Global thrombosis test for assessing thrombotic status and efficacy of antithrombotic diet and other conditions.

Because of the high mortality from myocardial infarction and stroke, there is a great demand for finding novel methods of diagnosis, prevention and treatment of these diseases. Most of the current tests measure important determinants of thrombosis such as platelet function, coagulation and fibrinolysis in isolation; therefore, a global test measuring the actual thrombotic status would be more useful in clinical conditions. We obtained considerable experience by using the global thrombosis test, which determines the actual thrombotic status by taking into account the measured platelet reactivity, coagulation and fibrinolytic activities. In animal experiments, we found significant correlation between the ex vivo global thrombosis test measurements and the in vivo thrombotic status. The published evidence for the benefit of an antithrombotic diet with regular physical exercise is also described.

Are all wines made from various grape varieties beneficial in the prevention of myocardial infarction and stroke?

AIM: Epidemiologic studies support the assumption (French paradox hypothesis) that drinking red wine is beneficial in the prevention of cardiovascular diseases. Our recent works however cast doubt on such claim. Earlier we have shown that the antithrombotic activity of various fruits and vegetables mainly depends on their varieties. For this reason, several varieties of red and white grapes were tested for antithrombotic effect in animal experiments. RESULTS: Antithrombotic effect of 45 red and white grape varieties were assessed in the present study. Out of the 45, one red grape variety showed antithrombotic effect, while the majority of red and white grape varieties enhanced thrombosis. CONCLUSION: Most red and white grape varieties enhanced thrombotic activity of blood.

Prevention of thrombotic disorders by antithrombotic diet and exercise: evidence by using global thrombosis tests.

Prevention of thrombotic disorders has priority over treatment. There are only two pathologically relevant tests which are suitable for measuring the overall thrombotic status both in experimental conditions and in humans. The Global Thrombosis Test (GTT) and the Global Parallel-Plate Thrombosis Test can detect the pathologically relevant global thrombotic status. These tests have been successfully used for monitoring the effect of antithrombotic drugs and for developing novel antithrombotic agents. By using GTT, varieties of fruits, vegetables, and regular physical exercise have been tested for the effect on global thrombotic status. This review discusses the published evidence for the benefit of diet of selected fruit and vegetable varieties and doing regular physical exercise on improving thrombotic status. Future clinical trials monitored by GTT or Global Parallel-Plate Thrombosis Test could decide on the effectiveness of an experimentally proven antithrombotic diet with regular physical exercise in the prevention of thrombotic diseases.

Reevaluation of antithrombotic fruits and vegetables: great variation between varieties.

In the quest for prevention of atherothrombotic diseases, an antithrombotic diet may offer a promising approach. The major stumbling block in finding an effective diet is the lack of pathophysiological relevant techniques to detect potential antithrombotic effects of various diet components. Platelet function and coagulation/fibrinolysis tests currently in use do not allow assessment of global thrombotic status and their value in screening diet-components for antithrombotic effects. Recently, we combined the point-of-care shear-induced ex vivo thrombosis test (Global Thrombosis Test-GTT) with the Flow-mediated Vasodilation (FMV) in vivo test and found that the combination improved the assessment of thrombotic status in humans and could be used for screening diet-components for antithrombotic effects. In the present experiments, a combination of GTT, hemostatometry, laser-induced thrombosis tests and FMV were employed for screening. The results show that the overall antithrombotic effect is determined by the effect on thrombus formation and endogenous thrombolytic activities. This study showed a great variation in the observed antithrombotic effect between the tested varieties. Antithrombotic activities were independent from polyphenolic content or antioxidant activities. The presented experimental techniques seem to be suitable for establishing an antithrombotic diet, which may be effective in the prevention of atherothrombotic cardiovascular diseases in humans.

Evaluation of antithrombotic effect: Importance of testing components and methodologies.

