Tomomi Taka

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.

High intensity exercise enhances platelet reactivity to shear stress and coagulation during and after exercise

Platelets play a crucial role in the pathogenesis of acute cardiac events, such as angina, myocardial infarction and sudden death. It is believed that regular low-intensity exercise can reduce, while high-intensity exercise may provoke acute cardiac events. The aim of the present study was to investigate the effect of acute exercise both at low and high intensities on the ventilatory threshold (VT), platelet reactivity and coagulation before and after exercise. Platelet reactivity and coagulation were measured under flow condition, using native blood, by hemostatometry. Seven healthy young men (age: 20–29 years) performed bicycle ergometer exercise for 30 min at intensities of 90% (Ex-VT90% or approximately 55% VO2max) and 130% (Ex-VT130% or 80% VO2max) of individual VT. Blood cell counts, hematocrit, blood lactic acid and plasma catecholamine levels were slightly but significantly increased after Ex-VT90% and markedly after Ex-VT130% after 30 min exercise. Subsequent to the exercise, the elevated blood cell counts decreased to the resting levels both at Ex-VT90% and at Ex-VT130%. Platelet reactivity to shear stress and dynamic coagulation were significantly enhanced immediately and 30 min after Ex-130%VT. In contrast, no significant changes occurred in those of Ex-90%VT. The present study suggests that high-intensity exercise-induced platelet hyperreactivity and hypercoagulable state may pose an increased risk for acute, sometimes fatal cardiac event. On the other hand, our findings support the view that low-intensity exercise does not present a risk of thrombosis.

A shear-induced in vitro platelet function test can assess clinically relevant anti-thrombotic effects

Morphological features of haemostatic plugs formed in vitro under high shear forces were investigated. Electron microscopy confirmed the relevance of such haemostatic plug to a platelet-rich arterial thrombus, which is formed in vivo . In rat blood samples, the effects of anticoagulants and various antiplatelet agents on platelet reactivity (rate of haemostatic plug formation) and subsequent coagulation of the flowing blood were investigated. Haemostasis did not occur in citrated blood, and heparin greatly inhibited the shear-induced platelet reaction. Aspirin (1 mM), a thromboxane A(2) receptor antagonist (5 microM), a stable prostacyclin (0.55 nM), a stable prostaglandin E(1) (141 nM) and a phosphodiesterase inhibitor (100 microM) were tested. All these agents exerted significant inhibitory effect on shear-induced platelet reaction, including the inhibition of the very first phase of platelet plug formation, due to aggregation of shear-activated platelets. Except for the phosphodiesterase inhibitor, which prolonged clotting time, none of the above agents affected dynamic coagulation. These results suggest that the employed in vitro shear-induced thrombosis/haemostasis test can reveal in vivo the antithrombotic effect of various agents independently of their mechanism of action.

A global platelet test of thrombosis and thrombolysis detects a prothrombotic state in some patients with non-insulin dependent diabetes and in some patients with stroke

