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5G polymorphism in plasminogen activator inhibitor-1 gene with stroke
International Congress Series 1251 (2003) 105 – 109
Lack of association of 4G/5G polymorphism in plasminogen activator inhibitor-1 gene with stroke Masaru Shoji a,*, Shoji Tsutaya b, Chiyako Oshikata a, Minoru Yasujima a a
Department of Laboratory Medicine, Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562, Japan b Department of Clinical Laboratory, Hirosaki University Hospital, Hirosaki, Japan
Abstract Disturbances in fibrinolytic activity have been linked with an increased risk for stroke. The insertion/deletion (4G/5G) variation in the plasminogen activator inhibitor-1 (PAI-1) gene promotor has been raised as a risk for cerebrovascular diseases because of its association with the transcription and the increased plasma PAI-1 levels. However, the association between the 4G/5G polymorphism and stroke has been controversial. Therefore, we evaluated the prevalence of PAI-1 genotype in 72 cerebral infarction, 60 cerebral hemorrhage and 94 healthy subjects in northern Japan. The specific genotypes for the 4G/5G polymorphism were isolated using restriction fragment length polymorphism. The distribution of 4G/5G genotypes in the healthy control group was 40.4% for 4G/4G, 45.8% for 4G/5G and 13.8% for 5G/5G, respectively. We found no association in the 4G/5G polymorphism of the PAI-1 gene with cerebral infarction or cerebral hemorrhage. Our results suggest that the PAI-1 4G/5G gene polymorphism may not be a genetic risk factor for stroke in northern Japan. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Association; Single nucleotide polymorphism; Genotype; Cerebral infarction; Cerebral hemorrhage
1. Introduction Plasminogen activator inhibitor-1 (PAI-1) plays a key role in the fibrinolytic homeostasis. Increased plasma levels of PAI-1 are associated with an increased risk of myocardial infarction and stroke. Recently, raised PAI-1 plasma levels have been related to the single * Corresponding author. Tel./fax: +81-172-39-5124. E-mail address: [email protected] (M. Shoji). 0531-5131/03 D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0531-5131(03)00541-7
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Table 1 The study population Parameter
NS
CI
CH
Number Age, years Sex, M/F
94 60.3 F 10.9 51/43
72 63.1 F 8.2 53/19
60 61.0 F 9.6 38/22
NS, healthy subjects; CI, patients with cerebral infarction; CH, patients with cerebral hemorrhage. Data are expressed as mean F S.D.
nucleotide insertion/deletion polymorphism (4G/5G) at the position of 675 in the PAI-1 gene [1]. A coronary ischemic episode had a higher number of homozygotes for the deleted allele (4G/4G) of the PAI-1 gene [2]. Also, variation in the PAI-1 gene promotor has been raised as a risk for stroke [3]. However, an association of PAI-1 4G/5G variation with cerebrovascular diseases has been controversial among studies. Therefore, we evaluated the association of this gene polymorphism with cerebrovascular diseases in northern Japan.
2. Subjects and methods A total of 226 subjects including 72 patients with cerebral infarction, 60 cerebral hemorrhage and 94 healthy subjects were enrolled in the present study. Cerebral infarction and cerebral hemorrhage for this study were diagnosed based on an acute neurologic and image evaluation with computer tomography and/or magnetic resonance imaging. DNA was extracted from the peripheral blood of each subject as previously reported [4]. The specific genotypes for 4G/5G polymorphism were isolated using restriction fragment length polymorphism. The sequences of PCR primers were 5V-cacagagagagtctggccacg-3V for forward and 5V-ccaacagaggactcttggtct-3Vfor reverse, respectively. The digestion of the PCR products was prosecuted using Bsl-1 according to the manufacturer’s guide. The differences in ages between groups were determined by the one-way analysis of variance followed by the Dunn’s multiple comparison tests. The differences in male/female ratio and the relationship between the gene polymorphism and stroke
Table 2 Genotypes of PAI-1 4G/5G polymorphism in healthy subjects (NS), cerebral infarction (CI) and cerebral hemorrhage (CH)
4G/4G 4G/5G 5G/5G vs. NS vs. CI
NS (n = 94)
CI (n = 72)
CH (n = 60)
38 (40.4) 43 (45.8) 13 (13.8)
24 (33.3) 37 (51.4) 11 (15.3) p = 0.6448
19 (31.7) 32 (53.3) 9 (15.0) p = 0.5414 p = 0.9737
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107
Table 3 Alleles of PAI-1 4G/5G polymorphism in healthy subjects (NS), cerebral infarction (CI) and cerebral hemorrhage (CH)
4G 5G vs. NS vs. CI
NS (n = 188)
CI (n = 144)
CH (n = 120)
119 (63.3) 69 (36.7)
85 (59.0) 59 (41.0) p = 0.4282
70 (58.3) 50 (41.7) p = 0.3829 p = 0.9091
were evaluated using v2 test. P values less than 0.05 were considered statistically significant.
