}. Before and after treatment with IgM anti-β2GP1 showed significant differences of TF mRNA levels {1.03 (0.11) versus 4.33 (1.98), p=0.008}, TM mRNA levels {0.93 (0.08) versus 0.33 (0.22, p=0.003}, and PAI1 mRNA levels {1.02 (0.10) versus 5.47 (2.64), p=0.01}. Conclusion: IgG anti-β2GP1 and IgM anti-β2GP1 increased TF and PAI-1 mRNA levels. However, IgG anti-β2GP1 and IgM anti-β2GP1 decreased TM mRNA levels. It proved that the mechanism of thrombosis in APS occurs through coagulation activation, reduction of fibrinolysis activity, and reduction of anticoagulant activity. Key words: APS, IgG/ IgM, anti-β2GP1, TF mRNA, TM mRNA, PAI-1 mRNA. INTRODUCTION Antiphospholipid syndrome (APS) is an autoimmune disorder characterized by venous or arterial thrombosis, recurrent fetal loss and the presence of persistent antiphospholipid antibodies.1,2 The antiphospholipid syndrome is the most common acquired risk factor of thrombosis.3 An international consensus on classification criteria for APS was held in Sapporo, and was revised in 2006 (Miyakis criteria) in Sydney. Antiphospholipid syndrome is present if at least one of the clinical criteria and one of the laboratory criteria are meet. The clinical criteria are vascular thrombosis or pregnancy morbidity. The laboratory criteria are lupus anticoagulant (LA) present in plasma, on two or more occasions at least 12 weeks apart, detected according to the guidelines of 32 the International Society on Thrombosis and Haemostasis; anticardiolipin (aCL) antibody of IgG and/or IgM isotype in serum or plasma, present in medium or high titer (i.e. >40 GPL or MPL, or >99th percentile), on two or more occasions, at least 12 weeks apart, measured by a standardized ELISA; anti-b2 glycoprotein-I antibody of IgG and/or IgM isotype in serum or plasma (in titer >the 99th percentile), present on two or more occasions at least 12 weeks apart, measured by a standardized ELISA, according to recommended procedures.4 The pathogenesis of thrombosis in APS remains unknown, although there are some hypotheses concerning this`matter. A condition predisposing to the development thrombosis is known as the prothrombotic state or hypercoagulable or thrombophilia state which Vol 47 • Number 1 • January 2015 Prothrombotic effect of anti-beta-2 glycoprotein-1 antibodies may result from hemostatic system imbalance.5 Endothelial cells are a major site of hemostatic regulation.6 The antiphospholipid antibodies are considered to have an important role in the mechanism of thrombosis as elevated antiphospholipid antibodies serum level is correlated with the incidence of thrombosis. A study by Arad et al.7 using mice injected with IgG anti-β2GP1 from APS patients showed thrombus in the blood vessels of the mice. Danowski et al.8 showed persistent positivity increased the association of IgG anti-β2GP1 with venous thrombosis and IgM anti-β2GP1 with arterial thrombosis. An experimental study by VegaOstegard et al.9 showed the increase of mRNA TF on endothelial cells after exposed by IgG from APS patients. Antiphospholipid antibodies are heterogeneous, and it is likely that more than one mechanism may be involved in causing thrombosis.9 Most antiphospholipid antibodies are directed against phospholipid binding plasma proteins, not anionic phospholipids. The two best characterized antigens are β2GP1 and prothrombin.10 Until now, the effect of anti-β2GP1 antibodies on coagulation system, natural anticoagulant and fibrinolytic system has not been completely understood. The aim of this study was to analyse the effects of IgG antiβ2GP1 and IgM anti-β2GP1 on the expression of TF, TM, and PAI-1 of endothelial cells in the messenger RNA (mRNA) levels. METHODS This experimental study in human umbilical vein endothelial cells (HUVEC) was conducted at Cipto Mangunkusumo Hospital/Faculty of Medicine Universitas Indonesia, from May 2012 to June 2013. Samples were purified IgG antiβ2GP1 from six APS patients serum and IgM anti-β2GP1 from six APS patients serum. For controls, purified IgG from six normal human serum (IgG-NHS) and purified IgM from six normal human serum (IgM-NHS) were used. HUVEC were treated with purified IgG antiβ2GP1, IgM anti-β2GP1, IgG-NHS, IgM-NHS. After four hours of incubation, TF, TM, and PAI-1 of HUVEC in mRNA relative expression levels (before and after treatment) were measured by real time reverse transcription polymerase chain reaction. Each subject has signed informed consent form. Purified IgG from the serum of 6 APS patients was obtained by affinity chromatography using MAb Trap Kit. 11 Purified IgM from the serum of 6 APS patients was obtained by affinity chromatography using HiTrap IgM Purification.12 Subsequently, purification of IgG or IgM anti-β2GP1 was obtained by using HiTrap N-hydroxysuccinimide-activated HP column. Purified human β2GPI protein was coupled to a HiTrap N-hydroxysuccinimide-activated HP column to produce β2GP1 affinity column. Purified IgG and IgM from sample was applied to the collumn according the manufacturer’s instructions. 