Pharmacological investigations on the adrenalin-thrombin-induced platelet aggregation in dogs

Adrenaline alone did not induce aggregation of canine blood platelets in citrated plasma in vitro; however, it potentiated the aggregation caused by thrombin concentrations which are too low to induce coagulation. Simultaneous infusion of adrenaline and thrombin resulted in accumulation of 111In-labelled platelets in the lung and in the heart as measured by means of a gamma camera. Separately infused adrenaline or thrombin did not change the distribution of radioactivity. In vitro and in vivo hirudin and cyproheptadine inhibited the adrenaline-thrombin-induced platelet aggregation. Dihydroergotamine exerted no in vivo effect at the doses used.     

The molecular nature and consequences of lipoprotein (a)'s association with platelets

Lipoprotein (a) (Lp (a)) may be pro-thrombotic in humans due to its apolipoprotein (a) (apo(a))-mediated decreases in fibrinolysis. Such decreased fibrinolysis arises putatively from interference with plasminogen conversion to plasmin due to the considerable homology between apolipoprotein (a) and plasminogen. However, in vitro, most studies have shown that human Lp (a) decreases agonist-stimulated platelet aggregation while in vivo it appears to decrease aggregation as implied by increased bleeding times with higher blood serum concentrations of Lp(a). Lp (a) binding to platelets mediated by apo (a) increases platelet intracellular c-AMP levels in resting platelets, and decreases platelet production of thromboxane A2 and fibrinogen binding to platelets all of which reduce platelet aggregation. One, though not the only, explanation of these conflicting data may be that Lp(a) self-regulates its interference with fibrinolysis by reducing platelet aggregation and platelet binding of fibrinogen and hence the degree of requirement for fibrinolysis. However, it is concluded more in vivo work needs to be done to fully understand whether, if at all, Lp(a) in varying concentrations and isoforms, favours reduced platelet aggregation or fibrinolysis.     

Effect of salvianolic acids from Radix Salviae miltiorrhizae on regional cerebral blood flow and platelet aggregation in rats.

This study was conducted to observe the effect of salvianolic acids (SA) on regional cerebral blood flow (rCBF) in rats and on platelet aggregation in vitro and in vivo. Cerebral ischemia was produced in rats by occluding of the right middle cerebral artery, together with the right common carotid artery. rCBF was monitored by H2 clearance method with a tissue blood-flow meter. Platelet aggregation induced by collagen, ADP, and AA was measured in vitro and in vivo by platelet aggregometer. Doses of SA at 6 and 10 mg/kg body wt. (i.v.) improved rCBF in rats after ischemia, but had no obvious effect on normal rCBF. In vitro, SA inhibited significantly the platelet aggregation induced by collagen, ADP, and AA with IC50 values of 0.197, 2.22 and 3.29 x 10(3) mg/l, respectively. In vivo, doses of SA at 6 and 10 mg/kg body wt. inhibited significantly the platelet aggregation induced by collagen, and SA at 10 mg/kg body wt. inhibited remarkably platelet aggregation induced by ADP. The results suggest that SA could improve rCBF in the ischemic hemisphere and inhibit platelet aggregation in rats.     

Effect of dipyridamole on prostaglandin generation by human platelets and vessel walls

These experiments were conducted to determine the effects of dipyridamole on human platelet aggregation, platelet thromboxane A2 (TXA2) and human vessel wall prostacyclin (PGI2) generation. Dipyridamole in varying concentrations (5 to 50 micrograms/ml) had no direct effect on ADP-induced platelet aggregation in vitro, but it potentiated PGI2-induced platelet aggregation inhibition at these concentrations. Dipyridamole also inhibited arachidonic acid-induced platelet TXA2 generation at these concentrations. In continuously perfused umbilical vein segments, dipyridamole treatment resulted in stimulation of PGI2 release determined by bioassay and by measurement of its stable metabolite 6-keto-PGF1 alpha. Minimum concentration of dipyridamole causing PGI2 release was 50 micrograms/ml. These in vitro studies suggest that anti-thrombotic effects of dipyridamole in man are mediated mainly by potentiation of PGI2 activity and to some extent by TXA2 suppression. Stimulation of PGI2 release by human vessels may not be seen in usual therapeutic concentrations.     

