Effect of PCR 4099 on ADP-induced calcium movements and phosphatidic acid production in rat platelets

Antiplatelet activity of PCR 4099, an analogue of ticlopidine, resides in its specific effect against exogenous as well as released ADP. This study investigated in rat platelets the effects of the drug on ADP-induced shape change, elevation of cytosolic free Ca2+ concentration ([Ca2+]i) and hydrolysis of inositol phospholipids, monitored as [32P]phosphatidic acid formation. Shape change and influx of Ca2+ ions across the plasma membrane were not modified after PCR 4099 administration using aspirin-treated platelets. On the other hand, phosphatidic acid formation and calcium mobilization from internal stores were strongly inhibited. These results suggest that PCR 4099 leaves intact the machinery involved in ADP-induced platelet shape change and influx of calcium ions, but inhibits an early step in the ADP-response coupling leading to inositol phospholipid hydrolysis and aggregation.     

Oncocalyxone A inhibits human platelet aggregation by increasing cGMP and by binding to GP Ibalpha glycoprotein

BACKGROUND AND PURPOSE: Oncocalyxone A (OncoA) has a concentration-dependent anti-platelet activity. The present study aimed to further understand the mechanisms related to this effect. EXPERIMENTAL APPROACH: Human platelet aggregation was measured by means of a turbidimetric method. OncoA (32-256 microM) was tested against several platelet-aggregating agents, such as adenosine diphosphate (ADP), collagen, arachidonic acid (AA), ristocetin and thrombin. KEY RESULTS: OncoA completely inhibited platelet aggregation with a calculated mean inhibitory concentration (IC50-microM) of 122 for ADP, 161 for collagen, 159 for AA, 169 for ristocetin and 85 for thrombin. The anti-aggregatory activity of OncoA was not inhibited by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). OncoA, at a concentration that caused no significant anti-aggregatory activity, potentiated sodium nitroprusside (SNP) anti-aggregatory activity (18.8+/-2.9%-SNP vs 85.0+/-8.2%-SNP+OncoA). The levels of nitric oxide (NO) or cAMP were not altered by OncoA while cGMP levels were increased more than 10-fold by OncoA in resting or ADP-activated platelets. Flow cytometry revealed that OncoA does not interact with receptors for fibrinogen, collagen or P-selectin. Nevertheless, OncoA decreased the binding of antibodies to GP Ibalpha, a glycoprotein that is related both to von Willebrand factor and to thrombin-induced platelet aggregation. CONCLUSION AND IMPLICATIONS: OncoA showed anti-aggregatory activity in platelets that was associated with increased cGMP levels, not dependent on NO and with blocking GP Ibalpha glycoprotein. This new mechanism has the prospect of leading to new anti-thrombotic drugs.     

Variation in the ability of glycoprotein IIb-IIIa antagonists to exert and maintain their inhibitory effects on the binding of fibrinogen

Tirofiban and eptifibatide dissociate rapidly from glycoprotein IIb-IIIa but have different dissociation constants (KD of tirofiban = 15 nmol/L, that of eptifibatide = 120 nmol/L). Binding of fibrinogen to glycoprotein IIb-IIIa is biphasic, forming an initial reversible complex (KD = 155-180 nmol/L) and a second more stable complex (KD = 20-70 nmol/L). To test whether a comparable extent of inhibition would be maintained by pharmacologic antagonists that exhibit a rapid rate of release, blood from 26 patients with symptomatic coronary artery disease was added to reaction tubes containing a concentration of either agent that had been shown to achieve optimal inhibition (for tirofiban 100 ng/mL, for eptifibatide 1.7 microg/mL) plus a platelet agonist (1 microM adenosine diphosphate [ADP] or 25 microM thrombin receptor agonist peptide [TRAP]), and fluorochrome labeled fibrinogen before analysis by flow cytometry. The extent of inhibition early on (30 seconds to 3 minutes) was similar. By contrast, the extent of inhibition 10 to 15 minutes later was maintained more effectively with tirofiban than eptifibatide (difference in slope P < 0.01). The differences are consistent with the biphasic binding of fibrinogen to GP IIb-IIIa. The clinical implications of this observation merit evaluation to potentially improve care of patients and to guide future drug development.     

