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(Circulation. 1997;96:3281-3286.)
© 1997 American Heart Association, Inc.

Articles

Coronary Artery Disease and Polymorphisms in a Receptor Mediating Shear Stress–Dependent Platelet Activation

Mitsuru Murata, MD; Yumiko Matsubara; Koichi Kawano, MD; Takeru Zama, MD; Nobuo Aoki, MD; Hideaki Yoshino, MD; Gentaro Watanabe, MD; Kyozo Ishikawa, MD; ; Yasuo Ikeda, MD

From the Department of Medicine, School of Medicine, Keio University (M.M., Y.M., T.Z., Y.I.), Tokyo; Second Department of Internal Medicine, Kyorin University (K.K., N.A., H.Y., K.I.), Tokyo; and Medical Center, Sakura Bank (G.W.), Tokyo, Japan.

Correspondence to Mitsuru Murata, MD, Department of Medicine, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160, Japan.

	Abstract

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Background Platelets play pivotal roles in coronary thrombosis, and antiplatelet therapies are widely used for coronary artery disease (CAD). However, the effects of genetic variation in platelets on CAD are poorly understood. We have assessed the association between CAD and polymorphisms in a platelet receptor for von Willebrand factor, the glycoprotein (GP) Ib/IX complex, which mediates shear stress–dependent platelet activation.

Methods and Results Genotypes of the -chain of the receptor (GP Ib, ¹⁴⁵Thr/Met) were determined in 91 patients with myocardial infarction (MI) or angina pectoris whose lesions were confirmed by coronary angiography as well as in 105 individuals from the general population with no history of angina or other heart diseases and normal resting ECGs. There was no homozygote for Met/Met in either the control or patient groups. The prevalence of the Thr/Met genotype (T/M) in all patients was not significantly different from that in the control group. However, the frequency of T/M was significantly higher in patients aged 60 years (31.8%) than in control subjects aged 60 years (16.0%; P<.05, odds ratio=2.5). An association was also demonstrated between CAD and the other polymorphism of GP Ib, a variable number of tandem repeats of a 13–amino acid sequence, which is known to be linked to the ¹⁴⁵Thr/Met polymorphism. There was an association between the frequency of the T/M genotype and the angiographic severity of CAD: 11.1% for Gensini score <40 versus 50.0% for Gensini score 40 (P=.0015). There was no difference in the distribution of GP Ib genotypes between patients with MI and those with angina pectoris.

Conclusions This study suggests that the presence of the Met allele in GP Ib is a risk factor for the prevalence and severity of CAD in individuals aged 60 years. The results need to be confirmed in a large-scale study of incident case subjects and matching control subjects.

Key Words: coronary disease • risk factors • platelets • glycoproteins • genetics

	Introduction

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Angiographic and angioscopic investigations have demonstrated the role of thrombus formation in the pathogenesis of acute coronary syndromes.^1,2 For coronary thrombosis, platelet function is believed to be a key factor.^3–6 Platelets may be also involved in the development of atherosclerosis.^7,8 Adhesion of platelets to damaged blood vessels initiates platelet thrombus formation and, by releasing growth factors, leads to proliferation of vascular cells. Recent studies have revealed that platelets under blood flow can be activated by high shear stress generated in stenosed arteries or arterioles, without chemical agonists. This shear-induced platelet activation is dependent on an adhesive ligand, vWF, and its platelet receptor, GP Ib/IX complex.^9–14 It is now believed that binding of vWF to GP Ib/IX initiates intracellular signals resulting in activation of another platelet receptor, GP IIb/IIIa complex, to form platelet clumps.^15–17

The roles of the vWF–GP Ib/IX interaction in the development of coronary stenosis and acute coronary syndromes have been implicated in recent reports. Animals that were congenitally devoid of vWF were less prone to develop atherosclerosis,^18,19 and agents that block either vWF or GP Ib/IX inhibited and delayed coronary occlusion in animal models.^20,21 Moreover, an elevated plasma level of vWF is a poor prognostic factor for coronary heart disease²² as well as an independent risk factor for subsequent acute coronary events in patients with angina pectoris.²³