13 IgG NHS or IgM NHS was isolated in a similar manner from the serum of control subjects. Human umbilical vein endothelial cells (HUVEC Lonza Clonetic) were cultured and maintaned in endothelial cell basal medium with endothelial cell growth medium. The cells were cultured at 370C in humidified air containing 5% CO2.14 HUVEC were passaged when they reached 90% confluency, using trypsin and neutralized with trypsin neutralizing solution.15 The cells were used at three passage. HUVEC were seeded on fibronectin-coated 96-well plates. Subsequently, 100 nM purified β2GP1 was added into the well. Human umbilical vein endothelial cells were treated with 100 μg/mL IgG antiβ2GP1, 100μg/mL IgM anti-β2GP1, IgG-NHS, IgM-NHS for four hours incubation. Total HUVEC RNA was extracted using Trizol reagent.16 The primer was designed using PRIMER design program to TF, TM, PAI-1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Moreover, TF, TM, PAI-1, and GAPDH in mRNA relative expression levels was measured by real time reverse transcription polymerase chain reaction method using super script III platinum SYBR green one-step qRTPCR kit.17 The result of mRNA examination was analyzed by measuring threshold cycle and the value was normalized with GAPDH using comparative quantification method.18 Data were expressed as the mean±SD. Statistical analysis 33 Rikarni Acta Med Indones-Indones J Intern Med Tabel 1. Subject characteristics Patients Diagnosis IgG anti-β2GP1 Concentration of anti-β2GP1 antibodies (SGU/mL) A Primer APS Sample 1 95.7 B Secondary APS Sample 2 165.2 C Primer APS Sample 3 94.5 D Primer APS Sample 4 60.6 E Primer APS Sample 5 49.7 F Secondary APS Sample 6 49.8 IgM antiβ2GP1 (SMU/mL) G Primer APS Sample 7 47.1 H Primer APS Sample 8 73.1 I Primer APS Sample 9 75.7 J Primer APS Sample 10 116.1 B Secondaey APS Sample 11 229.9 E Primer APS Sample 12 214.9 was carried out using the software SPSS version 20. Comparisons were made by parametric testing with student’s paired T-test. P values less than 0.05 were considered significantly. RESULTS Samples were purified immunoglobulin (Ig) G anti-β2GP1 from six APS patients serum and IgM anti-β2GP1 from six APS patients serum. For controls, purified IgG from six normal human serum (IgG-NHS) and purified IgM from six normal human serum (IgM-NHS) were used. Subject characteristics showed in Table 1. Table 2. The value of tissue factor mRNA, thrombomodulin mRNA, and plasminogen activator inhibitor-1 mRNA levels in HUVEC after treated with IgG anti-β2GP1 compared to IgG-NHS mRNA levels *(ratio, fold to control) The value of TF mRNA levels in HUVEC after treated with IgG anti-β2GP1 (n=6) compared to IgG-NHS were (3.03)-, (3.83)-, (4.19)-, (3.72), (2.01)-, and (2.08)- fold, respectively. (Table 2) In other hand, after treated with IgM anti-β2GP1 compared to IgM-NHS (n=6), mRNA levels of TF were (2.22)-, (2.50)-, (4.35)-, (7.67)-, (4.11)-, and (5.13)-fold, respectively. (Table 3) Before and after treatment with IgG anti-β2GP1 showed significant differences of TF mRNA levels {1.09 (0.76) versus 3.14 (0.93), p=0.003}. Before and after treatment with IgM anti-β2GP1 showed significant differences of TF mRNA levels {1.03 (0.11) versus 4.33 (1.98), p=0.008}. (Table 4) Table 3. The value of tissue factor mRNA, thrombomodulin mRNA, and plasminogen activator inhibitor -1 mRNA levels in HUVEC after treated with IgM anti-β2GP1 compared to IgM-NHS mRNA levels *(ratio, fold to control) PAI-1 mRNA Sample IgM antiβ2GP1 TF mRNA TM mRNA PAI-1 mRNA 0.41 8.20 1 2.22 0.33 7.62 0.18 8.90 2 2.50 0.51 3.53 4.19 0.41 3.55 3 4.35 0.10 4.63 3.72 0.45 2.75 4 7.67 0.17 9.51 4.11 0.67 2.41 Sample IgG antiβ2GP1 TF mRNA TM mRNA 1 3.03 2 3.83 3 4 5 2.01 0.24 5.82 5 6 2.08 0.14 2.75 6 5.13 0.18 5.17 Mean (SD) 3.14(0.93) 0.31(0.13) 5.33(2.75) Mean (SD) 4.33 (1.98) 0.33 (0.22) 5.47 (2.64) *The