Effects of age, sex and the oral contraceptive on the platelet membrane fibrinogen binding site (glycoprotein IIb/IIIa).

1. We have studied the effects of age, sex and the oral contraceptive pill on platelet fibrinogen 'receptor' density (Bmax) and affinity (Kd). 2. [125I]-fibrinogen binding to gel-filtered platelets was assessed after stimulation with adenosine 5-diphosphate (ADP) and thrombin in 60 normal subjects; 37 females and 23 males. 3. For both agonists there was no significant difference in Bmax or Kd between the sexes. A trend towards increasing affinity (reduced Kd) with age was noted in the female group following ADP stimulation. This was not considered to be physiologically relevant. In the group as a whole no significant relationship of Bmax and Kd with age was demonstrated. 4. A further group of eight women taking low dose oestrogen combined oral contraceptive pill were studied on days 7, 14, 21 and 28 of the treatment cycle and compared with eight women with regular ovulatory cycles. No significant variations in Bmax or Kd were detected within or between cycles in these two groups.     

Differences in the serum protein binding of prazosin in man and rat

The serum protein binding of prazosin in man and rat has been studied in vitro by equilibrium dialysis. Prazosin was more extensively bound in human serum than in rat serum with binding ratios (B/F) of 14.3 +/- 3.4 and 4.4 +/- 0.2 (corresponding to 93.4 and 81.4% bound), respectively. This difference in binding between the species was partly due to qualitative differences between human and rat serum albumin, but also to the lower concentration of albumin in rat serum. Rat serum albumin (RSA) apparently showed two different classes of binding sites for prazosin, one with high (KD = 5.78 X 10(-6) M) and one with low (KD = 1.1 X 10(-4) M) affinity; the former is suggested as representing alpha 1-acid glycoprotein (alpha 1-AGP) with one binding site for prazosin per molecule, the latter as representing RSA with 0.28 binding sites per molecule. Human serum albumin (HSA) and human alpha 1-AGP both showed one class of binding sites with KD values of 2.7 X 10(-5) and 1.95 X 10(-6) M, respectively. HSA possessed 0.5 and human alpha 1-AGP 1 binding site for prazosin per molecule. The binding parameters obtained for the isolated serum proteins overestimated to some degree the total serum protein binding of prazosin in man. This was explained by a specific deviation from the law of mass action. HSA was the major binding protein in human serum at therapeutic concentrations, with ca. 60% of the total binding, the remaining 40% being bound to alpha 1-AGP. Anticipating that the high affinity binding site on the RSA preparation represents the binding of prazosin to alpha 1-AGP, then this protein accounts for 70% of the binding in rat serum, while rat serum albumin accounts for approximately 23%. The binding of prazosin to lipoproteins was insignificant in both species. The observed differences between man and rat in the serum protein binding of prazosin implicate differences in the two species with respect to prazosin pharmacokinetics and the pharmacological effect.     

Sex difference in the effect of aspirin on rat platelet aggregation and arachidonic acid metabolism

The rat platelet aggregation induced by collagen was stronger in males than in females. The platelet malondialdehyde (MDA) production was more in males than in females, and the platelet cyclooxygenase activity was higher in males than in females. Aspirin at a dose of 10 mg/kg inhibited the collagen-induced aggregation in males, but not in females. Aspirin at a dose of 5 mg/kg blocked the MDA production only in males, but aspirin at a dose of 10 mg/kg inhibited the MDA production in both sexes. The effect of aspirin on the cyclooxygenase activity was only in males, but aspirin at a dose of 10 mg/kg inhibited the MDA production in both sexes. The effect of aspirin on the cyclooxygenase activity was similar to that on the MDA production. In gonadectomized rats, the MDA production and the cyclooxygenase activity were decreased by castration, and they were increased by ovariectomy. Aspirin at a dose of 5 mg/kg failed to inhibit them in castrated rats. Besides, in in vitro experiments, aspirin also inhibited the MDA production and the aggregation. Nevertheless, there was no sex difference in the content of arachidonic acid, a substrate of platelet cyclooxygenase. It is suggested that there is a sex difference in rat platelet cyclooxygenase activity, and it is closely related to the sex difference in the antiplatelet effect of aspirin.     

