Junichiro Yamamoto

Adiponectin Acts as an Endogenous Antithrombotic Factor

Objective—Obesity is a common risk factor in insulin resistance and cardiovascular diseases. Although hypoadiponectinemia is associated with obesity-related metabolic and vascular diseases, the role of adiponectin in thrombosis remains elusive. Methods and Results—We investigated platelet thrombus formation in adiponectin knockout (APN-KO) male mice (8 to 12 weeks old) fed on a normal diet. There was no significant difference in platelet counts or coagulation parameters between wild-type (WT) and APN-KO mice. However, APN-KO mice showed an accelerated thrombus formation on carotid arterial injury with a He-Ne laser (total thrombus volume: 13.36±4.25×107 arbitrary units for APN-KO and 6.74±2.87×107 arbitrary units for WT; n=10; P<0.01). Adenovirus-mediated supplementation of adiponectin attenuated the enhanced thrombus formation. In vitro thrombus formation on a type I collagen at a shear rate of 250 s−1, as well as platelet aggregation induced by low concentrations of agonists, was enhanced in APN-KO mice, and recombinant adiponectin inhibited the enhanced platelet aggregation. In WT mice, adenovirus-mediated overexpression of adiponectin additionally attenuated thrombus formation. Conclusion—Adiponectin deficiency leads to enhanced thrombus formation and platelet aggregation. The present study reveals a new role of adiponectin as an endogenous antithrombotic factor.

Tomatoes have natural anti-thrombotic effects

The prevention of arterial thrombotic diseases has a high priority in developed countries. An inappropriate diet may be an important risk factor for thrombotic events. The daily intake of an anti-thrombotic diet may offer a convenient and effective way of prevention. The aim of the present study was to test tomato extracts for anti-thrombotic effects and to identify those varieties that have such an effect. A shear-induced platelet-function test (haemostatometry) was used to test anti-thrombotic potential in vitro. Extracts from those tomato varieties that showed a significant anti-thrombotic activity in vitro were further assessed in vivo, using a laser-induced thrombosis test in mice. One tomato variety (KG99-4) showed significant anti-thrombotic activity both in vitro and in vivo. KG99-4 inhibited not only platelet-rich thrombus formation but also had a thrombolytic effect. It is concluded that haemostatometry can detect and classify the anti-thrombotic potential of fruits and vegetables and offers a simple way of screening for such effects.

Protective Effects of Ticlopidine and Aspirin, Administered Alone and in Combination, on Thrombus Formation in Rat Cerebral Vessels

The protective effects of ticlopidine and d,l-lysine acetylsalicylate (L-ASA), used alone and in combination, on the pathogenesis of thrombosis in cerebral blood vessels were investigated in a rat animal model using a He-Ne laser method. Ticlopidine and L-ASA, given orally at a concentration from 100 mg/kg, inhibited thrombus formation in a dose-dependent manner. Ticlopidine (300 mg/kg p.o.) inhibited thrombosis in arterioles and venules for 3 days after administration. The inhibitory activity of L-ASA (300 mg/kg p.o.) was less prolonged than that of ticlopidine and was observed for only approximately 24 h. Combined administration of ticlopidine and L-ASA significantly enhanced and prolonged the antithrombotic effects of either drug given alone. The results demonstrate that ticlopidine and L-ASA have potent antithrombotic properties in rat cerebral blood vessels in vivo.

