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Indian Journal of Medical Sciences, Vol. 58, No. 9, September, 2004, pp. 381-388 Original Article Combination of thrombophilia markers in acute myocardial infarction of the young Khare Amit, Ghosh Kanjaksha, Shetty Shrimati, Kulkarni Bipin, Mohanty Dipika Institute of Immunohaematology (I.C.M.R.), 13th floor,
New Bldg., KEM Hospital, Parel, Mumbai - 400 012 Code Number: ms04066 ABSTRACT BACKGROUND: The pathogenesis of arterial thrombotic disease involves multiple genetic and environmental factors related to atherosclerosis and thrombosis. But, there have been very few studies in India which have investigated some of the thrombophilia markers. AIM: To look for combined thrombophilia in MI patients. SETTINGS AND DESIGN: One hundred twenty patients of myocardial infarction (age below 40 yrs.) were recruited 8-10 weeks after stabilization. Hundred age and sex-matched healthy controls were also recruited in the present study. METHODS AND MATERIAL: Following thrombophilia markers were screened in these patients- plasma fibrinogen, protein C, protein S, antithrombin III, factor V Leiden, PT G20210A polymorphism, MTHFR C677T, homocysteine, fibrinogen b448 Arg/Lys polymorphism and CBS T833C mutation. STATISTICAL ANALYSIS: Statistical analysis was done using Statistical Package for Social Sciences (SPSS) version 10.0, SPSS Inc., Chicago, USA. RESULTS AND CONCLUSION: Elevated fibrinogen levels, homocysteine (p<0.001 and homocysteine with odds ratio 6.26) and factor V Leiden (p=0.038) were independently associated with MI in our patients. A total of 37 patients (42.5%) had the presence of more than one thrombophilia markers in combination. Out of these, 10 had the presence of three markers in combination and 1 had five thrombophilia markers in combination. Only 2 controls had prothrombotic markers in combination. Combined prothrombotic risk factors were significant in cases in comparison to controls (p<0.001). Further larger studies on a nationwide basis recruiting a large number of young MI patients should be done to substantiate these findings. Key Words: Homocysteine, Fibrinogen, Combined thrombophilia, Myocardial infarction, India. Myocardial infarction (MI) is one of the commonest causes of death in the developing and developed world. MI in Indian people is claiming a large number of lives even before the patients are reaching their fortieth birthday.[1] Various studies in India have revealed a consistent increase in the prevalence of CAD in urban and rural populations[2] with an alarming increase mainly in young age groups. The well-documented risk factors in Indians are smoking, higher levels of cholesterol, apolipoprotein B, blood glucose and lower levels of HDL cholesterol.[3],[4] In recent years there is rapidly growing literature on the relationship between the hemostatic system and arterial thrombosis. Various studies in Western countries on MI cases have comprised of variation in genes for some of the blood coagulation factors, inhibitors, fibrinolytic system and platelet membrane receptors. They have shown that some of the thrombophilia markers may be significant in a subset of patients (e.g. young patients, patients lacking conventional risk factors). But, in India there have been scattered studies which have investigated some of the thrombophilia markers in arterial thrombosis. Most frequently studied of these is Lp(a). Several case-control studies have shown that patients with CAD have significantly higher Lp(a) levels[5],[6],[7] and a recent study failing to find an association.[8] Study from Chennai found raised Protein C levels in patients with previous MI.[9] Among the coagulation factors, elevated levels of fibrinogen have been found to be a risk factor for CAD in Indians.[10],[11],[12] Studies of homocysteine in Indians have yielded conflicting results.[11],[13],[14],[15],[16],[17] In India, the prevalence of factor V Leiden is quite low[18],[19] and prothrombin gene polymorphism has not been reported in the Indian population.[18],[20] There is a paucity of data about the thrombophilia status in our young MI patients. In the West, studies have also investigated the possible role of combined thrombotic factors in patients with venous thrombosis and have reported the presence of combined risk factors in nearly 1/3rd of the patients.[21] But, to date, there have been case reports which have described the presence of combined prothrombotic factors in arterial thrombosis[22] and stroke.[23] However no such study from India has looked for combined thrombophilia in any thrombotic cases. All these factors prompted us to undertake the present study. METHODS One hundred twenty consecutive patients of MI (age< 40 years) were recruited 8-10 weeks after stabilization after MI from the out-patients department, Department of Cardiology, KEM Hospital, Mumbai over a period of two-and half years. The exclusion criteria were diabetes and hypertension. This study was approved by the ethics committee and an informed patient consent was obtained in all the cases before blood collection. Unequivocal evidence of MI was obtained by ECG, cardiac enzymes and cardiac stress test. Coronary angiography was done only in patients who had positive stress test. Detailed pro-forma was filled up with special emphasis on smoking, alcoholism, family history and hyperlipidemia. In addition, 100 age and sex-matched voluntary blood donors were recruited as healthy controls in the study. These control subjects did not have any past history of any cardiovascular diseases and had no family history of CAD. The socio-economic status of the patients did not differ from the controls.Blinding was achieved with the clinicians not knowing the laboratory investigation results and the laboratory personnel unaware of the clinical history of the patient. An overnight fasting blood sample was collected in 3.13% tri-sodium citrate. Platelet poor plasma was separated within 1 hour of collection and stored at -80oC until assayed. The lymphocytes were frozen at -80oC for DNA extraction. In addition, 3 ml of blood was collected in a plain tube and serum was separated within 30 minutes of collection and stored at -80oC until assayed. Assay of Protein C (PC), Protein S (PS) was done by ELISA (Diagnostica Stago, France) and Antithrombin III (AT III) by chromogenic substrate method (Diagnostica Stago, France). Fibrinogen levels were determined by the method of Clauss (Diagnostica Stago, France). DNA extraction was done by phenol-chloroform method.