The beneficial antithrombotic effect of some dietary components may offer the most promising approach of prevention of cardiovascular diseases and arterial thrombosis. The major stumbling block in finding effective dietary components is the lack of physiologically relevant techniques which can detect potential antithrombotic effect in humans. The presently used platelet function and coagulation tests do not allow the assessment of global thrombotic status and their value in screening dietary components for antithrombotic effect is questionable. Most of these in vitro tests ignore the effect of flow and shear stress, thrombin generation and vascular endothelium, the major contributors to arterial thrombogenesis in humans. As a gold standard, we employed the helium-neon (He-Ne) laser-induced thrombosis test in murine carotid artery and mesenteric microvessels, as the pathomechanism of this test closely reflects arterial thrombogenesis in humans. Results obtained with laser thrombosis test were compared with various shear-induced in vitro platelet function tests which use native blood (Haemostatometry, Thrombotic Status Analyser, Global Thrombosis Test-GTT). Contribution of vascular endothelium to thrombogenesis was assessed by measuring flow-mediated vasodilation (FMV) in vivo. The combination of the two shear-induced ex vivo thrombosis tests (Haemostatometry and GTT) with FMV correlated most closely with the laser-thrombosis test. Our findings suggest that combining the commercially available point-of-care GTT with the FMV test could provide a better assessment of the overall thrombotic status than either of the two tests alone.

Short- and long-term treatment with folic acid suppresses thrombus formation in atherogenic mice in vivo.

In the present study, we examined the effects of short- and long-term treatment with folic acid (FA) on thrombus formation in vivo in atherogenic mice to explore a novel agent for the prevention of atherothrombotic disease. Apolipoprotein E and low-density lipoprotein receptor double deficient (ApoE(-/-)LDLR(-/-)) mice were orally administrated a single bolus of FA (20mg/kg) or fed an atherogenic diet with or without FA (0.02, 0.5, and 1.5mg/kg) for 12 weeks. Thrombus formation and endothelial function were assessed in vivo using the He-Ne laser-induced carotid artery thrombus formation test and the flow-mediated vasodilation method. Platelet reactivity was assessed ex vivo using haemostatometry. Short-term treatment with FA markedly increased plasma folate levels and significantly suppressed laser-induced thrombus formation in apoE(-/-)LDLR(-/-) mice. Short-term treatment with FA suppressed platelet reactivity in apoE(-/-)LDLR(-/-) mice, but FA treatment did not affect endothelial function or plasma homocysteine levels. Long-term treatment with FA increased plasma folate levels dose-dependently. Thrombus formation and endothelial dysfunction were suppressed by treatment with 0.5 and 1.5mg/kg of FA, respectively, but not with 0.02mg/kg of FA, whereas platelet reactivity was not altered by treatment with any dose of FA. Long-term treatment with all doses of FA decreased the plasma homocysteine levels in apoE(-/-)LDLR(-/-) mice, although this result was not consistent with its anti-thrombotic action. In conclusion, our data showed that short- and long-term treatment with FA could suppress in vivo thrombus formation in an atherogenic setting, independent of its hypohomocysteinemic action.

Antithrombotic effect of taurine in healthy Japanese people may be related to an increased endogenous thrombolytic activity.

INTRODUCTION: Prevention of arterial thrombotic diseases has high priority in developed countries. Taurine (2-aminomethylsulfonic acid), which is rich in sea foods, showed antithrombotic effect in animal models of thrombosis. The present study aimed to investigate such effect in healthy human volunteers. METHODS AND RESULTS: In 101 healthy Japanese people the overall thrombotic status was accessed from non-anticoagulated blood sample by the Global Thrombosis Test (GTT). There was no significant correlation between taurine concentration in urine samples and GTT-Occlusion Times (OT; mainly reactivity of platelets). In contrast, a significant inverse correlation was demonstrated between urine taurine concentrations and GTT-Lysis Times (LT; showing spontaneous thrombolytic activity). CONCLUSIONS: Our findings suggest that taurine enhances endogenous thrombolytic activity which could be a mechanism of the earlier observed cardioprotective and antithrombotic effect.

First direct comparison of platelet reactivity and thrombolytic status between Japanese and Western volunteers: possible relationship to the "Japanese paradox".