Platelet aggregation and spontaneous thrombolytic activity were assessed in patients with non-insulin dependent diabetes and stroke using a shear-induced and agonist-induced platelet aggregation test. The Thrombotic Status Analyser (TSA), induces platelet-rich thrombus formation solely by shear forces, while whole blood platelet aggregometry measures platelet reactivity to different agonists. These tests were employed in the present study because in earlier studies they both demonstrated that platelet aggregability in healthy volunteers was unchanged with age. On the other hand, it is known that thrombolytic activity decreases with age in males, but not in females. In diabetic patients shear-induced platelet aggregability varied according to the stage of nephropathy but platelet aggregation to collagen was suppressed at all stages. Platelet reaction to shear stress was enhanced in stroke patients with haemorrhagic episodes but not in patients with lacunar infarction. In contrast, platelet reactivity to collagen was suppressed and changes in ADP-induced platelet aggregability were inconsistent. Suppressed thrombolysis was observed only in diabetes with minor renal defect. Fibrinogen was increased in diabetes with stage III and IV nephropathy. Fibrinopeptide A (FPA) and D-dimer were increased in stroke. Thus, the observed increase in fibrinogen, FPA and D-dimer is inconsistent with changes in platelet aggregability. Our present findings suggest that a shear-induced platelet aggregation test is superior to other tests such as agonist-induced platelet aggregation and thrombotic markers such as fibrinogen, FPA and D-dimer in detecting a prothrombotic state. It is concluded that elderly males may have a prothrombotic state not because of platelet hyper-aggregability but because of suppressed thrombolytic activity. On the other hand, a prothrombotic state in patients with non-insulin dependent diabetes and after stroke may be due to changes in age-independent platelet aggregability.Platelet aggregation and spontaneous thrombolytic activity were assessed in patients with non-insulin dependent diabetes and stroke using a shear-induced and agonist-induced platelet aggregation test. The Thrombotic Status Analyser (TSA), induces platelet-rich thrombus formation solely by shear forces, while whole blood platelet aggregometry measures platelet reactivity to different agonists. These tests were employed in the present study because in earlier studies they both demonstrated that platelet aggregability in healthy volunteers was unchanged with age. On the other hand, it is known that thrombolytic activity decreases with age in males, but not in females. In diabetic patients shear-induced platelet aggregability varied according to the stage of nephropathy but platelet aggregation to collagen was suppressed at all stages. Platelet reaction to shear stress was enhanced in stroke patients with haemorrhagic episodes but not in patients with lacunar infarction. In contrast, platelet reactivity to collagen was suppressed and changes in ADP-induced platelet aggregability were inconsistent. Suppressed thrombolysis was observed only in diabetes with minor renal defect. Fibrinogen was increased in diabetes with stage III and IV nephropathy. Fibrinopeptide A (FPA) and D-dimer were increased in stroke. Thus, the observed increase in fibrinogen, FPA and D-dimer is inconsistent with changes in platelet aggregability. Our present findings suggest that a shear-induced platelet aggregation test is superior to other tests such as agonist-induced platelet aggregation and thrombotic markers such as fibrinogen, FPA and D-dimer in detecting a prothrombotic state. It is concluded that elderly males may have a prothrombotic state not because of platelet hyper-aggregability but because of suppressed thrombolytic activity. On the other hand, a prothrombotic state in patients with non-insulin dependent diabetes and after stroke may be due to changes in age-independent platelet aggregability.

Effects of Clopidogrel on Platelet Activation and Coagulation of Non-Anticoagulated Rat Blood under High Shear Stress

Clopidogrel is a new thienopyridine derivative similar to ticlopidine, which inhibits adenosine diphosphate-induced platelet aggregation. The in vitro effects of clopidogrel on shear-induced platelet activation and coagulation were assessed after oral administration to rats, by subjecting non-anticoagulated blood to haemostatometry. Clopidogrel significantly inhibited shear-induced platelet activation and coagulation 2 h after administration at doses of 7.5 and 15 mg/kg. Both ticlopidine (200 mg/kg) and aspirin (200 mg/kg) inhibited shear-induced platelet activation, but not coagulation. The peak inhibition of plaetelet activation by clopidogrel occurred 2 h after oral administration, but significant inhibition persisted even after 24 h. These results suggest that clopidogrel could be a more potent antithrombotic agent than ticlopidine or aspirin, and also that ADP plays an important role in shear-induced platelet activation.

Detection of a prothrombotic state after acute aerobic exercise

Angina, myocardial infarction, and sudden cardiac death are associated with thrombus formation in coronary arteries. It is generally believed that these conditions benefit from long-term exercise. However, the evidence for such amelioration or prevention is inconclusive and so is the mechanism through which long-term exercise exerts its beneficial effect on various ischaemic conditions. Because of the thrombotic events, the effects of exercise on platelet reactivity, blood coagulation and fibrinolysis have been extensively studied, but the findings are not consistent (1-9). This is mainly due to methodological problems. Analysis of platelet response to a variety of agonists and the dozen of coagulation and fibrinolysis variables makes the assessment of the overall platelet function, coagulation and fibrinolysis status extremely difficult.