[24] Analysis of factor V Leiden, Prothrombin G20210A polymorphism, MTHFR C677T, fibrinogen b-448 Arg/Lys polymorphism, CBS T833C mutation was done by molecular biology techniques as previously described[25],[26],[27],[28],[29] and serum homocysteine by ELISA (Bio-Rad Laboratories, USA). Statistical analysis Multivariate analysis, chi-square and t-test were the different statistical tests employed for data analysis. Statistical analysis was done using a Statistical Package for Social Sciences (SPSS) version 10.0, SPSS Inc., Chicago, USA.RESULTS The clinical profile of the patients is shown in [Table - 1]. 92% of the patients were male. The most common site of infarction was the anterior wall of the heart. In the patients who had undergone a coronary angiography owing to a positive stress test, majority had single vessel disease (76%). Smoking as risk factor was present in 35 (31%) patients, alcoholism in 8 (7%) whereas 10 (8%) had a positive family history of coronary artery disease. A total of 22 patients (19%) had hyperlipidemia.[Table - 2] shows the distribution of thrombophilia markers in MI cases and controls. The mean homocysteine levels (mean ± S.D.) were 23.49 ± 16.11 in MI patients as compared to 14.08 ± 8.28 in controls. A photograph of 10% polyacrylamide gel for the analysis of factor V Ledien mutation is shown in [Figure - 1]. A total of 87 patients (72.5%) out of 120 in the present study had the presence of at least one thrombotic risk factor. Out of these, 37 patients (42.5%) had the presence of more than one thrombophilia markers in combination [Table - 3]. From these 37 patients, 10 had three thrombophilia markers in combination while one had five thrombophilia markers in combination. In the controls, only 2 individuals had combined prothrombotic markers in combination. Both these individuals had hyperhomocysteinemia in combination with heterozygosity for MTHFR C677T. Thus, combined thrombophilia was significantly present in MI cases as compared to controls (p<0.001). The patient who had five thrombophilia markers in combination had an anterior wall infarct, single vessel disease and LVEF of 55% on 2-D echocardiography. [Table - 4] shows the comparison of increased severity (denoted by a positive stress test or abnormal coronary angiogram) of MI among patients having combined thrombophilia markers as compared to patients lacking them in combination or negative for these markers. There was a significant association of severity with combined thrombotic factors (p=0.047). Only 14 out of the 37 patients having more than one thrombophilia marker in combination had the presence of conventional risk factors. The most common was smoking. DISCUSSION The event leading to MI is the formation of a thrombus. The present study investigates some of the thrombophilia markers in young MI patients from India since there is a paucity of data in Indian patients. There have also been reports which show that cardiovascular disease has reached alarming proportions in India and it cannot be neglected.[30],[31] Since MI in the young age groups occurs mainly in males, it is unlikely that our results will be altered if we remove the 10 female MI patients from the current series.Conventional risk factors like smoking, positive family history and hyperlipidemia were present in a very small proportion of cases. The prevalence of factor V Leiden was significantly higher in cases as compared to controls (p=0.038) in contrast to a recent study.[32] However, our findings are consistent with previous studies.[19],[33],[34] Prothrombin gene polymorphism was absent from both cases as well as controls. Previous Indian studies[18],[19],[20],[33] also showed absence of this mutation in Indian population. The presence of MTHFR C677T, CBS T833C and fibrinogen b448 Arg/Lys polymorphism was not significantly increased in our cases. Fibrinogen levels were significantly elevated in MI patients in agreement with previous Indian studies.[10],[11],[12] The presence of Protein C, Protein S and Antithrombin III deficeincy was seen in 3 cases each in agreement with previous studies which have reported isolated case reports of MI with Protein C, Protein S and Antithrombin III deficiencies.[35] Homocysteine levels were significantly elevated in the patients as compared to controls with an odds ratio of 6.26. This is in agreement with a recent study from Kanpur.[14] Other previous reports from India failed to find an association of plasma homocysteine levels with CAD or MI.[11],[15],[16],[17] The reason for this finding is not completely clear. Our study comprised of patients from Western India and an important point is that all the patients were studied only after stabilization. Although heterozygosity for MTHFR C677T is associated with mild increase in homocysteine levels, in the present study we found hyperhomocysteinemia in 4 patients heterozygous for MTHFR C677T. This may be explained by the fact that these patients may have folic acid deficiency or a second polymorphism- MTHFR A1298C in which case, individuals doubly heterozyogus for both these polymorphisms had 50-60% of the enzyme activity.[36] Some scattered studies in India over the past few years have focussed on the hemostatic aspects in CAD but they have not been able to determine the significance of thrombophilia markers in our young patients. Like in the West, combined thrombophilia markers may also be possible in our MI patients wherein the conventional factors play a minor role in the thrombotic episode. 1/3rd of the patients in the present series had the presence of combined thrombotic risk factors and a large proportion lacked any conventional risk factors. Also, combined prothrombotic risk factors were significant in cases (p<0.05). Lastly, there was an association of severity of MI with combined prothrombotic markers (p=0.047). CONCLUSION Thrombophilia markers- hyperhomocysteinemia, high fibrinogen concentration and factor V Leiden seem to be important in Indian CAD patients. Thus, it can be said that thrombotic markers in combination may contribute to MI in a proportion of the young individuals with increased severity of the disease. Further larger studies on a nationwide basis recruiting a large number of young MI patients should be done to substantiate these findings.REFERENCES
Copyright 2004 - Indian Journal of Medical Sciences The following images related to this document are available:Photo images[ms04066t4.jpg] [ms04066t3.jpg] [ms04066f1.jpg] [ms04066t2.jpg] [ms04066t1.jpg] |
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