OBJECTIVE: To determine and compare thrombotic and endogenous thrombolytic status in Japanese and Western populations. BACKGROUND: Incidence of coronary heart disease (CHD) and AMI in Japan remains lower than in Western countries. Primary genetic effects are unlikely, given the increased CHD in Japanese migrants. For men, cholesterol and blood pressure have been similar in Japan and the U.S. Dietary factors are implicated, but how these effect CHD is unclear. We postulated that differences in thrombotic and/or thrombolytic status may contribute. METHODS: We measured thrombotic and thrombolytic status in 100 healthy Japanese (J) from Japan and 100 healthy Westerners (W) from the U.K. using the Global Thrombosis Test (GTT). The GTT employs non-anticoagulated blood to create platelet-rich thrombi under high shear (occlusion time OT; seconds), and then measures the restart of blood flow, due to spontaneous thrombolysis (lysis time LT; seconds). RESULTS: OT was longer in (J) compared to (W) (545 vs. 364, p<0.0001). LT was longer in (J) than in (W) (1753 vs. 1052, p<0.0001). Distribution of LT in (J) did not conform to a normal population, with markedly impaired thrombolytic status (LT>3,000 s) in 18%, compared to none of the Westerners (p<0.0001). CONCLUSIONS: There are marked differences in thrombotic and thrombolytic status, with (J) having less prothrombotic (longer OT) but less favourable endogenous thrombolytic profile (longer LT). This may be important in the aetiology of thrombotic events. Since platelets and thrombolysis were both inhibited in (J) and yet incidence of AMI is lower, OT would seem more important than LT as a determinant of overall thrombotic risk in this population.

Impaired endothelial function may be due to decreased aortic tetrahydrobiopterin, assessed by a new flow-mediated vasodilation in vivo in hypercholesterolemic/atherogenic mice.

Tetrahydrobiopterin (BH4) is an important cofactor for endothelial nitric oxide synthase activity. The relationship between endothelial function in vivo and aortic BH4 level is not fully understood, however. In the present study, we aimed to clarify whether reduction of aortic BH4 levels contributes to endothelial dysfunction in vivo using spontaneously hyperlipidemic mice. To estimate endothelial function in vivo and in real-time state, we developed a flow-mediated vasodilation (FMV) method in mice, which measured changes in the diameter of the femoral artery in response to increased blood flow. C57BL/6 mice and apoE/low-density lipoprotein receptor double knock-out mice were fed a low-fat diet (LFD) or a high-fat diet (HFD) for 12 weeks from 6 weeks of age. HFD feeding impaired FMV in double knock-out mice, but not in C57BL/6 mice. Furthermore, HFD feeding reduced plasma NOx concentration and aortic BH4 level in double knock-out mice. Conversely, exogenous injection of BH4 (2 mg/kg) markedly increased aortic BH4 levels and restored endothelial function. In conclusion, we demonstrated that HFD feeding impaired nitric oxide-mediated endothelial function and reduced BH4 level in vivo, and that acute augmentation of aortic BH4 levels improved endothelial function. These findings indicate that BH4 is a critical determinant of nitric oxide-mediated endothelial function in hypercholesterolemia.

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.

The antithrombotic effects of carrot filtrates in rats and mice.

The prevention of arterial thrombotic disease has a high priority in developed countries. An inappropriate diet is known to enhance the risk for acute thrombotic events and a regular diet with proven antithrombotic effects might be a beneficial way to prevent disease. The present study was undertaken as a part of a series of investigations to examine vegetables and fruits for antithrombotic activity. A shear-induced in vitro platelet reactivity/thrombolysis method [Global Thrombosis Test, formerly Gorog Thrombosis Test] was used to screen 15 different varieties of carrot for antiplatelet and thrombolytic activities. Effective varieties were further investigated using an in vivo, laser-induced thrombosis model in mice. The different carrot varieties demonstrated a variable effect on thrombosis in vitro and in vivo. In particular, a variety designated SAKATA-0421 exerted an antithrombotic effect in vivo independent from heat treatment of the filtrate at 100 degrees C for 10 min. There was no significant correlation between antithrombotic activity and the levels of polyphenolics and any other biochemical parameter, including antioxidant activity, alpha-carotene and beta-carotene, alpha-tocopherol and ascorbic acid. Different varieties of carrot demonstrated a range of antithrombotic and prothrombotic activities. After oral intake, the particular heat resistant variety (SAKATA-0421) showed antithrombotic effect in vivo possibly due to antiplatelet reactivity and/or spontaneous thrombolytic activity. The present study added a new variety to the list of antithrombotic fruits and vegetables.

A rice variety (HYJA-Ri4) enhances arterial thrombus formation in an animal model of thrombosis.