3. Results The ages and male/female ratios of study groups were shown in Table 1. There were no significant differences in these parameters among the three groups of healthy subjects, cerebral infarction and cerebral hemorrhage. As shown in the Table 2, the distribution of 4G/5G genotypes in the healthy control group was 40.4% for 4G/4G, 45.8% for 4G/5G and 13.8% for 5G/5G, respectively. We found no difference in the frequencies of the 4G/5G genotypes among the three groups. As shown in the Table 3, the distribution of 4G/5G allele frequencies in the healthy control group was 63.3% for 4G and 36.7% for 5G, respectively. There were no differences in the allele frequencies among the three groups.
4. Discussion In PAI-1 4G/4G genotype, plasma PAI-1 levels were reported higher than those in 5G/ 5G homozygotes [1]. Increased PAI-1 activity is well known to result in the atherothrombotic diseases. Thus, it is likely that PAI-1 4G/5G variation can be a genetic marker of stroke [3]. In the present study, however, we found the lack of association of PAI-1 4G/ 5G variation cerebral infarction or cerebral hemorrhage. It is also noteworthy that there was no opposite accumulation of the specific genotypes between atherothrombotic stroke and hemorrhagic stroke. Namely, the frequencies of genotypes as well as alleles of PAI-1 4G/5G polymorphism were identical between patients with cerebral infarction and patients with cerebral hemorrhage. Association of stroke and PAI-1 polymorphism has been controversial. We confirmed the negative association reported by Nowak-Gottl et al. [5], Akar et al. [6] and Hindorff et al. [7]. However, our results were inconsistent with several positive reports [8 –10]. The reason for the discrepancy has been uncertain. A possibility includes the importance of interrelationship with tissue plasminogen activator (tPA) genotype [3]. Because plasma tPA and PAI-1 levels have opposite effects but are highly intercorrelated. In the present study we did not evaluate the genotype of tPA gene. Therefore, we could not exclude the possible role of the genotype interrelationship between PAI-1 and tPA in the pathogenesis
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of stroke. There also remains a possibility that PAI-1 genetic variation has an effect through obesity and/or insulin resistance. Since there is evidence that plasma PAI-1 levels and genotypes are mutually related to obesity [11] and insulin resistance [12]. In Japan, there has been little evidence available as to the association between PAI-1 genotype and stroke. Our study number was not large; it was rather small. However, the frequencies of 4G/5G genotype in the present study were not different from the prevalence in Japanese subjects reported by Iwai et al. [13] and Matsubara et al. [14]. Therefore, it appears that the subjects in the present study may represent the common Japanese population. Even though, more comprehensive studies will be required to exclude the possibility of false-negative results and to establish the precise role of PAI-1 in stroke. In conclusion, we found no association of the 4G/5G polymorphism in the PAI-1 gene with cerebral infarction or cerebral hemorrhage. Our results suggest that the PAI-1 4G/5G gene polymorphism may not be a genetic marker of stroke.