Two genetic polymorphisms have been reported in the coding sequence of the gene encoding the -chain of GP Ib (GP Ib).^14,24–26 The first polymorphism, a C/T transition at nucleotide 1018 (numbers according to Wenger et al²⁷), results in an amino acid dimorphism (Thr/Met) at residue 145 of GP Ib, which is located within the vWF-binding domain of the receptor. This polymorphism is a known molecular basis of a platelet alloantigen system, HPA 2a/2b, and is involved in the development of platelet transfusion refractoriness.^24,25 The second polymorphism is the variable number (one to four) of 13–amino acid sequence repeats.¹⁴ This "size polymorphism" is known to be strongly associated with the first polymorphism, alleles with one or two repeats being linked to ¹⁴⁵Thr whereas alleles with three or four repeats are linked to ¹⁴⁵Met.²⁸ However, the functional consequences of these polymorphisms in the receptor are still unknown.

Therefore, we sought to assess the relationships between these polymorphisms and the prevalence and severity of CAD. We have evaluated the frequencies of the genotypes in patients with angiographically proven CAD and control subjects.

	Methods

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Patients and Control Subjects
Blood samples were collected consecutively from 91 genetically unrelated Japanese patients with either MI (n=66) or angina pectoris (n=25) at an outpatient clinic of Kyorin University Hospital (Tokyo, Japan) if the patient was judged to be eligible for this study. All patients had been diagnosed as having CAD, and all were alive at the time of genotyping. The criteria for entry were as follows: for MI, diagnosis was based on clinical symptoms, appropriate new onset of ECG changes, and elevated serum creatine phosphokinase levels. Only patients whose diagnosis of MI had been made at this hospital were entered into the study. All but 8 patients with MI were evaluated by coronary angiography. For angina pectoris, diagnosis was made according to clinical symptoms and ECG findings, and only patients whose coronary lesions had been confirmed by coronary angiography were eligible for entry into the study. Patients having an affected vessel with >50% reduction of lumen size (75% stenosis by American Heart Association classification) were entered into the study. Patients enrolled in the study were considered prevalence cases. For patients with MI, their age at first event was recorded, whereas for patients with angina pectoris, the age at which coronary angiography was performed was recorded.

One hundred five control subjects recruited at Hibiya Medical Center (Tokyo, Japan) for their regular checkup were consecutively enrolled into the study. They were a genetically unrelated Japanese population without any symptoms who had no history of angina or other heart diseases and who had normal resting ECGs. Clinical data including smoking history, blood pressure, serum total cholesterol level, triglyceride level, HDL cholesterol level, and diabetes status were collected from medical records of patients. These data of control subjects were collected from regular checkup sheets.

Documentation of CAD Severity
To determine the severity of CAD, we used two systems: Gensini's coronary artery scoring method²⁹ and affected vessel number. In the former scoring system, the geometrically increasing severity of lesions, the cumulative effects of multiple obstructions, the significance of their locations, the modifying influence of the collaterals, and the size and quality of the distal vessels were taken into consideration.²⁹ In the latter system, the severity of CAD was expressed simply by affected vessel numbers (one-vessel, two-vessel, or three-vessel disease). A lesion at the left main coronary artery was regarded as two-vessel disease.