result of mRNA examination was analyzed by measuring threshold cycle and the value was normalized with GAPDH using comparative quantification method.18 34 *The result of mRNA examination was analyzed by measuring threshold cycle and the value was normalized with GAPDH using comparative quantification method.18 Vol 47 • Number 1 • January 2015 Prothrombotic effect of anti-beta-2 glycoprotein-1 antibodies Table 4. The value of tissue factor mRNA, thrombomodulin mRNA, plasminogen activator inhibitor mRNA levels in HUVEC with medium (before treated), after treated with IgG anti β2GP1, and with IgM anti β2GP1 HUVEC with medium (before treated, n=6) Mean (SD) HUVEC were treated by IgG anti β2GP1, n=6) mean (SD) p mRNA TF 1.09 (0.76) 3.14 (0.93) 0.003 mRNA TM 0.91 (0.1) 0.3 (0.13) 0.001 mRNA PAI-1 0.93 (0.3) 5.33 (2.75) 0.013 HUVEC with medium (before treated, n=6) mean (SD) HUVEC were treated by IgM anti β2GP1 (n=6) Mean (SD) p mRNA TF 1.03 (0.11) 4.33 (1.98) 0.008 mRNA TM 0.93 (0.08) 0.33 (0.22) 0.003 mRNA PAI-1 1.02 (0.10) 5.47 (2.64) 0.010 Variables Variables HUVEC = human umbilical vein endothelial cells mRNA TF = messenger ribo nucleid acid - tissue factor mRNA TM = messenger ribo nucleid acid –thrombomodulin mRNA PAI-1= messenger ribo nucleid acid- plassminogen activator inhibitor The value of thrombomodulin mRNA levels in HUVEC after treated with IgG anti-β2GP1 (n=6) compared to IgG-NHS were (0.41)-, (0.18)-, (0.41)-, (0.45)-, (0.24)-, and (0.14)fold, respectively (Table 2). In other hand, after treated with IgM anti-β2GP1 compared to IgMNHS (n=6), mRNA levels of thrombomodulin were (0.33)-, (0.51)-, (0.10)-, (0.17)-, (0.67)-, (0.18)-fold, respectively (Table 3). Before and after treatment with IgG anti-β2GP1 showed significant differences of TM mRNA levels {0.91 (0.11) versus 0.31 (0.13), p=0.001}. Before and after treatment with IgM anti-β2GP1 showed significant differences of TM mRNA levels{0.93 (0.08) versus 0.33 (0.22), p=0.003}. (Table 4) The value of PAI-1 mRNA levels in HUVEC after treated with IgG anti-β2GP1 (n=6) compared to IgG-NHS were (8.2)-, (8.9)-, (3.55)-, (2.75)-, (5.82)-, and (2.75)-fold, respectively. (Table 2) In other hand, after treated with IgM anti-β2GP1 compared to IgM-NHS (n=6), mRNA levels of PAI-1 were (7.62)-, (3.53)-, (4.63)-, (9.51)-, (2.41)-, and (5.17)-fold, respectively. (Table 3) Before and after treatment with IgG anti-β2GP1 showed significant differences of PAI-1 mRNA levels {0.93(0.13) versus 5.33(2.75), p=0.013}. Before and after treatment with IgM anti-β2GP1 showed significant differences of PAI-1 mRNA levels {1.02 (0.10) versus 5.47 (2.64), p=0.01}. (Table 4) DISCUSSION In this study, we found that the mean values of TF mRNA levels in HUVEC after treated with purified IgG anti-β2GP1 were higher than after treated with IgG-NHS. The mean value of TF mRNA levels in HUVEC after treated with purified IgM anti-β2GP1 were higher than after treated with IgM-NHS. Before and after treatment with IgG antiβ2GP1 showed significant differences of TF mRNA levels (1.09±0.76 versus 3.14±0.93, p=0.003). Before and after treatment with IgM anti-β2GP1 showed significant differences of TF mRNA levels (1.03±0.11 versus 4.33±1.98, p=0.008). The previous study, Vega-Ostegard et al.9, investigated the effect of IgG from APS patients on endothelial cell showed increased of TF mRNA and the involvement of p38 MAPK (mitogen-activated protein kinases). Hamid C et al.19 examined the gene expression induced by IgG anti-β2GP1 from five APS patients showed gene expression in endothelial cells, including apoptosis/antiapoptosis genes, adhesion molecule genes, cytokines, chemokines, transcription factors, and tissue factor gene. It proved that one of mechanism for development of thrombosis in APS is through activation of coagulation. In normal conditions, endothelial cells have antithrombotic characteristics as they synthesize several substances such as prostacyclin, TM 35 Rikarni and tissue plasminogen activator (tPA). When endothelial cells get injured or activated, their antithrombotic properties become prothrombotic as they express TF, which will induce coagulation cascade through extrinsic pathway. In addition, the activated endothelial cells also produce PAI-1 that will inhibit tPA and subsequently reduce fibrinolysis.6,10,20 Increased TF will induce coagulation process that lead to prothrombotic state that facilitate the development of thrombosis.5,21 The present study