Acknowledgements The current study was supported in part by Grants-in-Aid for Scientific Research (Nos. 12672236 and 13672412) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
References [1] C.W. Francis, Plasminogen activator inhibitor-1 levels and polymorphisms, Arch. Pathol. Lab. Med. 126 (2002) 1401 – 1404. [2] P. Eriksson, B. Kallin, F.M. van ’t Hooft, P. Bavenholm, A. Hamsten, Allele-specific increase in basal transcription of the plasminogen-activator inhibitor 1 gene is associated with myocardial infarction, Proc. Natl. Acad. Sci. U. S. A. 92 (1995) 1851 – 1855. [3] M. Roest, Y.T. van der Schouw, J.D. Banga, M.J. Tempelman, P.G. de Groot, J.J. Sixma, D.E. Grobbee, Plasminogen activator inhibitor 4G polymorphism is associated with decreased risk of cerebrovascular mortality in older women, Circulation 101 (2000) 67 – 70. [4] M. Shoji, S. Tsutaya, R. Saito, H. Takamatu, M. Yasujima, Positive association of endothelial nitric oxide synthase gene polymorphism with hypertension in northern Japan, Life Sci. 66 (2000) 2557 – 2562. [5] U. Nowak-Gottl, R. Strater, A. Kosch, A. von Eckardstein, R. Schobess, P. Luigs, P. Nabel, H. Vielhaber, K. Kurnik, R. Junker, The plasminogen activator inhibitor (PAI)-1 promoter 4G/4G genotype is not associated with ischemic stroke in a population of German children, Eur. J. Haematol. 66 (2001) 57 – 62. [6] N. Akar, E. Akar, E. Yilmaz, G. Deda, Plasminogen activator inhibitor-1 4G/5G polymorphism in Turkish children with cerebral infarct and effect on factor V 1691 A mutation, J. Child Neurol. 16 (2001) 294 – 295. [7] L.A. Hindorff, S.M. Schwartz, D.S. Siscovick, B.M. Psaty, W.T. Longstreth, A.P. Reiner Jr., The association of PAI-1 promoter 4G/5G insertion/deletion polymorphism with myocardial infarction and stroke in young women, J. Cardiovasc. Risk 9 (2002) 131 – 137. [8] G. Endler, W. Lalouschek, M. Exner, G. Mitterbauer, D. Haring, C. Mannhalter, The 4G/4G genotype at nucleotide position 675 in the promotor region of the plasminogen activator inhibitor 1 (PAI-1) gene is less frequent in young patients with minor stroke than in controls, Br. J. Haematol. 110 (2000) 469 – 471. [9] C.O. Bang, H.K. Park, M.K. Ahn, H.K. Shin, K.Y. Hwang, S.Y. Hong, 4G/5G polymorphism of the plasminogen activator inhibitor-1 gene and insertion/deletion polymorphism of the tissue-type plasminogen activator gene in atherothrombotic stroke, Cerebrovasc. Dis. 11 (2001) 294 – 299. [10] L.A. Hindorff, S.M. Schwartz, D.S. Siscovick, B.M. Psaty, W.T. Longstreth, A.P. Reiner Jr., The association
M. Shoji et al. / International Congress Series 1251 (2003) 105–109
[11]
[12]
[13]
[14]
109
of PAI-1 promoter 4G/5G insertion/deletion polymorphism with myocardial infarction and stroke in young women, J. Cardiovasc. Risk 9 (2002) 131 – 137. J. Hoffstedt, I.L. Andersson, L. Persson, B. Isaksson, P. Arner, The common 675 4G/5G polymorphism in the plasminogen activator inhibitor-1 gene is strongly associated with obesity, Diabetologia 45 (2002) 584 – 587. L.J. McCormack, D.K. Nagi, M.H. Stickland, M.W. Mansfield, V. Mohamed-Ali, J.S. Yudkin, W.C. Knowler, P.J. Grant, Promoter (4G/5G) plasminogen activator inhibitor-1 genotype in Pima Indians: relationship to plasminogen activator inhibitor-1 levels and features of the insulin resistance syndrome, Diabetologia 39 (1996) 1512 – 1518. N. Iwai, H. Shimoike, Y. Nakamura, S. Tamaki, M. Kinoshita, The 4G/5G polymorphism of the plasminogen activator inhibitor gene is associated with the time course of progression to acute coronary syndromes, Atherosclerosis 136 (1998) 109 – 114. Y. Matsubara, M. Murata, I. Isshiki, R. Watanabe, T. Zama, G. Watanabe, K. Watanabe, Y. Ikeda, Genotype frequency of plasminogen activator inhibitor-1 (PAI-1) 4G/5G polymorphism in healthy Japanese males and its relation to PAI-1 levels, Int. J. Hematol. 69 (1999) 43 – 47.