Preparation of Genomic DNA and Determination of GP Ib Genotypes
Blood was obtained from peripheral veins after informed consent was obtained from patients and control subjects. Genomic DNA was isolated from leukocytes as described.³⁰ To genotype the first polymorphism, a 591-bp DNA fragment of GP Ib that contains the dimorphism at nucleotide 1018 (amino acid residue 145) was amplified by PCR with the use of a DNA thermal cycler (Perkin Elmer, Takara Biomedicals) as described previously.²⁵ Briefly, the reaction was performed in a final volume of 100 µL containing 1 µg of genomic DNA, 10 mmol/L Tris (pH 8.3), 50 mmol/L KCl, 1.5 mmol/L MgCl₂, 0.01% gelatin, 0.2 mmol/L of each deoxynucleotide triphosphate, and 2.5 U of Taq polymerase. The two oligonucleotide primers used were 5'-GGACGTCTCCTTCAACCGGC (nucleotide number 899 to 918) and 5'-GCTTTGGTGGGGAACTTGAC (1470 to 1489). After a 30-cycle PCR, each cycle consisting of 94°C for 1 minute, 63°C for 1 minute, and 72°C for 1 minute, with a final extension at 72°C for 7 minutes, the PCR product was digested with restriction enzyme Bbi II (Takara Shuzo) at 37°C for 3 hours followed by a 2% agarose gel electrophoresis. Bbi II digestion of the amplified material from the allele containing ATG (M) at codon 145 would result in visible DNA fragments of 387 and 201 bp, whereas PCR products from ACG (T)–containing alleles would be cut into visible fragments of 271, 201, and 116 bp. For the second polymorphism, two oligonucleotide primers, 5'-ACACTTCACATGGAACTCCAT (nucleotide number 1626 to 1645) and 5'-GGGTCATTTCTGGAGCTCTC (1995 to 2014) were synthesized. PCR was performed as described above except that 0.1 mmol/L 7-deaza-2'-deoxyguanosine-5'-triphosphate was added for 30 cycles, each consisting of 96°C for 1 minute for denaturation, 51°C for 1 minute for annealing, and 72°C for 1 minute for extension. The amplified DNA fragments would be 389, 428, 467, and 506 bp if the genomic DNAs contained alleles with one, two, three, and four tandem repeats, respectively.

Statistics
Statistical analyses of frequency counts were performed with the use of the ² test or Fisher's exact test for small samples. Differences in the frequency of a GP Ib genotype (T/M) between patients and control subjects were considered to be statistically significant when the probability values were <.05. The OR was used as a measure of the risk of CAD in patients having the T/M genotype versus those having the T/T genotype. Variability in sampling associated with the estimated OR was assessed by two-sided 95% CI. An OR (95% CI) >1 was considered significant. A logistic regression analysis was performed to evaluate the interaction between the GP Ib genotypes and other variables in relation to the prevalence of CAD. Independent variables included in the analysis were age (quantitative), sex (male or female), smoking (yes or no), hypertension (yes or no), diabetes mellitus (yes or no), hypercholesterolemia (yes or no), and hypertriglyceridemia (yes or no). The analysis was executed by the SPSS statistical program version 7.5 for Windows.

	Results

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The 91 patients ranged in age from 41 to 74 years (60.7±8.7 years, mean±SD); there were 64 men (70.3%) and 27 women (29.7%, Table 1). When only patients aged 60 years were analyzed (n=44, of whom there were 36 men [81.8%]), the mean age was 53.9±6.6 years. The patient group had a higher prevalence of selected coronary risk factors (smoking, hypertension, diabetes mellitus, and hypertriglyceridemia) than control subjects. The patient group consisted of 66 MI patients and 25 angina patients.

View this table: [in this window] [in a new window]   Table 1. Characteristics of Control Subjects and Patients

Genotype frequencies of the patients and control subjects are shown in Table 2. There was no homozygote for M/M either in patients or the control group. The frequency of heterozygotes (T/M) was 16.2% in the control group, which was very comparable to the frequency reported previously.³¹ The frequency of the T/M genotype was higher in the patient group (22.0%), but the difference was not statistically significant (P=.30; OR=1.5; 95% CI, 0.7 to 3.2). However, when only patients aged 60 years were analyzed, the frequency of the T/M genotype was 31.8%, which was significantly higher than that of the control group (16.0%; P<.05; OR=2.5; 95% CI, 1.1 to 5.8).

