December 2007 Vol 8 After Meeting.indd Antiphosphatidyl Serine Autoantibodies and Premature Coronary Events *Hisham Y M Ali1 and Zainalabideen A Abdullah2 املبكرة القلبية واألزمات للفوسفاتيدل سيرين الذاتية املضادات اهللا عبد العزيز عبد العابدين زين ، علي محمد يوسف هشام الطريقة: من املرضى. مختارة مجموعة املبكرة لدى القلبية األزمات حدوث في سيرين ــفاتيدل الذاتية للفوس املضادات دور ــة امللخص: الهدف: دراس ودهوك/شمال اربيل, املوصل, من مدن جمعهم مت كمجموعة ضابطة ــخصا سليما 30 ش و قلبية بأزمات مصابا 50 مريضا ــة الدراس هذه ــملت ش تتكون ــنة) س (5.9 ± 39.6 ــنة ــني س اخلمس أعمارهم دون التي كانت املرضى مجموعة .2005 آذار/مارس 2004 إلى آذار/مارس من للفترة العراق الفحوصات .أجريت معروفة خطورة ــل عوام تكن لديهم ولم الصدرية ، بالذبحة مصابا 27 مريضا و القلب ــة عضل ــاء باحتش مصابا 23 مريضا ــن م املناعية األنزمية املقايسة ــتخدام فحص باس وذلك ،(IgM) م نوع و (IgG) ج نوع من ــيرين س ــفاتيدل للفوس الذاتية املضادات وجود ملعرفة الالزمة املضادات النتائج: شخصت الدراسة. في املشاركني لدى (IgM) م نوع و (IgG) ج نوع من ــيرين س ــفاتيديل للفوس املضادات الذاتية لقياس كمية معتد بفرق الضابطة ــة اموع لدى (3.3%) 1/30 ــت كان بينما املرضى من (20%) 10/50 ــي ف (IgG- ج (نوع ــيرين س ــفاتيدل للفوس ــة الذاتي دون فقط املرضى من (6%) 3/50 لدى عنه ــف مت الكش IgM فقد املضاد أما .(p < 0.05 ;9.1-odds ratio of 3.2 - 95%CI, 1.1) إحصائيا ــاف معدل اكتش يعني أن وهذا (IgG ) ج للنوع ــبة بالنس موجبة احلاالت الثالثة تلك وكانت إحصائيا. معتد فرق بدون الضابطة اموعة وجوده لدى (66.7%) 12/8 في وجدت الواصمة املضادات هذه فان لذلك . إضافة IgG لنوع املعدل نفس IgM هو نوع سيرين للفوسفاتيدل الذاتية املضادات املرضى املصابني باجللطة لدى توجد ، ولم ــتقرة املس الذبحة الصدرية مرضى من (13.3%) 15/2 وفي ، ــتقرة املس غير الصدرية مرضى الذبحة من عند املبكرة خاصة القلبية األزمات حدوث في أهمية IgG ذات الذاتية نوع سيرين الفوسفاتيدل مضادات بان الدراسة اخلالصة: تبني هذه القلبية. املستقرة. غير القلبية بالذبحة املصابني الشريان التاجي. ، احلوادث ، الذاتية املضادات سيرين الذاتية ، الفوسفاتيدل الكلمات: مضادات مفتاح 1College of Medicine, University of Dohuk, Kurdistan Region, Iraq; 2College of Medicine, University of Mosul, Iraq *To whom correspondence should be addressed. Email: halsinde@yahoo.com SULTAN QABOOS UNIVERSITY MEDICAL JOURNAL DECEMBER 2007 VOL 7, NO. 3, P. 227-232 SULTAN QABOOS UNIVERSITY© SUBMITTED - 9TH APRIL 2007 ACCEPTED - 12TH SEPTEMBER 2007 C L I N I C A L A N D B A S I C R E S E A R C H ABSTRACT Objectives: To determine whether antiphosphatidyl serine autoantibodies (aPS) are associated with increased risk of occurrence of coronary events in selected patients. Methods: This study compared 50 patients with coronary events with 30 controls, recruited from the cities of Mosul, Erbil, and Dohuk cities, Northern Iraq, between March 2004 and March 2005. The patient group consisted of 23 individuals with myocardial infarction and 27 with angina. We evaluated the presence of aPS antibodies (IgG and IgM isotypes) by an enzyme-linked immunosorbent assay. The studied cases were less than 50 years of age (mean ± SD, 39.6 ± 5.9) and had no recognizable risk factors. Results: The frequency of detecting IgG aPS was 0/50 (20%) among patients and /30 (3.3%) among controls, with significant difference and with adjusted odds ratio (OR) of 3.2 (95%CI, .-9.; p < 0.05). The IgM aPS frequency was 3/50 (6%) among patients and zero in the controls, with non-significant difference. The three cases were also IgG positive (i.e. the frequency rate for detection of aPS of IgM was the same as for IgG). Moreover, this marker (aPS) was detected in 8/2 (66.7%) of cases with unsta- ble angina, in 2/5 (3.3%) with stable angina, and in none of the cases with myocardial infarction. Conclusion: IgG aPS autoantibodies are associated with increased risk of coronary events especially angina of unstable subset. Key words: Antiphosphatidyl Serine; Autoantibodies; Events, coronary. Advances in Knowledge