Koichi Kawano

A 192Arg Variant of the Human Paraoxonase (HUMPONA) Gene Polymorphism Is Associated With an Increased Risk for Coronary Artery Disease in the Japanese

Recent reports have suggested that polymorphisms in the human paraoxonase (HUMPONA) gene may be a genetic risk factor for coronary artery disease (CAD) in white populations. However, this association has not yet been confirmed in other ethnic populations. We studied 75 Japanese patients with CAD, whose coronary lesions were confirmed by angiography, and 115 Japanese control subjects with no history of CAD and a normal resting electrocardiogram. The assays for genotyping the two polymorphisms in the HUMPONA gene (192Arg/Gln and 55Leu/Met) were based on changes in restriction enzyme digestion patterns. For codon 192, the frequencies of the Arg-coding allele (B allele) in both patients and control subjects were much higher than those from published results of whites (.26 to .31), and the difference between patients (.74) and control subjects (.59) was statistically significant (P = .002). The patient group had a higher proportion of Arg/Arg (B/B) homozygotes (52.0% vs 32.2%, P = .006). For codon 55, the frequencies of the Leu-coding allele in control subjects and patients were much higher (.91 and .93, respectively) than those published results for whites, but there was no difference between Japanese control subjects and Japanese patients. When subjects with the 55Leu/Leu genotype only were analyzed, 192Arg/Arg homozygotes were still significantly more frequent in the patients than in the control subjects (55.4% vs 37.2%, P = .024), and the frequency of the 192Arg allele was also higher in patients than control subjects (P = .013). Logistic regression analysis including conventional coronary risk factors revealed that 192Arg is an independent risk factor for CAD. Thus, in the Japanese, the association of CAD with the 192Arg variant of HUMPONA (B-type enzyme) is similar to that reported for whites, although the allele frequencies for 192Arg and 55Leu are much higher in the former than the latter population.

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

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 alpha-chain of the receptor (GP Ib alpha, 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 < or = 60 years (31.8%) than in control subjects aged < or = 60 years (16.0%; P<.05, odds ratio=2.5). An association was also demonstrated between CAD and the other polymorphism of GP Ib alpha, 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 > or = 40 (P=.0015). There was no difference in the distribution of GP Ib alpha genotypes between patients with MI and those with angina pectoris. CONCLUSIONS This study suggests that the presence of the Met allele in GP Ib alpha is a risk factor for the prevalence and severity of CAD in individuals aged < or = 60 years. The results need to be confirmed in a large-scale study of incident case subjects and matching control subjects.

Genotype distribution of estrogen receptor polymorphisms in men and postmenopausal women from healthy and coronary populations and its relation to serum lipid levels

The cardiovascular protective effects of estrogen are known to be mediated by its beneficial effects on lipid metabolism and its direct actions on the vessel wall. The latter can be mediated by a specific receptor for estrogen present on smooth muscle cells and endothelial cells. The gene for the receptor (the classic estrogen receptor [ER]) has three known polymorphisms, Pvu II, Xba I, and B-variant polymorphisms, which are reportedly associated with receptor expression and altered receptor function and with some disorders including breast cancer, hypertension, and spontaneous abortion. However, the significance of genetic variations of the ER in vascular diseases has not been reported. We have examined the association between coronary artery disease (CAD) and the three polymorphisms in ER. Genotypes (P1/P2, X1/X2, and B-wild type/B-variant type) were determined in 87 men and postmenopausal women with myocardial infarction or angina pectoris whose lesions were confirmed by coronary angiography, as well as from 94 control individuals from the general population with no coronary heart disease and normal resting ECG. For B-variant polymorphism, all individuals examined had B-wild type, which contrasts with the reported allele frequency for B-variant type (0.1) in the white population. Genotype distributions and allele frequencies of Pvu II or Xba I polymorphisms were not significantly different between control subjects and patients (P > .05 for Pvu II or Xba I genotypes; P > .05 for Pvu II or Xba I allele frequencies). When the allele frequencies were analyzed separately by sex, there was still no statistically significant difference for both polymorphisms (P > .05 for men; P > .05 for women). No association was found between the polymorphisms and the angiographic severity of CAD. Total cholesterol, triglyceride, or HDL-cholesterol levels were not significantly different among ER genotypes. These findings suggest that the three polymorphisms in ER are not associated with the prevalence and severity of CAD and that the polymorphisms are unrelated to the serum lipid levels in control subjects and patients.