found that the mean value of TM mRNA levels in HUVEC after treated with IgG antiβ2GP1 were lower than after treated with IgG-NHS.The mean value of TM mRNA levels in HUVEC after treated with IgM anti-β2GP were lower than after treated with IgM-NHS. Before and after treatment with IgG anti-β2GP1 showed significant differences of TM mRNA levels (0.91±0.11 versus 0.31±0.13, p=0.001). Before and after treatment with IgM anti-β2GP1 showed significant differences of TM mRNA levels (0.93±0.08 versus 0.33±0.22, p=0.003). It proved that one of mechanism for development of thrombosis in APS is through inhibition of protein C pathway. In the previous study, Liestol et al.22 showed increased of activated protein C (APC) resistance related with thrombosis in APS patients. Antiphospholipid antibodies can induce APC resistance and interfere the protein C pathway. Endothelial cells express TM, which will develop a complex with thrombin to activate protein C into activated protein C. Activated protein C is a physiological anticoagulant through its potential to inactivate clotting factors Va and VIIIa, which results in inhibition of thrombin formation. Protein S amplifies the activity of APC. The activated protein C with the help of protein S will inactivate coagulation factors (F)Va and FVIIIa, therefore, prevent thrombin formation. Activated protein C also has profibrinolytic activity, which is produced from the ability of APC to neutralize the activity of PAI-1.21,23 Thrombomodulin also competes with fibrinogen to bind with thrombin and prevent the development of fibrin. Low levels of TM might result in impaired C activation that lead to prothrombotic state.21 36 Acta Med Indones-Indones J Intern Med The present study found that the mean value of PAI-1 mRNA levels on HUVEC after treated with IgG anti-β2GP1 were higher than after treated by IgG NHS. The mean value of PAI1 mRNA levels in HUVEC after treated with IgM antiβ2GP1 were higher than after treated with IgM-NHS. Before and after treatment with IgG anti-β2GP1 showed significant differences of PAI-1 mRNA levels (0.93±0.13 versus 5.33±2.75, p=0.013). Before and after treatment with IgM anti-β2GP1 showed significant differences of PAI-1 mRNA levels (1.02±0.10 versus 5.47±2.64, p=0.01). It proved that one of mechanism for thrombosis in APS can be through inhibition of fibrinolysis. The process of fibrinolysis is started by releasing t-PA from endothelial cells; subsequently, t-PA will activate plasminogen into plasmin. Plasmin will degrade fibrin deposit into fibrin degradation product (FDP). The main inhibitors of this system are PAI-1 inactivating tPA and α2-antiplasmin neutralising plasmin activity. Increased levels of PAI-1 can reduce fibrinolysis and would allow the development of thrombosis.23 We suggest to examine the tissue factor, thrombomodulin, and plasminogen activator inhibitor -1 levels in plasma to determine the prothrombotic state in patients with APS, and treat the patients to prevent thrombosis. Strategies are focused on preventing the induction of procoagulant substances by antiphospholipid antibodies. Statin have a direct effect on the endothelial cells, providing an additional therapeutic tool. Activations on endothelial cells and thrombogenicity of antibodi antifosfolipid invivo can be reversed by statin. Dilazep, an antiplatelet agent, is also known to exert cytoprotective and antioxidant effects on endothelial cells. It can inhibit TF expression in endothelial cells, and may be a good candidate in the treatment of APS.24 CONCLUSION IgG anti-β2GP1 and IgM anti-β2GP1 increased TF and PAI-1 mRNA levels. However, IgG anti-β2GP1 and IgM anti-β2GP1 decreased TM mRNA levels. It proved that the mechanism of Vol 47 • Number 1 • January 2015 Prothrombotic effect of anti-beta-2 glycoprotein-1 antibodies thrombosis in APS occured through coagulation activation, reduction of fibrinolysis activity, and reduction of anticoagulant activity. REFERENCES 1. Giannakopoulos B, Passam F, Rahgosar S, Krilis SA. Current concept on the pathogenesis of the antiphospholipid syndrome. Blood. 2007;109:422-30. 2. Oku K, Amengual, Atsumi T. Pathophysiology of thrombosis and pregnancy morbidity in the antiphospholipid syndrome. Eur J Clin Invest. 2012; 42(10):1126-35. 3. Previtali E, Bucciarelli P, passamonti IM. Risk factors for venous and arterial thrombosis. Blood Transfus. 2011;9:120-38. 4. 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