褐藻酸钠硫酸酯对血液流变学和血小板聚集的影响

褐藻酸钠硫酸酯(Sodium Alginate Sulfates SAS)是以褐藻酸为原料经磺化、酯化而成的硫酸酯多糖的钠盐系列物(Ⅰ、Ⅱ、Ⅲ、Ⅳ)。本文研究了SAS对兔和大鼠血液流变学和血小板聚集的影响,结果发现:SAS静注5mg/kg能够显著降低高切、低切下的全血比粘度,降低血细胞压积,降低红细胞聚集指数,提高红细胞沉降率,从而改善血液的流变特性。实验中还发现,SAS体内外均可缩短大鼠红细胞电泳时间,体内还可以缩短大鼠血小板电泳时间,而体外则可延长血小板电泳时间。SAS体外可诱导兔、大鼠血小板聚集,能够促进ADP诱导的血小板聚集,而对AA诱导的血小板聚集则无促进作用。结果提示:静注SAS能够改善血液的流变特性,增加红细胞和血小板表面电荷,抑制红细胞及血小板间的粘附和聚集,从而使其具有一定的抗血栓作用。同时还提示,SAS在体内循环和代谢的过程中,结构可能发生了变化,从而使其体内、体外的作用特征出现了相对立的结果。     

Pyrazolidine derivatives: a comparative study of their effects on platelet aggregation.

Quantitative studies were carried out of the in vitro and ex vivo effects of phenylbutazone and 3-oxoalkyl substituted diphenyldioxopyrazolidines (kebuzone, tribuzone, benzopyrazone) on platelet aggregation. The specified pyrazolidine derivatives exhibited in vitro inhibitory effects on secondary platelet aggregation (induced by adrenaline and collagen), commensurable with the effects of sulfinpyrazone. The ex vivo efficacy was markedly influenced by the height of the drug level in blood and by differences in the elimination kinetics of the pyrazolidine derivatives in human organism. Inhibitory activities against primary aggregation (induced by ADP and thrombin) were found in vitro mainly in the phenyloxoalkyl derivative of diphenyldioxopyrazolidine (benzopyrazone) and its analogues. By substitution on the phenyl attached to its alkyl side chain (for example, by a halogen in the meta position), compounds were obtained which also possessed higher activities inhibiting secondary platelet aggregation.     

Effect of homocysteine, cysteine, and their derivatives on the platelet link of hemostasis system

The influence of homocysteine, homocysteine thiolactone, cysteine and their derivatives on activation and aggregation of human platelets was investigated using the model systems in vitro. It was established that homocysteine and cysteine increased platelet aggregation induced by ADP, epinephrine, or collagen. Their action began in a range of concentrations such as their physiological blood levels (10 microM) and was increasing with the rise of their concentrations. Cysteine increased ADP-induced platelet aggregation, hardly any affect on epinephrine-induced platelet aggregation and depressed collagen-induced platelet aggregation in the highest concentration (1000 microM). Their disulfides and thioethers did not influence platelet aggregation.     

Characterization of liposomes carrying von Willebrand factor-binding domain of platelet glycoprotein Ibalpha: a potential substitute for platelet transfusion.

Platelet glycoprotein (GP) Ib/IX/V complex is a receptor for von Willebrand factor (vWf), which plays a crucial role in primary hemostasis by mediating platelet adhesion to injured blood vessels. We have expressed in CHO cells a fragment of GPIba that retained a vWf-binding function. The recombinant fragment (rGPIba) was incorporated into liposomes and evaluated their functions in vitro. rGPIba on the liposome surface was detectable by flow cytometric analysis. Addition of vWf and ristocetin caused specific agglutination of rGPIbalpha-liposomes, as evaluated by an aggregometer or a fluorescent microscopy. When ristocetin was added to platelet-rich plasma (PRP) pre-mixed with rhodamine-labeled rGPIbalpha-liposomes, platelets aggregated and rhodamine-fluorescence was strongly positive in the platelet thrombi, suggesting that heterologous aggregation (attachment of liposomes to platelets) occurred. Platelet aggregation in PRP at low platelet concentration (20-80 x 10(6)/ml) was enhanced by rGPIbalpha-liposomes in a dose-dependent manner. Thus, rGPIbalpha-liposomes may accumulate on vWf-exposed subendothelial tissues and enhance platelet function in vivo, supporting hemostasis in thrombocytopenic individuals.     