The effects of vincristine on platelet aggregation studied by a filter loop technique in the rat.

1 A method for measuring aggregation of platelets of adenosine diphosphate (ADP) is described using a filter inserted into the flowing aortic blood in the rat. 2 Repeated infusions of ADP resulted in a fall in the calculated aggregation index without significant changes in the platelet count. 3 Vincristine (0.05 mg/kg) intravenously caused significant inhibition of ADP-induced platelet aggregation. 4 Infusion of ADP caused some peripheral vasodilatation though it is unlikely that this contributed to the effects seen to any great extent.     

Effects of CDP-choline on platelet aggregation and the antiaggregatory activity of arterial wall in the rat

The effects of the acute (250 mg/kg) and chronic (250 mg/kg for two weeks) treatments with CDP-choline on platelet aggregation and thromboxane formation and on the platelet antiaggregatory activity of thoracic aorta, have been studied in the rat. The acute administration resulted mainly in reduced platelet reactivity to aggregating agents, with no change of platelet thromboxane formation. The antiaggregatory activity of aortic walls was also concomitantly reduced. After chronic treatment, the major effect was a greater antiaggregatory activity of the vessel wall in respect of the control values, whereas platelet function was not affected. CDP-choline treatment, thus exerts favourable effects especially in the acute treatment, by reducing platelet reactivity.     

The change of serum leptin and its relationship with platelet membrane glycoprotein Ib in patients with coronary heart disease

The aim of this paper was to investigate the change of serum leptin and its relationship with platelet membrane glycoprotein Ib (GP Ib) in patients with coronary heart disease (CHD). The enrolled included 50 patients with CHD (CHD group) and 30 patients without CHD (control group) who were diagnosed by coronary angiography. The positive percentage and the average fluorescence intensity of platelet membrane GP Ib were detected by full-blood flow cytometry. Serum leptin was detected by enzyme linked immunosorbent assay. The positive percentage and the average fluorescence intensity of platelet membrane GP Ib in the CHD group were significantly lower than those in the control group (P < 0.05). After correcting the differences of systolic blood pressure, body mass index (BMI), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), fasting glucose, PPBS, fasting insulin and quantitative insulin sensitive index, serum leptin level in the CHD group was significantly higher than that in the control group (P < 0.05). Single factor correlative analysis revealed that serum leptin in CHD patients was negatively correlated with the average fluorescence intensity of platelet membrane GP Ib (P < 0.05). Multifactorial stepwise regression analysis showed that serum leptin in CHD patients was independently negatively correlated with the average fluorescence intensity of platelet membrane GP Ib (P < 0.05). Logistic analysis demonstrated that serum leptin was independently correlated with the risk of CHD (P < 0.05). Hyperleptinemia was verified in CHD patients. The increase of serum leptin could affect blood platelet activation. Hyperleptinemia may play an important role in the pathogenesis of CHD. __________ Translated from Tianjin Medical Journal, 2007, 35(2): 81–83 [译自: 天津医药]     

The roles of prostaglandin endoperoxides, thromboxane A2 and adenosine diphosphate in collagen-induced aggregation in man and the rat.