Platelet-Dependent Thrombin Generation in Patients With Hyperlipidemia

OBJECTIVES We evaluated coagulability as determined by platelet-dependent thrombin generation in hypercholesterolemic patients before and after treatment with pravastatin and in hypertriglyceridemic patients to investigate the usefulness of coagulability as an index of atherosclerosis and to determine the importance of treating hyperlipidemia. BACKGROUND An understanding of the interaction between platelets and the plasma coagulation system is important for clarifying the mechanism of the procoagulant process. METHODS We assessed coagulability in 58 patients with hypercholesterolemia (serum total cholesterol level > or = 220 mg/dl, age 56.5 +/- 1.5 years [mean +/- SEM]), 37 patients with hypertriglyceridemia (serum triglyceride level > or = 200 mg/dl, age 59.5 +/- 1.7 years), 13 patients with hypercholesterolemia plus hypertriglyceridemia (age 51.4 +/- 3.1 years) and 75 normal subjects (age 52.2 +/- 1.7 years). We also studied platelet-dependent thrombin generation in patients with hypercholesterolemia before and after treatment with pravastatin. Calcium chloride was added to 0.5 ml of platelet-rich plasma (150 x 10(9)/liter) to initiate coagulation. Ten microliters of the sample was transferred into 90 microliters of 3.8% sodium citrate at 10-min intervals for 30 min. A chromogenic substrate, S-2238, was added to each sample, and absorbance was measured spectrophotometrically at a wavelength of 405 nm to determine thrombin generation. RESULTS Platelet-dependent thrombin generation was increased in patients with hypercholesterolemia and patients with hypercholesterolemia plus hypertriglyceridemia (p < 0.01) compared with patients with hypertriglyceridemia and control subjects. Treatment with pravastatin normalized thrombin generation. CONCLUSIONS Hypercholesterolemia, but not hypertriglyceridemia, was associated with increased platelet-dependent thrombin generation. Pravastatin normalized the generation of thrombin.

Mental stress and physical exercise increase platelet-dependent thrombin generation

Abstract Thrombin generation is an important factor in the pathogenesis of thrombogenic disorders and acute coronary syndromes. Increase in mental stress has been associated with the initiation of the acute coronary syndromes, but the exact mechanism is not known. The present study examined the effects of physical exercise and mental stress on platelet-dependent thrombin generation. Twelve healthy men (mean age 34.2 ± 2.4 years) underwent a treadmill exercise test and a mental stress test by performing mental arithmetic. Platelet-dependent thrombin generation and plasma concentrations of catecholamines, thrombin-antithrombin III complex (TAT), plasmin-α2 plasmin inhibitor complex (PIC), and plasminogen activator inhibitor-1 (PAI-1) were measured before, immediately after, and at 10 and 30 min after stress. Thrombin generation increased significantly immediately after exercise, followed by rapid normalization. Mental stress caused a significant increase in thrombin generation 10 min after stress. While plasma concentrations of epinephrine, norepinephrine, and dopamine were elevated immediately after exercise, and rapidly returned to baseline, only plasma norepinephrine increased immediately after mental stress. TAT and PIC concentrations did increase immediately after exercise; however, PAI-1 remained unchanged. The increase in thrombin generation with exercise and mental stress was unaffected by treatment with 81 mg/day of aspirin of 7 days. However, it was inhibited by a single oral 40-mg dose of metoprolol. Both exercise and mental stress cause an increase in platelet-dependent thrombin generation, which was suppressed by β-blocker therapy, but not by aspirin.

Human platelet activation by thrombolytic agents: effects of tissue-type plasminogen activator and urokinase on platelet surface P-selectin expression.

The mechanisms that underlie reocclusion during thrombolytic therapy have not yet been clarified. The purpose of this study was to investigate the activating effects of tissue-type plasminogen activator and urokinase and the inhibitory effects of acetylsalicylic acid by measuring platelet surface P-selectin as a marker of platelet activation. After addition of urokinase (final concentration 192 U/ml, 1920 U/ml, or 19,200 U/ml) or tissue-type plasminogen activator (final concentration 120 U/ml, 1200 U/ml, or 12,000 U/ml) to platelet-rich plasma from 12 healthy persons, platelet surface P-selectin expression was measured by means of flow cytometry with an anti-CD62 monoclonal antibody. The presence of urokinase and tissue-type plasminogen activator increased platelet surface P-selectin expression in a concentration-dependent manner. In the next step, either 160 mg/day (n = 6) or 660 mg/day (n = 6) acetylsalicylic acid was administered to the 12 healthy persons, and venous blood samples were collected after 7 days of treatment. Platelet surface P-selectin expression was measured with the method used earlier and after addition of tissue-type plasminogen activator or urokinase. Although the effect of acetylsalicylic acid at 160 mg/day on P-selectin expression was minimal, a dose of 660 mg/day suppressed platelet P-selectin expression and inhibited the platelet activating effects of tissue-type plasminogen activator and urokinase in a statistically significant way. Platelets were activated by tissue-type plasminogen activator or urokinase, and this platelet activation was suppressed with administration of acetylsalicylic acid at 660 mg/day.