Effect of ethyl alcohol on selected parameters of the hemostasis system in vitro]

Alcohol produces several disorders in all components of hemostasis system. However, its mechanism of action is not clear. Therefore, an effect of ethyl alcohol on the selected parameters of both platelet and plasma hemostasis has been examined in vitro. Blood aggregation induced by ADP and PAF and platelet-leucocytic aggregates have been determined in vitro in the group of 45 healthy volunteers. Out of plasma parameters the selected factors of blood coagulation and fibrinolysis have been examined. Results suggest that the examined concentrations of ethyl alcohol mainly affect platelet function decreasing platelet aggregation. Alcohol does not affect significantly blood coagulation and fibrinolysis occurring in vitro.     

Blood platelet aggregation and personality traits.

Changes in blood platelet aggregation may precipitate episodes of arterial occlusive diseases. Little is known, however, regarding the influence of psychological traits, emotional states and other behavioral stressors on platelet aggregation phenomena. This study examined 46 healthy college men at rest and after submaximal treadmill exercise. Associations were found between the duration of platelet aggregation and a number of scores from the California Psychological Inventory and self-administered anxiety scales. The more socially adequate, poised and dominant persons--those with more mature ego development and less overt anxiety--had platelets with more prolonged aggregation reactions to the in vitro introduction of noradrenalin. Irreversible aggregation of platelets occurred more regularly to lower in vitro concentrations of noradrenalin in platelet samples drawn from subjects who were less anxious and tended to be more rigidly defensive. It is premature to attempt to derive clinical implications from this exploratory work, but some implications for the design of future research are discussed.     

Effects of novel plant antioxidants on platelet superoxide production and aggregation in atherosclerosis.

Superoxide anion is produced in human platelets predominantly by Nox2-dependent NADPH oxidases. In vitro experiments have shown that it might play a role in modulating platelet functions. The relationship between platelet superoxide production and aggregation remains poorly defined. Accordingly, we aimed to study superoxide production and aggregation in platelets from subjects with significant cardiovascular risk factors (hypertension, hypercholesterolemia, smoking and diabetes mellitus) and from control individuals. Moreover, we studied the effects of novel polyphenol-rich extracts of Aronia melanocarpa (chokeberry) berries on platelet function in vitro. Superoxide production was significantly increased in patients with cardiovascular risk profile when compared to controls, while platelet aggregation in response to either collagen or thrombin were borderline higher, and did not reach statistical significance. Interestingly, no relationship was observed between platelet aggregation ex vivo and platelet superoxide production in either of studied groups. No correlation was found between endothelial function (measured by FMD) and platelet aggregation ex vivo either. Polyphenol-rich extracts of A. melanocarpa berries caused a significant concentration dependent decrease in superoxide production only in patients with cardiovascular risk factors, while no effect was observed in the control group. A. melanocarpa extracts abolished the difference in superoxide production between risk factor patients and controls. A. melanocarpa extracts exerted significant concentration dependent anti-aggregatory effects in both studied groups, which indicated that these effects may be independent of it's ability to modulate superoxide production. The anti-aggregatory effects of chokeberry extracts were similar irrespective of aggregation inducing agent (collagen or thrombin). Moreover, they appear to be independent of platelet NO release as NOS inhibition by L-NAME did not lead to their abrogation.     

Exogenously added free phosphotyrosine induces aggregation of circulating platelets in rabbits.

When free phosphotyrosine is injected into rabbits, circulating aggregated platelets are readily observed in concomitance with altered electrocardiographic profiles. Since phosphotyrosine is also able to induce platelet aggregation in vitro, altogether these results suggest that free phosphotyrosine in blood could be meaningful for in vivo platelet activation.     