Recently, it was found that antiphosphatidyl serine (aPS) antibodies detection correlated more specifically with APS than anti-cardiolipin (aCL) antibodies in APS. Since, cardiolipin is present in intracellular membranes and the dose not becomes exposed to coagulation proteins in vivo, it was hypothesized that tests for antibodies against antiphosphatidyl serine, which is normally present in the inner leaflet of the plasma membrane, may be more H I S H A M Y M A L I A N D Z A I N A L A B I D E E N A A B D U L L A H 228 THE ANTIPHOSPHOLIPID SYNDROME (APS) is an autoimmune disease characterized by existence of antiphospholipid antibodies (APLAs). This syndrome is characterized by suscep- tibility to vascular thromboembolism and fetal loss.1 The lupus anticoagulant (LA) and aCL antibodies are associated with clinical events related to APS with varying severities.2 Anti- β 2- glycoprotein I (aβ-2- GPI) had been identified in patients with APS and found to play an important role in the pathogenesis of this syndrome.3 The APLAs (LA, aCL, and aβ-2-GPI) have been studied frequently and reported to be associated with an increased risk of thromboembolic phenomena such as ischaemic stroke.2, 4-6 The aPS antibodies have also been reported to be prevalent in adult stroke patients.7 There has been no study assessing the strength of their association with coronary events in the general pop- ulation. The aim of this study was to determine the prevalence of aPS in a highly selected group of patients with coronary events, who were below 50 years of age and without any conventional risk factors and had not been diagnosed as systemic lupus erythematosus (SLE) cases. Moreover, we compared the results of aPS obtained in this study to those of aCL autoantibodies from a previous study performed on the same group of patients. M E T H O D S Individuals in this study were a subset of those who participated in a PhD study disclosing the role of APLAs in patients with cardio-cerebrovascular dis- eases in the region.8 This study was carried out on 50 patients suffering from different coronary events and 30 healthy individuals, recruited from the cities of Mosul, Erbil, and Dohuk in Northern Iraq, during the period March 2004 and March 2005. The cases were selected to be under 50 years of age and having no rec- ognizable risk factors including hypertension ( systolic blood pressure > 140 mm Hg and/or diastolic of > 90 mm Hg ), diabetes mellitus (fasting blood glucose of ≥ 126 mg/dl and/or 2-hours post-prandial glucose of ≥ 140 mg/dl ), dyslipidemia ( total cholesterol level of > 240 mg/dl and HDL of < 35 mg/dl ), non-smoking (current or previous), and lastly not overweight (body mass index of less than 25). The cases were not pre- viously diagnosed as SLE. The purpose of the study was discussed in detail with the participants of both groups and obligatory ethical agreement was obtained from the volunteer cases accepted for inclusion in this study. The age range of the studied cases was 29-49 years (mean ± SD, 39.6 ± 5.9) with premature coronary oc- clusive and ischaemia diseases. Twenty-three patients had myocardial infarction (MI), 15 stable angina (SA) and 12 unstable angina (UA). These cases were diag- relevant pathophysiologically in this syndrome. The phosphatidyl serine is also exposed on syncytialized cells, on apoptotic cells and on activated platelets and their antibodies interfere more with anticoagulant cascade. The aPS, the anionic non-cardiolipin phospholipid, have recently reported to be associated with an increased risk of stroke in young patients and positive association was reported among ischaemic stroke patients. Therefore, the risk associated with APLAs in APS may be underestimated if the testing of these autoantibodies is not included in the list of APLAs used currently. Consequently, the aPS IgG isotype showed to