Antithrombotic papain-hydrolyzed peptides isolated from pork meat

INTRODUCTION Atherothrombotic disease such as coronary artery disease and stroke is one of major causes of death. Platelets play important role in these arterial diseases. Suppression of platelet activity by materials may decrease incidence of the disease. The present study aimed to examine the peptides from pork meat with antithrombotic activity. MATERIAL AND METHODS Peptide fraction or hydrolyzate of defatted pork meat by papain was used as the starting material. Antithrombotic activity of the fraction was assessed by a share-induced platelet function test in vitro (haemostatometry) using non-anticoagulated rat blood, followed by a helium-neon laser-induced mouse carotid artery thrombosis test in vivo. RESULTS AND DISCUSSION The starting peptide fraction with mean molecular weight 2500 showed antithrombotic activity in vivo after oral administration to mice at 210 mg/kg body weight. The fraction with mean molecular weight 2517 further purified by cation exchange chromatography showed antithrombotic activity after oral administration at 70 mg/kg body weight. Antithrombotic activity of the purified peptide fraction was equivalent to that of aspirin at 50 mg/kg body weight. It is possible this pork peptide could be beneficial to prevent atherothrombosis.

Effect of trans-resveratrol on the thrombogenicity and atherogenicity in apolipoprotein E-deficient and low-density lipoprotein receptor-deficient mice.

Resveratrol is one of the major polyphenolics in red wine that has been shown to exert the preventive effects against cardiovascular diseases. The effect of trans-resveratrol (t-RES) administered as an ingredient of the diet on the atherothrombotic tendency was assessed in genetically hypercholesterolemic mice after laser-induced damage on endothelium. Mice lacking both apolipoprotein E and low-density lipoprotein receptor (apoE–/–/LDLR–/–) were fed with a high-fat diet with or without t-RES (9.6 and 96 mg/kg diet) for 8 weeks. The atherosclerotic tendency was morphometrically analyzed in their aortae. The thrombotic tendency was determined by inducing thrombus by the irradiation of a helium–neon laser on carotid arteries of these mice with injection of Evans blue. Atherosclerotic area and thrombus size were evaluated by image analyzing in a computer system. Even though the plasma concentrations of lipids (total cholesterol and triacylglycerol) did not change in the control and t-RES groups, a significant decrease (∼ 30%) in the formation of atheroma was observed in the aortae of the t-RES group. The size of laser-induced thrombus that mostly consisted of platelet aggregates was significantly reduced (∼ 25%) in the t-RES group compared with that in the control group. Thus, t-RES orally administrated with a high-fat diet in apoE–/–/LDLR–/– mice significantly suppressed atherosclerosis in their aortae and reduced the laser-induced thrombosis in their carotid arteries.

Anti-thrombotic effect of strawberries.

Because of the high mortality, prevention of arterial thrombotic disease has top priority in developed countries. As inappropriate diet is known to predispose to acute thrombotic events, regular intake of an anti-thrombotic diet may offer a convenient and effective method of prevention. As part of a systematic investigation into the anti-thrombotic effect of fruits and vegetables, strawberry varieties were tested in this study. An in vitro platelet function test (haemostatometry) was used for screening strawberry filtrates. Those that showed significant antiplatelet effect were further assessed with a laser-induced thrombosis test in mice. Measurement of flow-mediated vasodilation in the femoral artery of mice reflected the effect on the vascular endothelium. Correlation between the effects on platelet reactivity in vitro and the antioxidant activity (hypoxanthine/xanthine oxidase test) or phenolic compound content was assessed. Strawberry varieties KYSt-4 (Nohime), KYSt-11 (Kurume IH-1) and KYSt-17 (Kurume 58) showed significant antiplatelet activity both in vitro and, after oral administration, in vivo. Both KYSt-11 and KYSt-17, but not KYSt-4, significantly reduced flow-mediated vasodilation; that is, caused endothelial dysfunction. Antiplatelet activities were heat stable. Significant correlation was found between antiplatelet and antioxidant activities (P = 0.049, R2 = 0.23) or total phenolic compounds (P = 0.0096, R2 = 0.36). Of the tested strawberry varieties, KYSt-4, KYSt-11 and KYSt-17 showed significant anti-thrombotic effect. The dual mechanism of the effect may involve a direct inhibition of both platelet function and antioxidant activities.