Lack of association of 4G/5G polymorphism in plasminogen activator inhibitor-1 gene with stroke Masaru Shoji a,*, Shoji Tsutaya b, Chiyako Oshikata a, Minoru Yasujima a a
Department of Laboratory Medicine, Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562, Japan b Department of Clinical Laboratory, Hirosaki University Hospital, Hirosaki, Japan
Abstract Disturbances in fibrinolytic activity have been linked with an increased risk for stroke. The insertion/deletion (4G/5G) variation in the plasminogen activator inhibitor-1 (PAI-1) gene promotor has been raised as a risk for cerebrovascular diseases because of its association with the transcription and the increased plasma PAI-1 levels. However, the association between the 4G/5G polymorphism and stroke has been controversial. Therefore, we evaluated the prevalence of PAI-1 genotype in 72 cerebral infarction, 60 cerebral hemorrhage and 94 healthy subjects in northern Japan. The specific genotypes for the 4G/5G polymorphism were isolated using restriction fragment length polymorphism. The distribution of 4G/5G genotypes in the healthy control group was 40.4% for 4G/4G, 45.8% for 4G/5G and 13.8% for 5G/5G, respectively. We found no association in the 4G/5G polymorphism of the PAI-1 gene with cerebral infarction or cerebral hemorrhage. Our results suggest that the PAI-1 4G/5G gene polymorphism may not be a genetic risk factor for stroke in northern Japan. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Association; Single nucleotide polymorphism; Genotype; Cerebral infarction; Cerebral hemorrhage
1. Introduction Plasminogen activator inhibitor-1 (PAI-1) plays a key role in the fibrinolytic homeostasis. Increased plasma levels of PAI-1 are associated with an increased risk of myocardial infarction and stroke. Recently, raised PAI-1 plasma levels have been related to the single * Corresponding author. Tel./fax: +81-172-39-5124. E-mail address: [email protected] (M. Shoji). 0531-5131/03 D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0531-5131(03)00541-7
106
M. Shoji et al. / International Congress Series 1251 (2003) 105–109
Table 1 The study population Parameter
NS
CI
CH
Number Age, years Sex, M/F
94 60.3 F 10.9 51/43
72 63.1 F 8.2 53/19
60 61.0 F 9.6 38/22
NS, healthy subjects; CI, patients with cerebral infarction; CH, patients with cerebral hemorrhage. Data are expressed as mean F S.D.
nucleotide insertion/deletion polymorphism (4G/5G) at the position of 675 in the PAI-1 gene [1]. A coronary ischemic episode had a higher number of homozygotes for the deleted allele (4G/4G) of the PAI-1 gene [2]. Also, variation in the PAI-1 gene promotor has been raised as a risk for stroke [3]. However, an association of PAI-1 4G/5G variation with cerebrovascular diseases has been controversial among studies. Therefore, we evaluated the association of this gene polymorphism with cerebrovascular diseases in northern Japan.
2. Subjects and methods A total of 226 subjects including 72 patients with cerebral infarction, 60 cerebral hemorrhage and 94 healthy subjects were enrolled in the present study. Cerebral infarction and cerebral hemorrhage for this study were diagnosed based on an acute neurologic and image evaluation with computer tomography and/or magnetic resonance imaging. DNA was extracted from the peripheral blood of each subject as previously reported [4]. The specific genotypes for 4G/5G polymorphism were isolated using restriction fragment length polymorphism. The sequences of PCR primers were 5V-cacagagagagtctggccacg-3V for forward and 5V-ccaacagaggactcttggtct-3Vfor reverse, respectively. The digestion of the PCR products was prosecuted using Bsl-1 according to the manufacturer’s guide. The differences in ages between groups were determined by the one-way analysis of variance followed by the Dunn’s multiple comparison tests. The differences in male/female ratio and the relationship between the gene polymorphism and stroke
Table 2 Genotypes of PAI-1 4G/5G polymorphism in healthy subjects (NS), cerebral infarction (CI) and cerebral hemorrhage (CH)
4G/4G 4G/5G 5G/5G vs. NS vs. CI
NS (n = 94)
CI (n = 72)
CH (n = 60)
38 (40.4) 43 (45.8) 13 (13.8)
24 (33.3) 37 (51.4) 11 (15.3) p = 0.6448
19 (31.7) 32 (53.3) 9 (15.0) p = 0.5414 p = 0.9737
M. Shoji et al. / International Congress Series 1251 (2003) 105–109
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Table 3 Alleles of PAI-1 4G/5G polymorphism in healthy subjects (NS), cerebral infarction (CI) and cerebral hemorrhage (CH)
4G 5G vs. NS vs. CI
NS (n = 188)
CI (n = 144)
CH (n = 120)
119 (63.3) 69 (36.7)
85 (59.0) 59 (41.0) p = 0.4282
70 (58.3) 50 (41.7) p = 0.3829 p = 0.9091
were evaluated using v2 test. P values less than 0.05 were considered statistically significant.