The effects of aspirin, carboxyheptylimidazole (CHI) and creatine phosphate/creatine phosphokinase (CP/CPK) on platelet aggregation and thromboxane B2 (TxB2) formation induced by collagen have been examined in vitro. Platelets from two species, man and the rat, have been used. In man, aspirin and CHI abolished TxB2 production but only partially inhibited aggregation. CP/CPK partially inhibited aggregation and TxB2 formation. In the rat, aspirin and CHI abolished TxB2 formation but had no effect on aggregation. CP/CPK completely inhibited aggregation and partially inhibited TxB2 generation. In man, collagen-induced aggregation is largely dependent on ADP and to a lesser extent on arachidonate metabolites whereas, in the rat, ADP alone mediates aggregation induced by this agonist. The results with CP/CPK suggest that TxB2 formation is dependent either on the prior release of platelet ADP or on aggregation itself rather than being responsible for the aggregation response.     

急性心肌梗死患者血小板活化分子标记物血小板激活复合物1及CD62p与纤维蛋白原的相关性研究

目的 探讨急性心肌梗死（AMI）患者血小板活化分子标记物血小板激活复合物1（PAC-1）、CD62p的变化及其与纤维蛋白原（Fg）的关系.方法 用三色全血流式细胞术测定36例AMI患者（AMI组）外周血中血小板PAC-1、CD62p的表达水平,并检测患者Fg水平,与32例健康体检者（健康对照组）的相关项目进行比较.结果 AMI组PAC-1、CD62p、Fg均高于健康对照组[（79.53±11.23）%比（47.02±5.98）%,（44.76±6.85）%比（29.13±4.38）%,（4.72±0.98）g/L比（3.38±0.48）g/L,P＜0.01].AMI组PAC-1与Fg呈正相关（r=0.56,P＜0.01）,CD62p与Fg呈正相关（r=0.42,P＜0.05）.结论 AMI患者血清Fg增加、血小板明显活化,其血小板活化程度与Fg关系密切.     

The role of the platelet glycoprotein IIb/IIIa in thrombosis and haemostasis

Haemostasis is a finely balanced and complex process ideally initiated only in response to disruption of the vascular endothelium as a means of preventing loss of blood from an injured vessel. Deviations from the ideal can lead to serious disease. Firstly, thrombosis, which arises as a consequence of inappropriate platelet-platelet interactions at a region of vessel damaged by atherosclerosis, can lead to occlusion of the affected vessel as in myocardial infarction or stroke. Secondly, loss of the ability of platelets to form aggregates leads to Glanzmann's thrombasthenia (GT) with a tendency to bleed for prolonged periods following injury. Glycoprotein IIb/IIIa (GPIIb/IIIa) plays a major role in the regulation of platelet adhesion and aggregation during haemostasis. Upon platelet activation by an agonist a signalling process is initiated, termed "inside-out" signalling, which gives rise to conformational changes within GPIIb/IIIa. These conformational changes increase the affinity of the receptor for its primary ligand, fibrinogen. Bound fibrinogen then acts as a bridging molecule facilitating the interaction of adjacent platelets. Upon fibrinogen binding GPIIb/IIIa undergoes further conformational changes and through a process termed "outside-in" signalling the receptor signals in to the platelet ultimately resulting in acceleration of the aggregation process. Qualitative or quantitative abnormalities in GPIIb/IIIa give rise to GT, a recessive bleeding disorder, and analysis of affected individuals has provided invaluable insights into the structure/function relationship of this receptor. Due to its critical role in mediating platelet aggregate formation GPIIb/IIIa has become a primary target for the development of antithrombotic agents.     

Genistein,an isoflavone included in soy,inhibits thrombotic vessel occlusion in the mouse femoral artery and in vitro platelet aggregation

Diet can be the most important factor that influences risks for cardiovascular diseases. Genistein included in soy is one candidate that may benefit the cardiovascular system. Here, we investigated the inhibitory effects of genistein on thrombotic vessel occlusion in the mouse femoral artery using a photochemical reaction, and in vitro platelet aggregation in whole blood measured by single platelet counting. Genistein (10 mg/kg), intravenously administered 10 min before the rose bengal injection, significantly prolonged the thrombotic occlusion time from 6.1+/-0.4 to 8.4+/-0.8 min (P<0.05). Genistein at doses higher than 30 microM significantly (P<0.01) inhibited in vitro platelet aggregation induced by collagen (1 and 3 microg/ml). When 10 mg/kg genistein was intravenously administered, ex vivo platelet aggregation induced by collagen (1 and 3 microg/ml) was significantly suppressed (P<0.01). In conclusion, genistein prevented in vivo thrombogenesis and suppressed in vitro platelet aggregation. These results suggest that dietary supplementation of soy may prevent the progression of thrombosis and atherosclerosis.     

