The effects of imidazoline agents on the aggregation of human platelets

Clonidine (2-[(2,6-dichlorophenyl)amino]-2-imidazoline), an imidazoline alpha(2)-adrenoceptor agonist, is known to exert complex effects on human platelet aggregation distinct from those of the catecholamines, which are non-imidazoline alpha-adrenoceptor agonists. This study has investigated the aggregatory/anti-aggregatory effects of various imidazolines on human platelets. Blood samples were taken from normal volunteers and platelet aggregation was assessed by a turbidimetric method using a Chronolog aggregometer. Noradrenaline (2 microM) and adenosine diphosphate (1 microM) were used as aggregating agents. The results showed that, with the exception of moxonidine, all of the imidazoline agents used (with or without alpha(2)-adrenoceptor activity) were able to inhibit noradrenaline-induced platelet aggregation. Compared with the non-imidazoline alpha(2)-adrenergic antagonist, yohimbine, the rank order of potency was: efaroxan (IC50 = 3.07 x 10(-8) M) > idazoxan (IC50 = 1.74 x 10(-7) M) > tolazoline (IC50 = 3.90 x 10(-7) M) > clonidine (IC50 = 1.49 x 10(-6) M) congruent with antazoline (IC50 = 1.77 x 10(-6) M) > yohimbine (IC50 = 3.19 x 10(-6) M) > rilmenidine (IC50 = 1.27 x 10(-5) M) > moxonidine (IC50 > 10(-4) M). Clonidine-displacing substance (CDS), a putative endogenous ligand at imidazoline receptors, was found to inhibit noradrenaline-induced platelet aggregation. Harmane, norharmane and agmatine, putative candidates for the active principle of CDS, had no effect on noradrenaline-induced platelet aggregation. In contrast to noradrenaline-induced aggregation, ADP-induced platelet aggregation was neither potentiated nor inhibited by the imidazoline agents, with the exceptions of clonidine and moxonidine. In conclusion, most imidazoline agents effectively inhibit noradrenaline-induced human platelet aggregation. The lack of effect of moxonidine and the proposed endogenous ligands suggested this effect was mediated by an 'atypical' non-adrenoceptor imidazoline-binding site. The results indicated an anti-aggregatory role of imidazoline compounds on noradrenaline-induced human platelet aggregation. In addition, CDS might be an endogenous modulator that prevented platelet hyper-reactivity to catecholamine stimulation.     

The stimulatory effects of cationic amphiphilic drugs on human platelets treated with thrombin

The actions of eight cationic amphiphilic drugs on human platelets displayed three different effects according to drug concentration ranges. At lower concentrations (below approximately 25 microM), the drugs stimulated secretory responses induced by 0.2 U/mL of thrombin, while at concentrations in the 25-50 microM range they inhibited these responses. Above 50-100 microM, the drugs caused permeabilization of the platelet plasma membrane as measured by leakage of cytoplasmic adenine nucleotides. The effects of these agents on phosphoinositide metabolism were monitored in platelets prelabeled with (32)P-inorganic phosphate, such that phosphatidic acid (PA), phosphatidylinositol 4-phosphate (PIP), and phosphatidylinositol 4,5-bisphosphate (PIP(2)), but not phosphatidylinositol (PI), were labeled to equilibrium. In unstimulated platelets, the level of labeled PA decreased slightly (about 25%), with corresponding increases in PIP(2) labeling up to drug concentrations of about 50 microM. In contrast to the relatively small changes in PI and PIP(2), the levels of labeled PIP, precursor to PIP(2), increased 2- to 4-fold in both resting and thrombin-treated platelets from 5 microM up to about 50-100 microM of drugs and remained elevated throughout the permeabilization concentrations. [(32)P]PA increased 20-fold over control upon thrombin activation and 5-30 microM of drugs caused [(32)P]PA to increase 30-37 times over that seen in control, resting platelets; the concentration of drugs that potentiated thrombin-induced [(32)P]PA elevation corresponded to that causing the potentiation of platelet secretion. Higher drug concentrations decreased [(32)P]PA elevation. [(32)P]PIP(2) levels increased about 25% in response to thrombin treatment alone; low concentrations of drugs led to another 25% elevation. A significant decrease in [(32)P]PIP(2) was seen above 30 microM, corresponding to inhibition of platelet secretion. Concentrations of 5-30 microM of several psychoactive agents, both neuroleptics and antidepressants, potentiated the thrombin-induced activation of platelets as measured by dense granule secretion and increased turnover of phosphoinositides. Remarkably, all of the drugs increased the levels of PIP even in resting platelets, indicating that they have common effects apart from the specific receptor interactions currently attributed to them. These common effects, e.g. an increase in membrane fluidity such as is known to be caused by amphipathic agents, may be in part responsible for the observed overlapping psychotropic effects of tricyclic antidepressants and phenothiazines.     