3. Results The ages and male/female ratios of study groups were shown in Table 1. There were no significant differences in these parameters among the three groups of healthy subjects, cerebral infarction and cerebral hemorrhage. As shown in the Table 2, the distribution of 4G/5G genotypes in the healthy control group was 40.4% for 4G/4G, 45.8% for 4G/5G and 13.8% for 5G/5G, respectively. We found no difference in the frequencies of the 4G/5G genotypes among the three groups. As shown in the Table 3, the distribution of 4G/5G allele frequencies in the healthy control group was 63.3% for 4G and 36.7% for 5G, respectively. There were no differences in the allele frequencies among the three groups.
4. Discussion In PAI-1 4G/4G genotype, plasma PAI-1 levels were reported higher than those in 5G/ 5G homozygotes [1]. Increased PAI-1 activity is well known to result in the atherothrombotic diseases. Thus, it is likely that PAI-1 4G/5G variation can be a genetic marker of stroke [3]. In the present study, however, we found the lack of association of PAI-1 4G/ 5G variation cerebral infarction or cerebral hemorrhage. It is also noteworthy that there was no opposite accumulation of the specific genotypes between atherothrombotic stroke and hemorrhagic stroke. Namely, the frequencies of genotypes as well as alleles of PAI-1 4G/5G polymorphism were identical between patients with cerebral infarction and patients with cerebral hemorrhage. Association of stroke and PAI-1 polymorphism has been controversial. We confirmed the negative association reported by Nowak-Gottl et al. [5], Akar et al. [6] and Hindorff et al. [7]. However, our results were inconsistent with several positive reports [8 –10]. The reason for the discrepancy has been uncertain. A possibility includes the importance of interrelationship with tissue plasminogen activator (tPA) genotype [3]. Because plasma tPA and PAI-1 levels have opposite effects but are highly intercorrelated. In the present study we did not evaluate the genotype of tPA gene. Therefore, we could not exclude the possible role of the genotype interrelationship between PAI-1 and tPA in the pathogenesis
108
M. Shoji et al. / International Congress Series 1251 (2003) 105–109
of stroke. There also remains a possibility that PAI-1 genetic variation has an effect through obesity and/or insulin resistance. Since there is evidence that plasma PAI-1 levels and genotypes are mutually related to obesity [11] and insulin resistance [12]. In Japan, there has been little evidence available as to the association between PAI-1 genotype and stroke. Our study number was not large; it was rather small. However, the frequencies of 4G/5G genotype in the present study were not different from the prevalence in Japanese subjects reported by Iwai et al. [13] and Matsubara et al. [14]. Therefore, it appears that the subjects in the present study may represent the common Japanese population. Even though, more comprehensive studies will be required to exclude the possibility of false-negative results and to establish the precise role of PAI-1 in stroke. In conclusion, we found no association of the 4G/5G polymorphism in the PAI-1 gene with cerebral infarction or cerebral hemorrhage. Our results suggest that the PAI-1 4G/5G gene polymorphism may not be a genetic marker of stroke.