Impaired Flow-Mediated Vasodilation in vivo and Reduced Shear-Induced Platelet Reactivity in vitro in Response to Nitric Oxide in Prothrombotic, Stroke-Prone Spontaneously Hypertensive Rats

Previous investigations using an He-Ne laser-induced thrombosis method have shown that stroke-prone spontaneously hypertensive rats (SHRSP) have an enhanced thrombotic tendency in vivo compared to normotensive, Wistar Kyoto rats (WKY). In addition, studies in the presence of acetylcholine have suggested the presence of endothelial dysfunction in SHRSP. In contrast, shear-induced platelet reactivity in vitro appeared to be depressed in SHRSP. The aim of the present study was to investigate endothelial function in SHRSP using a new physiological in vivo model, and to determine the response of platelets to nitric oxide (NO) in non-anticoagulated blood using a shear-induced platelet function in vitro method (haemostatometry). Endothelial function was estimated by measuring flow-mediated vasodilation (FMV) of the femoral artery. Vessels were exposed and blood flow was arrested using a silicone-coated arterial clamp. Vasodilation was measured by computer-assisted image analysis 3 min after release of stasis. Arterial vasodilation was observed in the femoral artery of WKY, but not in SHRSP. Vasodilation was seen in both WKY and SHRSP; however, in response to the NO donor, 1-hydroxy-2-oxo-3-(3-aminopropyl)-3-isopropyl-1-triazene (NOC 5). In contrast, 100 µM NOC 5 did not affect platelet reactivity in SHRSP. The NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide, sodium salt (carboxy-PTIO) and the NO synthase inhibitor, NG-nitro-L-arginine methyl ester, hydrochloride (L-NAME), did not affect shear-induced platelet reactivity. NOC 5 at 10 µM (final concentration) inhibited shear-induced platelet reactivity in WKY. These results confirm the presence of endothelial dysfunction in SHRSP and indicate that platelets are non-responsive to NO in this hypertensive model. The data suggest that defective endothelial reactions or disturbed thrombogenic mechanisms outweigh the platelet hyporeactivity and contribute to the prothrombotic status in SHRSP.

Effects of cyclosporine and FK506 on in vitro high shear-induced platelet reactivity in rat and human non-anticoagulated blood

BACKGROUND The immunosuppressants cyclosporine and FK506 have been used successfully in clinical transplantation, but both agents have various side effects. We have previously found that cyclosporine is prothrombotic and that FK506 is antithrombotic in an in vivo system. The aim of the present study was to assess the effects of these agents on platelet reactivity and coagulation using an in vitro shear-induced hemostatic platelet plug-forming instrument, the hemostatometer. METHODS A purpose-built hemostatometer was constructed in our laboratory. The effects of cyclosporine and FK506 on platelet reactivity and coagulation were assessed under high shear stress using non-anticoagulated rat and human blood. RESULTS FK506 significantly inhibited both platelet reactivity and coagulation. Cyclosporine also significantly inhibited coagulation, but a proaggregatory effect was observed at a final blood concentration of 0.05 mg/ml. CONCLUSIONS The present in vitro results support our previous in vivo findings regarding the prothrombotic and antithrombotic effects of cyclosporine and FK506, respectively.

Platelet reactivity in spontaneously diabetic rats is independent from blood glucose and insulin levels

Diabetes is associated with thrombotic disorders. Chemically induced and spontaneously induced diabetic animals and various in vitro tests have been used to reveal the prothrombotic state of diabetic patients. However, the results are not consistent. In the present study, platelet reactivity of spontaneously diabetic Goto-Kakizaki rats in vivo and in vitro was evaluated by laser-induced and shear-induced thrombosis models, which are physiologically relevant to thrombosis. Both results showed platelet hyperreactivity.

Adjuvant Effect of Antibodies against von Willebrand Factor, Fibrinogen, and Fibronectin on Staphylokinase-Induced Thrombolysis as Measured Using Mural Thrombi Formed in Rat Mesenteric Venules

The change in thrombus mass during thrombolytic therapy is thought to be the difference between its growth and its degradation induced by thrombolytic agents. Platelets play a pivotal role in arterial thrombosis and bind to each other and to exposed subendothelial matrices via adhesive proteins such as von Willebrand Factor, fibrinogen, and fibronectin. The aim of the present study was to assess whether administration of antibodies against these adhesive proteins, in conjunction with plasminogen activator, could enhance the degradation of platelet-rich thrombus. Mural platelet-rich thrombi were formed in rat mesenteric venules using He-Ne laser irradiation. Recombinant staphylokinase was infused continuously and polyclonal antibodies against adhesive proteins were given by bolus injection. The thrombolytic process was analysed using computer-enhanced image analysis software. Administration of each of the antibodies enhanced staphylokinase-induced thrombolysis.