carry an independent risk factor associated with ischaemic events especially among cases with unstable angina. On the other hand, many clinical events related to APS were observed to be positive for this marker alone, in the absence of other markers. Application to patient care It is generally assumed that an association does exist between APLAs and ischaemic coronary events. This assump- tion is based mainly upon the results obtained from different studies. The evidence supporting this association needs to be strong enough since the presence of these autoantibodies influences the management approaches of such pa- tients. Furthermore, the clinicians who diagnose such cases should realize that these cases are prone to recurrences and long-term anticoagulants need to be prescribed. Therefore, the recent trend of including APLAs tests in cases with premature ischaemic events may totally change the therapeutic options from those used in traditional ischaemic cases. The diagnosis of APS attributed to detec- tion of aPS autoantibodies or other APLAs may justify the need for more prolonged or even lifelong management to prevent recurrences. A N T I P H O S P H AT I D Y L S E R I N E A U T O A N T I B O D I E S A N D P R E M AT U R E C O R O N A R Y E V E N T S 229 nosed by a cardiac specialist depending on the acute clinical presentation, ECG changes, elevated serum cardiac marker such as creatine phosphokinase (CPK), exercise study, and ECHO study. The age range of the control group was 26-48 years (mean ± SD, 36 ± 6.5), with no history of any thromboembolic manifestations and without recognizable risk factors such as smok- ing, hypertension, diabetes mellitus, hyperlipidemia, or overweight. The individuals were recruited from the Central Blood Bank, Infertility Unit, Outpatient clinics, and volunteers. A blood sample of 10 ml was obtained from each participant and tested. A second blood sample taken after 6 weeks or more was collected from APLAs posi- tive cases. Enzyme-immunoassay kits (Orgentic Di- agnostika from Mainz, Germany) were used for the detection of IgG or IgM aPS by indirect solid phase en- zyme immunoassay (ELISA) and the tests performed in the Postgraduate Study Laboratory of Dohuk Medi- cal College. The IgG and IgM isotype results were as- sessed in IgG phospholipid (GPL) and IgM phospholi- pid (MPL) units, with one unit equal to 1µg/ml of IgG or IgM. They were considered positive if the titer was > 10 GPL units and > 10 MPL units for both isotypes, as suggested by the ELISA kit manufacturer. A significant relative risk factor was considered when the OR was > 1. The proportions were compared using the Chi-square test. The 95% CI of proportions were calculated according to the binomial distribu- tion. R E S U L T S Table 1 demonstrates the prevalence of IgG and IgM aPS autoantibodies in the studied cases and controls. A persistence positive IgG aPS titer, in both samples, was present in 20% of patients (10/50) and 3.3% of controls (1/30), with significant difference and OR of 3.2 (95%CI, 1.1 - 9.1; p > 0.05). Moreover, a positive IgM a PS titer was detected in 6% of patients (3/50) in initially tested samples and in 4% (2/50) in second test- ed samples and in none of the controls, with non-sig- nificant difference. These IgM aPS positive cases were also IgG positive (i.e. the frequency rate for aPS of any isotype were the same as for IgG). Eight out of the 10 IgG aPS positive cases had unstable angina, while, two IgG aPS positive cases had stable angina. However, none of the studied cases with a history of MI were revealed to be positive for any isotype of aPS antibod- ies. The mean titers of IgG isotype was 64.1µg/ml among patients with UA and 12.9µg/ml in patients with SA. However, the mean titer of IgM aPS in the 2 persistence positive cases was 29µg/ml with UA. In comparison, the frequency of