The effects of monovalent and divalent cations on the alpha-adrenoceptor of intact human platelets.

1 We have examined the effects of monovalent and divalent cations and purine nucleotides on the binding of agonists and antagonists to the alpha-adrenoceptor of intact human platelets. 2 Replacement of Na+ (150mM) by NH4+ (150mM) in the incubation medium significantly reduced the binding affinity of [3H]-dihydroergocryptine (P less than 0.05) but did not alter the binding capacity. The competitive binding affinity of adrenaline and noradrenaline was unaltered. 3 The addition of Ca2+ (1mM) or Mg2+ (1mM) to the platelet suspension significantly reduced the platelet alpha-adrenoceptor capacity as indicated by either [3H]-dihydroergocryptine (P less than 0.05) or [3H]-yohimbine (P less than 0.01; Ca2+ only). 4 The addition of Ca2+ (1mM) or Mg2+ (1mM) had no effect on the binding affinity of [3H]-dihydroergocryptine but significantly reduced that of [3H]-yohimbine (P less than 0.05). The competitive affinity of adrenaline and noradrenaline determined by inhibition of [3H]-dihydroergocryptine binding, was unchanged in the presence of either cation. 5 Addition of the purine nucleotides ADP, ATP, GDP or GTP (final concentration 10 microM), either alone or in the presence of 1 mM Ca2+ or 1 mM Mg2+, had no effect on the binding of [3H]-dihydroergocryptine or on the competitive affinity of adrenaline or noradrenaline. 6 We conclude that the alpha-adrenoceptor of intact human platelet displays the binding characteristics of the alpha 2L form of the receptor previously identified in the platelet lysate preparation.     

磷酯酶C对家兔血小板聚集和超微结构的影响

目的　应用电镜研究磷酯酶C(phospholipaseC ,PLC)对家兔血小板聚集和超微结构的影响 ,以探讨用药后PLC抗血小板聚集作用的机制。方法　家兔颈动脉插管取血 ,制备PRP ,将其分为 4组 :①空白对照组 ;②生理盐水组 ;③ 0 5UPLC组 ;④ 2 5UPLC组。 2～ 4组分别用ADP诱导聚集 ,测出其聚集抑制率 ,各组分别制成超薄切片样品进行电镜观察、摄片。结果　 0 5、2 5UPLC明显抑制血小板聚集反应 ,聚集抑制率分别为 86 0 3 %± 12 6%和 82 47%±5 49% ;0 5UPLC抑制血小板形成伪足 ,α颗粒和致密颗粒较多 ,OCS管腔较小 ,其超微结构较生理盐水组变化小 ;2 5UPLC处理血小板的形态结构和超微结构与空白对照组无差异 ,血小板呈圆形或椭圆形 ,边缘光滑 ,无伪足样突起 ,α颗粒、致密颗粒、OCS等正常分布于胞质中。结论　PLC明显抑制ADP诱导的血小板聚集反应及其超微结构的改变 ,这可能是PLC抗血小板聚集作用的重要原因之一     

Cytotoxic effects of imipramine on platelets

Incubation for 30 min at 30° with 1 mM imipramine, chlorpromazine or phencyclidine produced increases in creatine phosphokinase (CPK) activity of rat platelet-rich plasma (PRP). Increases in lactic dehydrogenase (LDH) activity of human PRP were produced by imipramine at 0.5 mM. Ouabain, diphenylhydantoin, calcium chloride, EDTA and thrombin did not affect CPK activity of rat PRP. None of the dugs tested except 10 mM diphenylhydantoin affected CPK or LDH activity in platelet-poor plasma. Increases in enzyme activity of PRP produced by 1 mM imipramine were associated with a decrease in platelet count. The increases were independent of temperature of incubation from 0° to 30° and were maximal after a 5-min incubation. The release of LDH and CPK caused by the above drugs is not related to platelet aggregation or the platelet release reaction. The increases in LDH and CPK activity in PRP appear to be the result of platelet destruction or damage to the platelet plasma membrane with release of these enzymes.     