Effect of dipyridamole of prostaglandin generation by human platelets and vessel walls

These experiments were conducted to determine the effects of dipyridemole on human platelet aggregation, platelet thromboxane A₂ (TXA₂) and human vessel wall prostacyclin (PGI₂) generation. Dipyridamole in varying concentrations (5 to 50 μg/ml) had no direct effect on ADP-induced platelet aggregation in vitro, but it potentiated PGI₂-induced platelet aggregation inhibition at these concentrations. Dipyridamole also inhibited arachidonic acid-induced platelet TXA₂ generation at these concentrations. In continuously perfused umbilical vein segments, dipyridamole treatment resulted in stimulation of PGI₂ release determined by bioassay and by measurement of its stable metabolite 6-keto-PGF_1α. Minimum concentration of dipyridamole causing PGI₂ release was 50 μg/ml. These in vitro studies suggest that anti-thrombotic effects of dipyridamole in man are mediated mainly by potentiation of PGI₂ activity and to some extent by TXA₂ suppression. Stimulation of PGI₂ release by human vessels may not be seen in usual therapeutic concentrations.     

Blood platelets and schistosome egg excretion

The eggs of helminths of the Schistosoma genus require to be extravasated in order to continue the life cycle of the parasite. The possible mode by which this takes place was investigated in a mouse model. Suppression of platelet activity in Schistosoma mansoni-infected mice by administering rabbit anti-mouse platelet serum or a selection of "antiplatelet drugs" resulted in a significant reduction of parasite egg excretion. This reduction was best achieved when antiplatelet agents were administered just before the onset of parasite egg excretion. The association between parasite eggs and platelets was illustrated in vivo and in vitro where platelet aggregates on egg surfaces were seen in both light and electron microscopy. In addition, eggs that had been isolated from infected mouse tissues induced platelet aggregation in whole mouse blood, and this was inhibited by preincubation with the beta-lactam antibiotic, ticarcillin. Isolated eggs were also capable of inducing ex vivo platelet aggregation in mice, which was dependent on presensitization with eggs. These data suggest a role for platelets in the extravasation and excretion of parasite eggs in schistosomiasis.     

Aggregation of platelets in damaged vessels

The only certain physiological function of platelets is their aggregation in injured vessel walls as haemostatic plugs. The association of thrombocytopenia with petechial haemorrhages suggests that platelets are somehow required for the functional integrity of small vessels, but no mechanism has yet been established. The pathological aggregation of platelets as thrombi in atherosclerotic arteries is commonly, if not always, initiated by haemorrhage. In artificial vessels, platelets tend to aggregate on the walls wherever blood flow is non-laminar. The mural aggregation of platelets is not prevented by unphysiologically high wall-shear forces. The facts suggest, on the contrary, that the process depends in some way on abnormal haemodynamic conditions. This contribution is mainly concerned with questions about how haemodynamic conditions in and around vascular leaks affect arriving platelets that aggregate there, and about the chemical agents responsible for making the platelets reactive. The effects of these agents are known mainly from in vitro experiments in which aggregation can be quantitatively correlated with biochemical effects by simple and reproducible methods; the relevance to their reactions in haemostasis and thrombosis is uncertain. It is difficult to devise quantitative methods for analysing these processes in vivo because of the very low concentrations at which endogenous agents can activate platelets and haemostasis factors in the plasma; the rapidity with which platelets aggregate in a damaged blood vessel; and the complexity and inconstancy of the haemodynamic situation. All these factors must be accounted for in hypotheses of haemostasis. New experimental approaches towards analysing the haemostatic mechanism in vivo are described.     

广西眼镜蛇毒L-氨基酸氧化酶体内外抗血小板聚集实验研究

目的 研究广西眼镜蛇中提取的L-氨基酸氧化酶(L-amino acid oxidase)在人体外及家兔体内的抗血小板聚集作用.方法 用比浊法测定广西眼镜蛇毒L-氨基酸氧化酶对二磷酸腺苷(ADP)、胶原、凝血酶、花生四烯酸(AA)在人体外及家兔体内引起的血小板聚集率的影响.结果 实验中,能明显抑制二磷酸腺苷(ADP)、胶原、凝血酶、花生四烯酸(AA)引起的血小板聚集,并呈明显的正相关.结论 广西眼镜蛇毒L-氨基酸氧化酶在体内外均有较强的抗血小板聚集活性.     