Preventive Effects of Hesperidin, Glucosyl Hesperidin and Naringin on Hypertension and Cerebral Thrombosis in Stroke‐prone Spontaneously Hypertensive Rats

The effects of hesperidin, glucosyl hesperidin (G‐hesperidin), a water‐soluble derivative of hesperidin, and naringin on blood pressure and cerebral thrombosis were investigated using stroke‐prone spontaneously hypertensive rats (SHRSP). Hesperidin, G‐hesperidin and naringin were mixed with diet and fed to the animals for 4 weeks. No effect was evident on body weight, but the supplements significantly suppressed the age related increase in blood pressure. Thrombotic tendency, as assessed using a He‐Ne laser technique in the cerebral blood vessels, was significantly decreased in the treated animals compared with the control animals. Measurements of 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG) demonstrated that the supplements had strong antioxidant activity. Furthermore, these supplements significantly increased the production of nitric oxide (NO) metabolites in urine measured with Griess reagent. Vasodilation induced by acetylcholine‐mediated NO production in the endothelium was assessed using thoracic aortic ring preparations and indicated that endothelial function was significantly improved by the administration of these supplements.

A new model to evaluate the diet-induced prothrombotic state, using He-Ne laser-induced thrombogenesis in the carotid artery of apolipoprotein E-deficient and low-density lipoprotein receptor-deficient mice.

Epidemiological studies suggest the high fat content of the Western diet to be responsible for atherosclerosis and its thrombotic complications. Despite such a prevailing view, few animal experiments have so far succeeded in demonstrating enhanced thrombogenicity due to a high fat diet. Even a high fat and a very high cholesterol (1%) diet has failed to demonstrate an enhanced thrombotic reaction in rodents and rabbits. The aim of the present study was twofold. First, we wanted to establish a new, sensitive and specific in vivo thrombosis model in mice, which can then be used to study the effect of diets. Second, we intended to employ such a thrombosis model in investigations into the effect of high or low fat diets on thrombosis. The technique described uses a laser to induce thrombus formation in the exposed carotid artery of apolipoprotein E-deficient and low-density lipoprotein receptor-deficient mice. Thrombus formation was recorded on video, analysed by computer, and the size of thrombus was calculated by image analysis software. Thrombotic status was evaluated by analysing a total of 61 individual images of the thrombotic reaction, which were taken over 600 s. The severity of atherosclerosis was assessed by image analysis of the stained elastic fibers. Two kinds of diets were used: the Western type, high fat diet contained 20% fat (w/w) and 0.05% cholesterol (w/w); the low fat diet contained 7% fat, without cholesterol. These diets were on the basis of AIN93G and were given to mice for 4 or 8 weeks. The high fat diet significantly enhanced both the thrombotic reaction and the development of atherosclerosis as compared with the low fat diet.

Effect of Physical Training on Thrombotic Tendency in Rats: Decrease in Thrombotic Tendency Measured by the He-Ne Laser-Induced Thrombus Formation Method

The effect of physical training on thrombotic tendency was assessed in rats. Exercise was done on a flat treadmill for 30 min at a rate of 1,400 m/h (submaximal speed), 5 times a week for either 1.5 or 3 months. The thrombotic tendency was measured by the He-Ne laser-induced thrombus formation method in microvessels of mesentery, i.e. measurement of the number of laser irradiations necessary to induce stasis of blood flow by occlusive thrombus formation. An increase in the number of irradiations necessary to induce occlusive thrombus formation was observed in arterioles, but not in venules after physical training for 1.5 and 3 months.

Effects of Ginkgo biloba extract (EGb 761) on cerebral thrombosis and blood pressure in stroke‐prone spontaneously hypertensive rats

1. An extract of Ginkgo biloba (EGb 761) has been reported to alleviate cerebrovascular problems. In the present study, we investigated the antithrombotic effects of EGb 761 in cerebral blood vessels of stroke‐prone spontaneously hypertensive rats (SHRSP/Izm).