Acknowledgements The current study was supported in part by Grants-in-Aid for Scientific Research (Nos. 12672236 and 13672412) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
References [1] C.W. Francis, Plasminogen activator inhibitor-1 levels and polymorphisms, Arch. Pathol. Lab. Med. 126 (2002) 1401 – 1404. [2] P. Eriksson, B. Kallin, F.M. van ’t Hooft, P. Bavenholm, A. Hamsten, Allele-specific increase in basal transcription of the plasminogen-activator inhibitor 1 gene is associated with myocardial infarction, Proc. Natl. Acad. Sci. U. S. A. 92 (1995) 1851 – 1855. [3] M. Roest, Y.T. van der Schouw, J.D. Banga, M.J. Tempelman, P.G. de Groot, J.J. Sixma, D.E. Grobbee, Plasminogen activator inhibitor 4G polymorphism is associated with decreased risk of cerebrovascular mortality in older women, Circulation 101 (2000) 67 – 70. [4] M. Shoji, S. Tsutaya, R. Saito, H. Takamatu, M. Yasujima, Positive association of endothelial nitric oxide synthase gene polymorphism with hypertension in northern Japan, Life Sci. 66 (2000) 2557 – 2562. [5] U. Nowak-Gottl, R. Strater, A. Kosch, A. von Eckardstein, R. Schobess, P. Luigs, P. Nabel, H. Vielhaber, K. Kurnik, R. Junker, The plasminogen activator inhibitor (PAI)-1 promoter 4G/4G genotype is not associated with ischemic stroke in a population of German children, Eur. J. Haematol. 66 (2001) 57 – 62. [6] N. Akar, E. Akar, E. Yilmaz, G. Deda, Plasminogen activator inhibitor-1 4G/5G polymorphism in Turkish children with cerebral infarct and effect on factor V 1691 A mutation, J. Child Neurol. 16 (2001) 294 – 295. [7] L.A. Hindorff, S.M. Schwartz, D.S. Siscovick, B.M. Psaty, W.T. Longstreth, A.P. Reiner Jr., The association of PAI-1 promoter 4G/5G insertion/deletion polymorphism with myocardial infarction and stroke in young women, J. Cardiovasc. Risk 9 (2002) 131 – 137. [8] G. Endler, W. Lalouschek, M. Exner, G. Mitterbauer, D. Haring, C. Mannhalter, The 4G/4G genotype at nucleotide position 675 in the promotor region of the plasminogen activator inhibitor 1 (PAI-1) gene is less frequent in young patients with minor stroke than in controls, Br. J. Haematol. 110 (2000) 469 – 471. [9] C.O. Bang, H.K. Park, M.K. Ahn, H.K. Shin, K.Y. Hwang, S.Y. Hong, 4G/5G polymorphism of the plasminogen activator inhibitor-1 gene and insertion/deletion polymorphism of the tissue-type plasminogen activator gene in atherothrombotic stroke, Cerebrovasc. Dis. 11 (2001) 294 – 299. [10] L.A. Hindorff, S.M. Schwartz, D.S. Siscovick, B.M. Psaty, W.T. Longstreth, A.P. Reiner Jr., The association
M. Shoji et al. / International Congress Series 1251 (2003) 105–109
[11]
[12]
[13]
[14]
109
of PAI-1 promoter 4G/5G insertion/deletion polymorphism with myocardial infarction and stroke in young women, J. Cardiovasc. Risk 9 (2002) 131 – 137. J. Hoffstedt, I.L. Andersson, L. Persson, B. Isaksson, P. Arner, The common 675 4G/5G polymorphism in the plasminogen activator inhibitor-1 gene is strongly associated with obesity, Diabetologia 45 (2002) 584 – 587. L.J. McCormack, D.K. Nagi, M.H. Stickland, M.W. Mansfield, V. Mohamed-Ali, J.S. Yudkin, W.C. Knowler, P.J. Grant, Promoter (4G/5G) plasminogen activator inhibitor-1 genotype in Pima Indians: relationship to plasminogen activator inhibitor-1 levels and features of the insulin resistance syndrome, Diabetologia 39 (1996) 1512 – 1518. N. Iwai, H. Shimoike, Y. Nakamura, S. Tamaki, M. Kinoshita, The 4G/5G polymorphism of the plasminogen activator inhibitor gene is associated with the time course of progression to acute coronary syndromes, Atherosclerosis 136 (1998) 109 – 114. Y. Matsubara, M. Murata, I. Isshiki, R. Watanabe, T. Zama, G. Watanabe, K. Watanabe, Y. Ikeda, Genotype frequency of plasminogen activator inhibitor-1 (PAI-1) 4G/5G polymorphism in healthy Japanese males and its relation to PAI-1 levels, Int. J. Hematol. 69 (1999) 43 – 47.