The accumulation of guanethidine by human blood platelets

1. When human blood platelets were incubated aerobically in plasma containing 2 x 10(-7) to 10(-3)M radioactive guanethidine for 10 min to 6 hr, the drug was accumulated against a concentration gradient until concentration ratios (platelet/plasma) of up to 80:1 were obtained.2. The decline in rate of uptake after 3 hr appeared to result from a decrease in platelet viability, because accumulation was reduced by prolonged incubation before addition of guanethidine.3. Uptake was energy-dependent because it was inhibited by cold and ouabain.4. Sodium ions were essential for guanethidine uptake and retention of 5-hydroxytryptamine (5-HT).5. Accumulation was inhibited by 5-HT, desipramine, cocaine, dexamphetamine, bretylium, tyramine and noradrenaline; bethanidine, p-chlorophenylalanine and (-)-alpha-methyldopa were inactive.6. Guanethidine was tightly bound to platelets, only 10% being lost from labelled cells during 60 min incubation in drug-free plasma; but efflux was increased by addition of amphetamine.7. The binding sites for guanethidine seemed to be different from those for 5-HT since guanethidine accumulation was independent of 5-HT levels, and neither guanethidine uptake or release were affected by reserpine.8. Guanethidine was not metabolized by platelets or plasma in vitro.9. We consider that, if our results regarding uptake, binding and release of guanethidine are confirmed in vivo, and also found to apply to other pharmacologically active agents, then the eventual loss of a platelet-bound substance may increase pharmacological action by raising plasma levels.     

Antiaggregation effect of alum on human platelets

OBJECTIVE: The purpose of this study was to evaluate the effect of alum on human platelet aggregation. METHODS: Platelet-rich plasma fractions were prepared from fresh blood drawn from a group of healthy male volunteers. Platelet aggregation was induced by various inducers. The percent aggregation was recorded in the absence and presence of various concentrations of alum. RESULTS: Alum at concentrations less than 1.5 mg/ml inhibited platelet aggregation induced by collagen, epinephrine. ADP and thrombin in a dose-dependent manner. The IC50s were 0.668, 0.324, 0.250 and 0.191 mg/ml of alum, for collagen-, epinephrine-, ADP- and thrombin-induced platelet aggregation, respectively. In contrast, alum at concentrations up to 1.5 mg/ml did not inhibit ristocetin-induced platelet aggregation. CONCLUSION: Alum has an anti-platelet action and should be used cautiously in the treatment of intractable intravesical hemorrhage. Alum is a cheap anti-platelet drug that needs further investigation.     

Nanoparticle size and surface charge determine effects of PAMAM dendrimers on human platelets in vitro

Blood platelets are essential in maintaining hemostasis. Various materials can activate platelets and cause them to aggregate. Platelet aggregation in vitro is often used as a marker for materials' thrombogenic properties, and studying nanomaterial interaction with platelets is an important step toward understanding their hematocompatibility. Here we report evaluation of 12 formulations of PAMAM dendrimers varying in size and surface charge. Using a cell counter based method, light transmission aggregometry and scanning electron microscopy, we show that only large cationic dendrimers, but not anionic, neutral or small cationic dendrimers, induce aggregation of human platelets in plasma in vitro. The aggregation caused by large cationic dendrimers was proportional to the number of surface amines. The observed aggregation was not associated with membrane microparticle release, and was insensitive to a variety of chemical and biological inhibitors known to interfere with various pathways of platelet activation. Taken in context with previously reported studies, our data suggest that large cationic PAMAM dendrimers induce platelet aggregation through disruption of membrane integrity.     

Beta-adrenoceptor antagonists and human platelets: relationship of effects to lipid solubility

Six beta-adrenoceptor antagonists (propranolol (+ and - isomers); ICI-118,551; oxprenolol; timolol; metoprolol; and practolol (+ and - isomers), chosen to represent a spectrum of physicochemical and pharmacological properties, inhibited the response of human platelets to all aggregating agents tested. For any given aggregating agent the extent of inhibition correlated with the lipid solubility of the beta-adrenoceptor antagonist and showed no relation to other properties of these compounds. Inhibition of aggregation by the beta-adrenoceptor antagonists was manifested as a parallel shift to the right in the dose-response curve. Analysis of these data according to Arunlakshana and Schild (Br. J. Pharmac. 14, 48-58 (1969] showed a dependence of the apparent pA2 on the agonist employed and gave a slope approximating unity when ADP, 9,11-epoxymethanoprostaglandin H2 (U-46619), adrenaline, 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (PAF) or arachidonate were used as agonists. Slopes significantly greater than unity, and approaching a value of 2, were obtained when this analysis was applied to data obtained using collagen in the presence or absence of aspirin, 12-O-tetradecanoylphorbol-13-acetate (TPA), or a divalent cation ionophore (A-23187) as agonists. Inhibition by (+/-) propranolol of secretion induced by collagen was manifested as a parallel shift to the right in the dose-response curve for collagen. The Schild plot of these data has a slope of unity. (+/-)-Propranolol inhibited thromboxane B2 production induced by collagen but over a similar concentration range had little effect on conversion of arachidonate to thromboxane B2. (+/-)Propranolol had no significant effect on the level of cyclic-3',5'-AMP (cAMP) in unstimulated platelets or on the increase in the level caused by addition of forskolin, but caused partial inhibition of the increase in platelet cAMP induced by prostaglandin E1. It also completely abolished inhibition by ADP of the increase in [cyclic-3',5'-AMP] induced by prostaglandin E1. These data are interpreted on the basis of a model in which interaction of propranolol with phosphatidylserine and phosphatidylinositol causes inhibition of phospholipases C and A2, inhibition of protein kinase C and alteration of membrane receptor properties as a consequence of distortion of their microenvironment.     