View this table: [in this window] [in a new window]   Table 2. Genotypes in Control Subjects and Patients

Association of the GP Ib genotype with CAD (standardized for age, sex, and other coronary risk factors including smoking, hypertension, diabetes mellitus, hypercholesterolemia and hypertriglyceridemia) was analyzed among subjects aged 60 years by the use of a logistic regression model. This model provided an OR of 5.8 (95% CI, 1.6 to 21.0; P=.0073) for the relation between CAD and the GP Ib T/M genotype adjusted for all other variables, suggesting that the GP Ib genotype is an independent risk factor for CAD.

We next analyzed the relationship between CAD and the second polymorphism, the variable number of repeats of a 13–amino acid sequence, which is known to be linked to the ¹⁴⁵Thr/Met polymorphism. The frequency of each allele in our control group was similar to that reported previously²⁶ (Table 3). However, the allele frequencies for three and four repeats were higher in our patient group than in our control group (4.8% for patients versus 0.6% for control subjects for three repeats; 17.7% for patients versus 5.7% for control subjects for four repeats). When the allele distribution was compared between patients and control subjects, the difference was statistically significant (P=.003; Table 3). Linkage between the two GP Ib polymorphisms was also analyzed. There was one control subject with the T/M genotype who had alleles with one repeat and two repeats, but all other subjects with the T/M genotype had either a three- or four-repeat allele, a finding compatible with previous studies.²⁸ In addition, there was one patient with the T/T genotype who had alleles with one and three repeats, but all other subjects with the T/T genotype were without three or four repeats (data not shown in tables).

View this table: [in this window] [in a new window]   Table 3. Allele Frequencies of the Size-Polymorphism of GP Ib in Patients and Control Subjects Aged 60 y

The relationship between the GP Ib genotype and disease status among patients aged 60 years is shown in Table 4. The frequency of the T/M genotype was 31.2% for patients with MI and 33.3% for angina patients (P=NS). On the other hand, we found a relationship between the genotype and the severity of CAD. The genotype frequency of T/M was 50.0% in patients with a G-score 40 and 11.0% for patients with a G-score <40 (P=.0015 when compared by use of a 2x3 contingency table). When CAD severity was classified by affected vessel number, 27.8% of patients had single-vessel disease, 30.8% had two-vessel disease, and 42.9% had three-vessel disease (OR=2.0, 2.3, and 3.9, respectively, compared with the control group), although the difference did not reach statistical significance (P=.18 by use of a 2x4 contingency table). This trend still existed even if individuals with diabetes mellitus were excluded; 25.0% had single-vessel disease, 33.3% had two-vessel disease, and 66.7% had three-vessel disease (OR=1.7, 2.6, and 10.3, respectively, compared with nondiabetic subjects in the control group).

View this table: [in this window] [in a new window]   Table 4. Disease Status and Genotypes in Patients Aged 60 y

	Discussion

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The present study provides clear evidence that polymorphisms in the platelet GP Ib/IX receptor are associated with increased risk for the prevalence and severity of CAD. We have found an age dependency in the frequency of the T/M genotype in the patient group: 12.8% for individuals aged >60 years and 31.8% for those aged 60 years, compared with 22.0% for all ages. If only individuals aged 50 years were analyzed, 42% of patients had the T/M genotype compared with 17% in the control group aged 50 years (data not shown in tables). Conversely, the frequency of the T/M genotype in our control group did not differ significantly when compared between different age groups. Thus, although the age distribution in our total control group was different from that in the total patient group, it is unlikely that this affected the results. Moreover, a logistic regression analysis revealed that T/M genotype was an independent risk factor for CAD for those aged 60 years, even after adjustment for age, sex, and other coronary risk factors.