persistent IgG aCL with a concentration of < 30 GPL units was detected in 14% of cases (7/50), with significant difference and OR of 9.8 (95%CI, 7.8 - 76.4; p > 0.01), while none of the control showed such a concentration. The results of transthoracic echocardiographic findings of the 10 aPS positive cases are summarized in Table 2. Normal results were reported in 40% of pa- tients (4/10), two with SA and another two with UA. Table 1: The frequencies of the different antiphospholipid autoantibodies in patients with coronary events. Groups Patients No. (%) Control No. (%) OR 95% CI P value* Test aPS (GPL units) < 10 40 (80) 29 (96.7) 3.2 1.1 – 9.1 P > 0.05 > 10 10 (20) 1 (3.3) aPS (MPL units) < 10 47 (94) 30 (100) 3.8 1.7 – 24.6 NS > 10 3 (6) 0 (0.0) aPS of IgG and/or IgM 10 (20) 1 (3.3) 3.2 1.1 – 9.1 P > 0.05 * Chi-square test NS: Not significant OR: Odds ratio 95% CI: Confidence interval H I S H A M Y M A L I A N D Z A I N A L A B I D E E N A A B D U L L A H 230 Abnormal findings were recorded in the remaining 60% (6/10), 4 in the form of hypokinetic ventricles (all were with UA) and three cases as heart valves abnor- malities with UA. D I S C U S S I O N The association of APLAs with coronary artery dis- eases has been shown in several studies but remains controversial.8, 9, 10 The mechanisms through which these APLAs can induce pathological changes and tissue necrosis in MI or initiating atherosclerotic changes are debatable.9 The association between car- diac events especially MI and IgG aCL has been sug- gested in other studies.10, 11 Billi et al.12 reported that in post-infarction patients elevated IgG aCL antibodies are an independent risk factor for recurrent cardiac events and patients with elevated IgG aCL and low IgM aCL antibodies have the highest risk. The aPS antibodies, members of the non-cardioli- pin APLAs, have been shown to be associated with increased stroke risk in SLE patients with a variety of APLAs to non-cardiolipin antigens.13 Another study by Toschi et al.14 of stroke and/or transient ischaemic attack patients, but with unselected patients, demon- strated also a positive correlation. Although these au- toantibodies have been associated with stroke events related to APS, their prevalence in coronary events among patients without conventional risk factors is unknown. In this study, although the number of the studied cases were relatively small, a positive association was demonstrated between IgG aPS in patients with coro- nary events especially those with UA. On the other hand, three of our patients who were IgG aPS positive were negative for IgG aCL, i.e. 6% of the studied pa- tients would have been characterized as APLAs nega- tive if only an aCL assay were used. Moreover, the estimated mean titer of IgG aPS was higher in patients with UA (64.1µg/ml) compared with cases with SA (12.9µg/ml). This finding could be attributed to the more intensified immune process go- ing on in patients with UA than in SA events related to APS. The absence of aPS autoantibodies in patients with MI may signal to the weak immune inducer effect of the tissue necrosis characterizing these events.9 An echocardiographic study of aPS positive cases revealed that 60% (6/10) have abnormal cardiac find- ings in form of hypokinetic ventricles and valvular infection and all cases were sufferings from UA. One case was with mitral valve prolapse and one with aor- tic insufficiency. Niaz and Butany’s study in 199815 revealed that 35% of patients with primary APS had valvular abnormalities, while Bouillanne et al., in 1996 16 reported that about 20% of cardiac patients with val- vular heart disease had evidence for APLAs compared with about 10% of matched control subjects. Depos- its of APLAs such aCL antibodies and of complement components are common in the affected valves of patients with primary or secondary APS.17 