Accumulation of quinidine by human blood platelets: effects on platelet ultrastructure and 5-hydroxytryptamine

1. We have studied the mechanism of quinidine uptake by normal human blood platelets.2. Platelets in plasma or Krebs solution took up quinidine extremely rapidly, the maximal drug concentration ratio of platelet to medium being 24 to 1 after 1 minute.3. The effects of metabolic inhibitors on uptake were equivocal. Ouabain had no effect but iodoacetate and dinitrophenol were effective. However, as accumulation was not inhibited by low temperature, it was probably not energy dependent.4. Interactions between quinidine and 5-HT were also investigated.5. Quinidine blocked 5-HT uptake, but conversely, neither 5-HT itself nor 5-HT uptake inhibitors, such as cocaine, dexamphetamine, and desipramine interfered with quinidine accumulation.6. Electronmicrographs of platelets incubated with 10(-5), 10(-4) or 2 x 10(-3)M quinidine in Krebs solution for 2 h showed no changes at the lower concentrations, but gross ultastructural damage, including disintegration of the plasma membrane, at 2 x 10(-3)M.7. Further evidence for intracellular penetration was obtained because quinidine released endogenous 5-HT and ATP, and also (14)C-5-HT from loaded cells.8. We conclude that quinidine is taken up by the platelet by a passive process, unrelated to the 5-HT transport mechanism. It is probably accumulated largely in the plasma membrane and outer protein layer, but intracellular penetration and ultrastructural damage may occur.9. Although the effects on 5-HT and ATP were not due to platelet damage, this may occur in vivo and be the cause of quinidine induced thrombocytopenic purpura.     

The effects of sympathomimetic agents on ADP aggregation of rat platelets in vitro

The rate of ADP-induced aggregation of platelets was increased markedly by 1-epinephrine or norepinephrine. Naphazoline, N-methyl epinephrine or d-epinephrine also produced significant increases in rate. In contrast, other α- or β-receptor agonists or indirectly acting agents (d-norepinephrine, phenylephrine, synephrine, metanephrine, tyramine, dopamine, isoproterenol, salbutamol) did not accelerate aggregation. None of the sympathomimetics tested was able to induce aggregation when used alone. Important structural requirements of epinephrine for acceleration of aggregation were R-configuration, a β-hydroxyl group, a catechol moiety, and the absence of large N-alkyl groups. The potentiating effect of epinephrine was blocked equally well by d- or 1-phenoxybenzamine and by propranolol; however, the latter compound did so at concentrations which inhibited ADP aggregation alone. These results with various agonists and antagonists indicate that the receptor involved in catecholamine acceleration of ADP-aggregation does not have all the usual characteristics of either the α- or β-adrenergic receptor.     

Comparison of vasodilator effects on human middle cerebral artery constriction induced by vasoconstrictors or activated platelets.

Experiments were performed on de-endothelized perfused human middle cerebral artery constricted with noradrenaline, serotonin, prostaglandin F2 alpha and collagen-aggregated platelets, all of which provoked similar vasoconstriction; however, the ability of dipyridamole, prostacyclin and nifedipine to reduce the constriction was significantly less marked when induced by activated platelets. The decrease in drug antispasmatic effects was less marked for nifedipine than for other vasodilators. It was suggested that the resistance of platelet-induced vasoconstriction to vasodilator action depends on platelets ability to release several vasoactive substances simultaneously.     

Differential effects of ganodermic acid S on the thromboxane A2-signaling pathways in human platelets.