The association between CAD and GP Ib was also evident when analyzed by another polymorphism, the variable number of tandem repeats of a 13–amino acid sequence within the macroglycopeptide domain of GP Ib, which is closely linked to the ¹⁴⁵Thr/Met dimorphism.²⁸ The frequencies of the four alleles (one to four repeats) in our control group were very comparable to that reported previously for the general Japanese population,²⁶ but the distribution was significantly different from that observed in our patient group, three or four repeats being more frequent in patients than in control subjects (P=.003; Table 3)

The frequency of the T/M genotype did not differ significantly between angina and MI patient groups. We have found, however, a strong relationship between T/M heterozygotes and severity of CAD in patients aged 60 years. As shown in Table 4, the frequency of the T/M genotype in individuals aged 60 years was 50.0% for patients with a G-score 40 and 11.0% for those with a G-score <40 (P=.0015), or 27.8% for single-vessel disease, 30.8% for two-vessel disease, and 42.9% for three-vessel disease, although in the latter classification, the difference did not reach statistical significance.

Because platelets are believed to play crucial roles in coronary thrombosis, we initially assumed that the association of the T/M genotype was stronger with MI than with angina. However, our results indicated that the genotype distributions were not significantly different between the two groups (frequency of the T/M genotype was 31.2% for MI compared with 33.3% for angina; Table 4). Rather, the polymorphism was associated with the angiographic severity of CAD. Although the role of platelets in the development of CAD is still not clearly understood, one can speculate that platelets, by adhering through the GP Ib/IX receptor to damaged endothelium or subendothelial tissues with subsequent release of growth factors, may participate in coronary atherosclerosis. The molecular basis that underlies the association of the GP Ib polymorphism and CAD is not known. The Thr/Met dimorphism at residue 145 of GP Ib was originally described as a molecular basis of a platelet alloantigen system. Kuijpers et al²⁴ and Murata et al²⁵ showed a relationship between the presence of the ¹⁴⁵Met-containing allele and the HPA 2b alloantigen on platelets, and the causative role of ¹⁴⁵Met for HPA 2b was confirmed by the expression of a ¹⁴⁵Met-containing GP Ib protein in heterologous mammalian cells.²⁵ Although residue 145 is located in the vWF-binding domain of the GP Ib/IX receptor, no functional difference has been reported between platelets with and without GP Ib ¹⁴⁵Met.^14,32 However, in vitro experiments reported thus far have been performed using nonphysiological agonists to induce vWF–GP Ib/IX interaction. Thus, a functional difference between the two forms of the GP Ib/IX receptor in vivo could still be possible. It could be speculated that the vWF binding function might differ between the ¹⁴⁵Thr- and ¹⁴⁵Met-containing receptors or that GP Ib with ¹⁴⁵Met (three or four repeats) is longer in size and thus places the vWF-binding global domain farther away from the platelet plasma membrane. In that case, vWF, which is a large, multimeric molecule, would be more easily accessible to the binding site on the receptor.

Recently, a polymorphism in GP IIb/IIIa, another platelet receptor relevant for platelet aggregation, has been shown to be associated with CAD,³³ although conflicting results have also been reported.^34,35 GP IIb/IIIa is the final common pathway of activation signals generated by various stimuli and is a key molecule for platelet aggregation and thus has been a recent target of antiplatelet therapy. However, because the Pl^A2 allele (the less frequent allele) is very rare in Japan,³¹ it is unlikely that this polymorphism contributed to the prevalence of CAD in the present study. On the contrary, we have provided new evidence that the other major platelet receptor, GP Ib/IX, is associated with CAD. This receptor is unique in that it mediates platelet activation specifically under high shear stress conditions that are generated in stenosed arteries or capillaries.^9,10

In conclusion, we have described a new genetic risk factor for CAD. However, the results need to be confirmed in a large study of incident cases and matching control subjects. Additional basic and clinical studies will elucidate the roles of GP Ib/IX in the development of CAD and acute coronary syndromes, as well as whether antiplatelet interventions targeting the platelet receptor could be of some benefit.

	Selected Abbreviations and Acronyms

 

bp = base pairs CAD = coronary artery disease GP = glycoprotein G-score = Gensini score MI = myocardial infarction OR = odds ratio PCR = polymerase chain reaction vWF = von Willebrand factor

Received April 8, 1997; revision received July 17, 1997; accepted August 1, 1997.

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