Another study of patients with SLE and primary APS did not find any relationship between increased aCL antibod- ies and valvular abnormalities.18 A small number of studies investigated patients with cardiac events and their correlation with APS. Some of them studied cases with MI and tested for aCL antibodies only 10, 11, 19 while others studied the relationship between APLAs and atherosclerosis in ischaemic heart disease using the aβ 2-GPI antibod- ies tests only.20, 21, 22 Over the last decade, many studies have been performed and suggest that aβ 2-GPI plays a corner stone role in the pathological consequences 23, 24, but studies of aPS and coronary events were lack- ing, The mechanisms by which these antibodies may influence thrombogenicity are incompletely un- derstood, but these have recently been reviewed comprehensively.14, 25 These mechanisms may include the stimulation of platelet aggregation, the recognition of vascular endothelial cells and the disruption of the annexin-V shield that forms a carpet protecting anion- ic phospholipids on endothelial cell surfaces from par- ticipating in coagulation reactions.1, 14, 25 This last pro- posed mechanism may be applicable to the role of aPS specifically because aPS have been shown to remove annexin-V from and facilitate binding of prothrombin to cell surfaces.26 Finding (s) No. (10) % Normal 4 40 Hypokinetic ventricles 4 40 Valvular abnormality 2* 20 * One case with mitral valve prolapse and the other with aortic valve insufficiency. Table 2: The echocardiographic findings in antiphosphatidyl serine autoantibodies positive patients with coronary events A N T I P H O S P H AT I D Y L S E R I N E A U T O A N T I B O D I E S A N D P R E M AT U R E C O R O N A R Y E V E N T S 231 C O N C L U S I O N The results of the current study demonstrate an in- creased risk and positive association between IgG aPS and coronary events of angina subsets among cases with UA. Moreover, our results revealed that testing for this antibody, along with other APLAs, may iden- tify more cases related to APS. This, therefore, justi- fyies the routine use of aPS as a serologic marker in the general population in suspected coronary events. R E F E R E N C E S 1. Rand JH. Antiphospholipid antibody syndrome: new insights on thrombogenetic mechanisms. Am J Med Sci 1998; 316:142–151. 2. The Antiphospholipid Antibodies in Stroke Study (APASS) Group. Anticardiolipin antibodies are an inde- pendent risk factor for first ischaemic stroke. Neurology 1993; 43:2069-2073. 3. McNeil HP, Simpson RJ, Chesterman CN, Krilis SA. Anti-phospholipid antibodies are directed against a complex antigen that includes a lipid-binding inhibitor of coagulation: beta 2-glycoprotein I (apolipoprotein H). Proc Natl Acad Sci USA 1990; 87:4120-4124. 4. Rauch J, Tannenbaum M, Janoff AS. Distinguishing plasma lupus anticoagulant from anti-factor antibodies using hexagonal (II) phase phospholipids. Thromb Hae- most 1999; 62:892-896. 5. Lopez-Soto A, Cerver AR, Font J, Bove A, Reverter JC, Munoz FJ, et al. Isotype distribution and clinical signifi- cance of antibodies to cardiolipin, phosphatadic acid, phosphotidylinositol and phosphatidylserine in system- ic lupus erthymatosus: prospective analysis of a series of 92 patients. Clin Exp Rheumatol 1997; 15:143-149. 6. Camerlingo M, Casto L, Censori B, Drago G, Frigeni A, Ferraro B, et al. Anticardiolipin antibodies in acute non- hemorrhagic stroke seen within six hours after onset. Acta Neurol Scand 1995; 92:69-71. 7. Toschi V, Motta A, Castelli C, Paracchini ML, Zerbi D, Gibelli A. High prevalence of antiphosphatidylinositol antibodies in young patients with cerebral ischaemia of undetermined cause. Stroke 1998; 29:1759–1764. 8. Ali Hisham YM. The Role of Antiphospholipid Au- toantibodies Syndrome in Patients with cardio-cerebro vascular diseases. PhD thesis submitted to College of Medicine, University of Mosul, Iraq 2006. 