Epidemiological evidence exists for a causative role of inappropriate diets in the development and clinical outcome of thrombotic diseases. Our previous studies have shown that some fruits and vegetables have antithrombotic activity. As rice is a principal ingredient of everyday food in Japan, the aim of the present study was to determine rice varieties with antithrombotic effect. Western-style high-fat diet containing flour prepared from whole-grain rice of different varieties was fed to atherosclerosis-prone (apolipoprotein E- and low density lipoprotein receptor-deficient) mice for 3 months. A laser-induced carotid-artery thrombosis model was used. None of the five tested rice varieties showed an antithrombotic effect. The effect of HYJA-Ri1, HYJA-Ri2, HYJA-Ri3 and HYJA-Ri5 rice on experimental thrombus formation did not differ significantly from the controls. However, HYJA-Ri4 rice significantly enhanced the thrombotic reaction (p = 0.0078), thus having a prothrombotic effect. Our findings suggest that HYJA-Ri4 rice should not be included in an antithrombotic diet.

Testing various fruits for anti-thrombotic effect: i. Mulberries.

Prevention of arterial thrombotic disease has high priority in developed countries. As inappropriate diet predispose to acute thrombotic events, regular intake of an antithrombotic diet may be a convenient and effective way of prevention. The present study was performed to examine antithrombotic effect of mulberry varieties. A shear-induced in vitro platelet reactivity/thrombolysis test (Gorog Thrombosis Test) was used to screen for antiplatelet and thrombolytic activities. In case of effectiveness, it was followed by an in vivo test of laser-induced thrombosis in mice. Antioxidant capacity was assessed by superoxide anion and radical scavenging activities. Total polyphenolics, anthocyanin and citrate contents were also measured. The tested varieties showed different effect in vitro on platelet reactivity and endogenous thrombolytic activity. Varieties inhibiting platelet reactivity were antithrombotic in vivo regardless inhibition or enhancement of thrombolysis. Those mulberry varieties, which enhanced platelet reactivity in vitro, were prothrombotic only if inhibitory activity on endogenous thrombolysis coexisted with the platelet effect. Antioxidant activities and polyphenolics content did not affect platelets and the overall thrombotic status. However, antioxidant activities and polyphenolics content significantly correlated with the endogenous thrombolytic activity. These data showed that mulberry varieties can be grouped into subclasses with either anti- or prothrombotic activities. Antioxidant activities and polyphenolic contents do not affect platelets but may enhance endogenous thrombolysis, thus causing an overall antithrombotic effect.

Effect of dietary vegetable and animal proteins on atherothrombosis in mice.

OBJECTIVE: It is believed that vegetable and fish, but not animal, proteins prevent thrombosis. The present study compared the effect of long-term intake of purified vegetable and animal proteins (casein, pork, egg white, chicken, white and red fish, soybean, and potato) and powders from whole vegetable and animal meats (soybean, pork, chicken, and horse mackerel) on thrombotic tendency. METHODS: Western-style high-fat diets with various proteins were given to mice deficient in apolipoprotein-E and low-density lipoprotein receptor for 8 or 12 wk. Thrombotic tendency was assessed by a laser-induced thrombosis test, severity of atherosclerosis was assessed by morphometric analysis of the aortic lesions, and reactivity of platelets was measured by a shear-induced platelet function test. RESULTS: There was no difference in thrombotic tendency between the vegetable and animal protein diets with the exception of pork protein, which showed a prothrombotic effect. The latter was not observed in animals kept on a high-protein diet. Thrombotic tendency was not affected by preparation of dietary powders from whole pork, chicken, or horse mackerel meats and casein (control), all of which contained the same percentages of protein and lipid. In contrast, soybean powder showed an antithrombotic effect, which was mainly due to an inhibition of atherogenesis rather than to platelet inhibition. CONCLUSION: Neither the purified animal nor the vegetable protein diet affected thrombotic tendency. Soybean powder inhibited atherothrombosis mainly by inhibiting the atherosclerotic process.

Dietary diacylglycerol extenuates arterial thrombosis in apoE and LDLR deficient mice.