Effect of a Paf antagonist, WEB 2086, on airway microvascular leakage in the guinea-pig and platelet aggregation in man.

1. The triazolodiazepine WEB 2086 has been evaluated as an antagonist of platelet-activating factor (Paf) by studying its effects on Paf-induced human platelet aggregation and microvascular leakage in guinea-pigs. 2. WEB 2086 inhibited Paf-induced platelet aggregation in platelet-rich plasma in vitro (IC50 = 117 +/- 35 nM, mean +/- s.d.) but had no effect on adenosine 3',5'-diphosphate-induced aggregation. 3. Paf-induced microvascular leakage, measured by the extravasation of intravenously-injected Evans blue dye, was inhibited in a dose-related fashion in the airways and other tissues by WEB 2086, achieving a maximal inhibitory effect at 10 micrograms kg-1, i.v. 4. However, WEB 2086 (10 micrograms kg-1, i.v.) did not inhibit a comparable increase in vascular permeability induced by ovalbumin in sensitized guinea-pigs. 5. We conclude that WEB 2086 is a potent antagonist of Paf and that Paf does not appear to be responsible for antigen-induced microvascular leakage.     

The ingestion of Ginkgo biloba extract (EGb 761) inhibits arachidonic acid-mediated platelet aggregation and thromboxane B2 production in healthy volunteers

Twelve non-diabetic volunteers (age = 39 +/- 13 years, BMI = 23.5 +/- 3.5) undertook a randomized double-blind placebo-controlled crossover study in which they ingested either 120 mg of EGb 761 or a placebo daily for 3 months and then switched to the other test capsules for the next 3 months. Platelet aggregation in platelet-rich plasma (PRP) was performed at the end of each 3-month arm, with or without 1-min incubation with graded doses of EGb 761. In the placebo cycles, AA-stimulated TXB2 production was 2581 +/-1337 pg/10(6) platelets (range 897-5485) compared to 1668 +/- 992 pg/10(6) platelets (range 6-1668) in the EGb 761 cycles (p < 0.005). Incubation of PRP with EGb 761 (150 microg/ml) completely inhibited platelet aggregation accompanied by inhibition of TXB2 synthesis in all subjects both in the placebo (<200 pg TXB2/10(6) platelets) and EGb 761 cycles (< 120 TXB2/10(6) platelets) (p < 0.0001). These results support EGb 761-mediated inhibition of platelet TXB2 synthesis in vivo.     

Heterogeneity of binding sites for glucocorticoid and the glucocorticoid-receptor complex in rat livers

Glucocorticoid binding to cytoplasmic and nuclear fractions and glucocorticoid-receptor complex binding to the nuclear fraction were investigated using rat liver. The glucocorticoid-receptor complex binding to the nuclear fraction was temperature-dependent, saturable, small in amount and of high affinity. The affinity and number of the glucocorticoid-receptor complex binding to the nuclear fraction were altered according to the glucocorticoid. Both the Bmax of nuclear glucocorticoid-receptor complex binding and the affinity of glucocorticoid to the cytoplasmic fraction were correlated with the relative anti-inflammatory potencies of glucocorticoids reported by Hynes and Murad (1980) and Fried et al. (1958). These results suggest that the number of nuclear binding sites of the glucocorticoid-receptor complex depends on the ligand steroid which is bound to the receptor of the cytoplasmic fraction and may be involved in physiological and pharmacological potencies of the glucocorticoid in addition to the affinity of the glucocorticoid to the receptor.