Ganodermic acid S (GAS) [lanosta-7,9(11),24-triene-3beta,15alpha-diacetoxy-26-oic acid], isolated from the Chinese medicinal fungus Ganoderma lucidum (Fr.) Karst (Polyporaceae), exerted a concentration-dependent inhibition on the response of human gel-filtered platelets (GFP) to U46619 (9,11-dideoxy-9alpha,11alpha-methanoepoxyprostaglandin F2alpha), a thromboxane (TX) A2 mimetic. GAS at 2 microM inhibited 50% of cell aggregation. GAS at 7.5 microM inhibited 80% of Ca2+ mobilization, 40% of phosphorylation of myosin light chain and pleckstrin, 80% of alpha-granule secretion, and over 95% of aggregation. GAS also strongly inhibited U46619-induced diacylglycerol formation, arachidonic acid release, and TXB2 formation. An immunoblotting study of protein-tyrosine phosphorylation showed that GAS inhibited the formation of phosphotyrosine proteins at the steps involving the engagement of integrin alphaIIbbeta3 and aggregation. However, GAS did not inhibit U46619-induced platelet shape change or the inhibitory effect of U46619 on the prostaglandin E1-evoked cyclic AMP level in GFP. It is concluded that GAS inhibits platelet response to TXA2 on the receptor-Gq-phospholipase Cbeta1 pathway, but not on the receptor-G1 pathway.     

地塞米松及C-反应蛋白增高对血小板输注疗效的影响

目的探讨血小板减少出血时,C反应蛋白(CRP)含量及输血前静脉注射地塞米松对输注血小板疗效的影响。方法观察45例无发热、脾肿大和弥散性血管内凝血(DIC)的白血病、再障、实体瘤化疗后,因出血需输注血小板的患者,根据CRP值分为CRP正常组和CRP增高组,各组再分地塞米松组(输血前静脉注射地塞米松10mg)和非地塞米松组。每例在输血前、输血后12～16h检测外周血小板计数(CRP)和临床出血。分析CRP及输血前静脉注射地塞米松后对输血小板效果的影响。结果输血小板后,外周血小板计数增加,在CRP正常组高于CRP增高组,分别为(33.2±9.2)×109/L和(25.4±10.4)×109/L,两者差值的差异有统计学意义(t值为4.42,P<0.01)。在CRP增高组中,地塞米松组的患者较非地塞米松组能显著提高外周血小板计数,分别为(30.8±9.4)×109/L和(20.3±10.0)×109/L,两组差值的差异有统计学意义(t值为3.53,P<0.01)。临床出血改善率达76%。结论CRP高是影响血小板输注效果的又一因素,对CRP增高的患者,输血前静脉注射地塞米松能提高血小板输注的效果。     

Endothelin-1 and human platelets

There is conflicting evidence regarding the effect of endothelin-1 (ET-1) on platelets. Some studies show that ET-1 activates platelets, others show platelet inhibition with ET-1 and some studies did not detect an effect of ET-1. These conflicting results may be due to complex interactions between platelet ET(A) and ET(B) receptors. ET-1 antagonism may emerge as an important therapeutic strategy in the management of several vascular disorders. However, to date the only prescribed ET-1 antagonist is bosentan for pulmonary arterial hypertension. Bosentan is a 'dual' ET-1 antagonist (i.e. it acts on both ET(A) and ET(B) receptors). Whether this action involves an effect on platelets remains to be established. In this review some of the studies describing the effect of ET-1 on human platelets are discussed. Vascular diseases where ET-1 is implicated are also considered.     

Dopamine receptors in human platelets

The expression of dopamine receptors by human platelets was investigated by Western blot analysis and immunocytochemical techniques using antibodies raised against dopamine D1-D5 receptor protein. The influence of dopamine D1-like and D2-like receptor agonists on adrenaline-induced platelet aggregation was also investigated. Western blot analysis revealed that platelet membranes bind anti-dopamine D3 or D5 receptor protein antibodies, but not anti-D1, D2 or D4 receptor protein antibodies. Cytospin centrifuged human platelets exposed to anti-dopamine D3 or D5 receptor protein antibodies developed a specific immune staining, whereas no positive staining was noticeable in platelets exposed to other antibodies tested. Both the D1-like receptor agonist 1-phenyl2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol hydrochloride (SKF 38393) and the D2-like receptor agonist 7-hydroxy-N,N-di-n-propyl-2-aminotetralin (7-OH-DPAT) dose-dependently inhibited adrenaline-induced platelet aggregation. These effects were decreased respectively by the D-like and D2-like receptor antagonists R(+)-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepin-7-ol hydrochloride (SCH 23390) and (-)sulpiride. The above findings indicate that human platelets express dopamine D3 and D5 receptors probably involved in the regulation of platelet function.