Effects of the extract from Conyza canadensis on human blood platelet aggregation

The effects of different parts of extract from medicinal plant Conyza canadensis, used to control bleeding, on human blood platelet aggregation in vitro were investigated. Aqueous extract of Conyza c. from young or old plants, glycoconjugate part, polysaccharide part and aglycon part at the concentrations above 0.75 mg/ml strongly inhibited platelet aggregation induced by collagen (2 microg/ml) in dose-dependent manner. Polysaccharide part isolated from plant extract had the strongest inhibitory effect on aggregation stimulated by collagen and seems to be responsible for antiaggregatory properties.     

In vitro platelet function in infantile autism

It has previously been demonstrated that patients with infantile autism demonstrate impaired in vivo platelet behaviour. Therefore, in 14 children (13 boys and 1 girl) with infantile autism (aged 2-14, mean 6 years) and 12 healthy control boys (aged 6-15, mean 11 years) we studied in vitro platelet reactivity using ADP- and collagen-induced platelet aggregation. In each child a total of 7 different final concentrations of ADP and 4 different concentrations of collagen were employed. At all concentrations of ADP and collagen used the autistic children consistently exhibited diminished platelet aggregability; the differences, however, did not reach statistical significance. Therefore a wider panel of in vitro tests is apparently required and a larger group of patients be studied to help elucidate the functional/metabolic platelet defect met in infantile autism.     

De-aggregatory action of prostacyclin in vivo and its enhancement by theophylline.

In the mixed venous blood of anaesthetized, heparinized cats prostacyclin de-aggregated platelet thrombi, which were formed on the surface of blood-superfused collagen strips or on the surface of blood-superfused aortic strips from atherosclerotic rabbits. The reversal of platelet aggregation by prostacyclin was still achieved 3 hrs after the formation of platelet clumps. After an intravenous injection of prostacyclin the ID50 for its de-aggregatory action was 7.5 microgram/kg. Theophylline ethyl-diamine (aminophylline), at a dose of 3 mg/kg i.v., did not reverse platelet aggregation but it enhanced the duration of the de-aggregatory action of prostacyclin; it had little effect on the hypotensive action of prostacyclin. It is concluded that prostacyclin disintegrates platelet clumps long after they are formed in heparinized blood in vivo and that its anti-platelet action, but not hypotensive action, is selectively potentiated by a phosphodiesterase inhibitor. The above experimental data indicate the possibility of the combined use of theophylline and prostacyclin in arterial thrombosis.     

滇丹参注射液对兔血小板功能的影响

观察云南产滇丹参对血小板聚集功能的影响.方法采用Bom氏比浊法,测定滇丹参体内、体外对抗ADP、PAF、AA诱导的兔血小板聚集的作用.体外每种药物浓度分别为40 g/L、20 g/L、10 g/L、5 g/L、2.5 g/L,体内实验分为7组,即生理盐水组、两种丹参低中高剂量组分别为5 g/kg、10 g/kg、20 g/kg,每组6只.结果与对照组相比,滇丹参体外显著抑制ADP、AA诱导的血小板聚集,抑制效应呈浓度-效应关系(P＜0.05,0.01),IC50为33.7 g/L(ADP)、18.1 g/L(AA).滇丹参也显著抑制PAF诱导的血小板聚集,其最大抑制率为36.8%;体内实验结果显示,滇丹参在高剂量时可显著抑制ADP、PAF和AA诱导的血小板聚集(P＜0.05,0.01),且均具有剂量依赖性.滇丹参在药后20 min开始显效,40 min达到最大抑制作用,抑制率分别为95.6%(ADP)、91.5%(PAF)和88.5%(AA).结论以上结果表明,滇丹参体外、体内显著抑制血小板聚集,且其抗血小板聚集作用优于丹参,为进一步开发和利用滇丹参提供了依据.