9. Vaarala O. Antiphospholipid antibodies and atheroscle- rosis. Lupus 1996; 5:442-447. 10. Zuckerman E, Toubi E, Shiran A, Sabo E, Shmuel Z, Golan TD, et al. Anticardiolipin antibodies and acute myocardial infarction in non-systemic lupus erythema- tosus patients: a controlled prospective study. Am J Med. 1996; 171:381-386. 11. Wu R, Nityanand S, Berglund L, Lithell H, Holm G, Lefvert AK. Antibodies against cardiolipin and oxida- tively modified LDL in 50 year old men predict myo- cardial infarction. Arterioscler Thromb Vasc Biol 1997; 17:3159-3163. 12. Bili A, Moss AJ, Francis CW, Zareba W, Watelet LF, Sanz I. Anticardiolipin antibodies and recurrent coronary events: a prospective study of 1150 patients. Thrombo- genic Factors and Recurrent Coronary Events Investiga- tors. Circulation 2000; 102:1258-1263. 13. Tuhrim S, Rand JH, Wu X, Horowitz DR, Weinberger J, Goldman ME, Godbold JH. Antiphosphatidylserine antibodies are independently associated with ischaemic stroke. Neurology 1999; 53:1523-1527. 14. Toschi V, Motta A, Castelli C, Gibelli S, Cimminiello C, Molaro GL, et al. Prevalence and clinical significance of antiphospholipid antibodies to non-cardiolipin in sys- temic lupus erythematosus. Haemostasis 1993; 23:275- 283. 15. Niaz A, Butany J. Antiphospholipid antibody syndrome with involvement of a bioprosthetic heart valve. Can J Cardiol. 1998; 14:951-954. 16. Bouillanne O, Millaire A, de Groote P, Puisieux F, Ces- bron JY, Jude B, et al. Prevalence and clinical significance of antiphospholipid antibodies in heart valve disease: a case-control study. Am Heart J 1996; 132:790-795. 17. Ziporen L, Goldberg I, Arad M, Hojnik M, Ordi-Ros J, Afek A, et al. Libman-Sacks endocarditis in the an- tiphospholipid syndrome: immunopathological find- ings in deformed heart valves. Lupus 1996; 5:196-205. 18. Gabrielli F, Alcini E, Prima MA, Masala C. Cardiac in- volvement in connective tissue diseases and primary antiphospholipid syndrome: echocardiographic assess- ment and correlation with antiphospholipid antibodies. Acta Cardiol 1996; 51:425-439. 19. Raghavan C, Ditchfield J, Taylor RJ, Haeney MR, Barnes PC. Influence of anticardiolipin antibodies on immedi- ate patient outcome after myocardial infarction. J Clin Pathol. 1993; 46:1113-1115. 20. Vaarala O, Manttari M, Manninen V, Tenkanen L, Puu- runen M, Aho K et al. Anticardiolipin antibodies and risk of myocardial infarction in a prospective cohort of middle-aged men. Circulation 1995; 91:23-27. 21. Farsi A, Domeneghetti MP, Fedi S, Capanni M, Giusti B, Marcucci R et al. High prevalence of anti-beta2 glyco- protein I antibodies in patients with ischaemic heart disease. Autoimmunity 1999; 30:93-98. 22. Brey RL, Abbott RD, Curb JD, Sharp DS, Ross GW, Stallworth CL, et al. Beta(2)-Glycoprotein 1-dependent anticardiolipin antibodies and risk of ischaemic stroke and myocardial infarction: the Honolulu heart program. Stroke 2001; 32:1701-1706. 23. Salemink I, Blezer R, Willems GM, Galli M, Bevers E, Lindhout T. Antibodies to β2-glycoprotein I associated H I S H A M Y M A L I A N D Z A I N A L A B I D E E N A A B D U L L A H 232 with antiphospholipid syndrome suppress the inhibi- tory activity to tissue factor pathway inhibitor. Thromb Haemost 2000; 84:653-656. 24. Crowther MA, Wisloff F. Evidence based treatment of the antiphospholipid syndrome II. Optimal anticoagu- lant therapy for thrombosis. Thromb Res 2005; 115:3-8. 25. Metz LM, Edworthy S, Mydlarski R, Fritzler MJ. The frequency of phospholipid antibodies in an unselected stroke population. Can J Neurol Sci 1998; 325:64–69. 26. Vogt E, Ng A, Rote NS. Antiphosphatidyl serine anti- body removes annexin V and facilitates the binding of prothrombin at the surface of a choriocarcinoma model of trophoblast differentiation. Am J Obstet Gynecol 1997; 177:964–972.