INTRODUCTION: Prevention of arterial thrombotic diseases has high priority in developed countries. An appropriate diet is thought to be the best way to prevent or reduce the risk of mortality from such diseases. The aim of the present study was to evaluate the effect of diacylglycerol (DAG)-rich diets on arterial thrombosis. MATERIAL AND METHODS: Diet-sensitive congenital apolipoprotein E (ApoE) and LDL receptor (LDLR) double deficient mice were used. Thrombosis was assessed by the rate and extent of thrombus formation in the carotid artery of mice after laser irradiation. Plasma total cholesterol and triglyceride levels were measured by enzymatic assays. Four kinds of diets were used: high fat (Western-style) diet contained 20% fat (w/w) and 0.05% cholesterol (w/w); the TAG-rich and the DAG-rich high fat diet contained 20% TAG or DAG oil (w/w) with very similar fatty acid composition and 0.05% cholesterol ; Low fat (Japanese-style) diet contained 7% fat, without cholesterol. These diets were on the basis of AIN93G and were given to mice for 8 weeks from 6 weeks of age. RESULTS: Western-style high fat and TAG-rich high fat diets significantly increased thrombogenicity compared with low fat diet. DAG-rich high fat diet showed the lowest value, and the extent of thrombogenicity was equivalent to the low fat diet group. Fasting plasma total cholesterol level of DAG-rich high fat and low fat diet groups were significantly lower than that of TAG-rich high fat and high fat diet groups. Fasting plasma triglyceride levels in DAG-rich high fat diet group were significantly lower than in the TAG-rich high fat diet group. CONCLUSIONS: Dietary DAG but not TAG oil extenuates arterial thrombus formation. The mechanism of this effect is unclear and further investigated.

Endothelial dysfunction precedes atherosclerotic lesions and platelet activation in high fat diet-induced prothrombotic state.

INTRODUCTION: Earlier we have demonstrated a prothrombotic state in spontaneously atherogenic rodents kept on Western-style high fat diet. The aim of the present study was to investigate the cellular mechanism of such prothrombotic state. MATERIALS AND METHODS: Two kinds of diets, Western-style high fat diet containing 20% fat (w/w) and 0.05% cholesterol (w/w) and low fat diet containing 7% fat without cholesterol based on AIN93G, were added to diet-sensitive apolipoprotein E and low-density lipoprotein receptor double deficient male mice for 12 or 18 weeks from 6 weeks of age. Atherosclerosis was assessed by morphometry of the aortic wall or lipid-stained lesions. Endothelial function was measured by flow-mediated vasodilation (FMV) of the femoral artery. Platelet reactivity was measured ex vivo by a shear-induced platelet aggregation test. RESULTS AND CONCLUSIONS: 12 weeks feeding of mice with high fat diet significantly impaired FMV, as compared with mice fed with low fat diet (P<0.05). In contrast, there was no significant difference in the lipid-stained areas and in the reactivity of platelets between the two groups. 18 weeks feeding with high fat diet significantly impaired FMV (P<0.05) and enhanced both lipid-stained areas (P<0.05) and platelet reactivity (P<0.01). These findings show that in high fat diet-induced prothrombotic state, endothelial dysfunction precedes both the morphologically detectable lesions and the enhancement of platelet reactivity.

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 x 10(7) arbitrary units for APN-KO and 6.74+/-2.87x10(7) 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. CONCLUSIONS: Adiponectin deficiency leads to enhanced thrombus formation and platelet aggregation. The present study reveals a new role of adiponectin as an endogenous antithrombotic factor.

Mechanism of the antithrombotic effect of dietary diacylglycerol in atherogenic mice.

INTRODUCTION: We have shown earlier that diacylglycerol (DAG) but not triacylglycerol (TAG) inhibited thrombus formation. The aim of the present study was to investigate the mechanism of this antithrombotic effect of DAG. MATERIALS AND METHODS: Four different diets, the (1) Western-style high-fat diet (HFD) containing 20% lipid and 0.05% cholesterol (w/w), (2) TAG-rich and (3) DAG-rich HFDs containing 20% lipid and 0.05% cholesterol, but all lipid replaced by TAG or DAG oil with very similar fatty acid composition and the (4) Japanese-style low-fat diet (LFD) containing 7% oil but no cholesterol were given to apolipoprotein E and low-density lipoprotein (LDL) receptor double-deficient mice. Atherogenicity was assessed by morphology, mapping the whole aorta and measuring the total area of lipid-stained lesions. Endothelial function was measured by the flow-mediated vasodilation test. Platelet reactivity was assessed from native blood sample by a shear-induced platelet function test (hemostatometry). Serum lipoprotein profile was measured by HPLC. RESULTS: Both the Western-style and the TAG-rich HFDs have accelerated atherosclerosis. In contrast, DAG-rich HFD inhibited the atherosclerotic process to an extent comparable with the Japanese-style LFD. There was no significant difference in platelet and coagulant activity between the studied diet groups. DAG-rich but not the TAG-rich HFD significantly suppressed serum LDL cholesterol level. CONCLUSIONS: The present findings suggest that the mechanism of antithrombotic and anti-atherogenic effect of DAG may involve the protection of the vascular endothelium from injury and lowered serum LDL cholesterol.