Anticoagulant effect and action mechanism of sulphated flavonoids from Flaveria bidentis

Quercetin 3-acetyl-7,3',4'-trisulphate (ATS) and quercetin 3,7,3',4'-tetrasulphate (QTS) obtained from Flaveria bidentis (Asteraceae) were investigated in vitro for anticoagulant activity. Three different concentrations of each flavonoid were assayed at different incubation times, showing at 1 mM significant prolongation on the activated partial thromboplastin time (APTT), less on the prothrombin time (PT), and no effect on the thrombin time (TT). In order to define the action mechanism of the anticoagulant activity, all coagulation factors were evaluated and no important activity decrease was observed, indicating that another mechanism is involved. Thus, thrombin inhibition mediated by antithrombin III (ATIII) and heparin cofactor II (HCII) activation was investigated in comparison to the physiological activators, heparin and dermatan sulphate (DS), respectively. As a conclusion, no activation on ATIII for neither flavonoids was observed. On the contrary, QTS much more than ATS produced an activation on HCII comparable to the one of DS, indicating that these flavonoids act as agonists of this inhibitor. A plausible explanation of the effects of both flavonoids could be due to the different degree of sulphation of these molecules. According to the results obtained, and taking in account the high solubility of these natural products in aqueous media and the nontoxic nature of this family of compounds, further investigation on the antithrombotic effects of these flavonoids are merited.     

The effects of glucosamine on platelet aggregation

The effects of glucosamine on platelet aggregation induced by a variety of agents were investigated. When compared with the effects of N-acetyl glucosamine (NAG), an acetylated derivative of glucosamine (1), it became apparent that the N-acetyl group was important in stimulating $\text{Staphylococcus aureus}$ induced platelet aggregation. The absence of the N-acetyl group from NAG changes it to glucosamine which is an inhibitor rather than a stimulator of $\text{S. aureus}$ induced platelet aggregation and release. Glucosamine also inhibits collagen, epinephrine, and ADP induced platelet aggregation and release.     

Action of some salicylate derivatives on in vitro platelet aggregation. Inhibitory and inhibition antagonistic effects

Twenty salicylate derivatives were tested for their antagonistic activity on the inhibitory effect of aspirin on platelet aggregation. The blocking effect was not limited to the salicylate but also characterised some of its substituted compounds. The substituant influence did not seem to be related to electronic or size parameters. This antagonistic activity of these derivatives decreased as concentrations increased, owing to the emergence of their own inhibitory activity: several salicylate derivatives showed dual inhibitory and inhibition antagonistic activity, with both properties present at the same concentration. A mechanism involving dissociated activities on the two enzymatic sites of cyclooxygenase is proposed.     

Effects of N-acetyl glucosamine on platelet aggregation

N-acetyl glucosamine (NAG) alone did not aggregate platelets but in low concentrations shortened the delay phase of the $\text{Staphylococcus aureus}$ induced platelet aggregation in a concentration dependent manner, which suggests that NAG may be one of the components of the $\text{S. aureus}$ cell wall responsible for platelet aggregation. NAG, on the other hand, inhibited collagen induced platelet aggregation and the secondary platelet aggregation induced by epinephrine and ADP. This inhibition appears to be due to impurities that occur at higher levels in some NAG lots than in others. NAG has also been shown to decrease the length of time before the release of ATP from platelets when $\text{S. aureus}$ is the aggregating agent.     

海风藤醇提取物对血小板活化因子诱导血小板聚集作用的初步研究

目的：观察不同浓度的海风藤醇提取物（ＡＥＰＫ）对血小板活化因子（ＰＡＦ）诱导血小板聚集的拮抗作用。方法：于兔富含血小板血浆（ＰＲＰ）中分别加入海风藤醇提取物（ＡＥＰＫ）,使后者达到不同的最终浓度;另设一份不加ＰＥＡＫ的对照ＰＲＰ。各份ＰＲＰ加入１０＾－８ＭＯｌ浓度的ＰＡＦ并血小板最大聚集率（ＭＡＲ）,且加以对比。结果：所有加ＡＥＰＫ的ＰＲＰ,其ＭＡＲ都比对照ＰＲＰ的ＭＡＲ低,且ＡＥＰＫ浓度越高,ＭＡＲ越低。结论：海风藤醇提取物对ＰＡＦ诱导血小板聚集有剂量依赖性抑制作用。     

Storage of platelets for tests of platelet function: effects of pH on platelet aggregation and liberation of beta-thromboglobulin

Platelet aggregation responses are influenced by conditions of storage of platelet-rich plasma (PRP). The aim of the present study was to further define the necessity for pH control during storage of PRP for tests of platelet function. Aliquots of citrated PRP were maintained at different pH levels by alteration of the CO2 content of the atmosphere in an incubation chamber. At intervals over 2-2 1/2 hours, plasma beta-thromboglobulin and 14C-serotonin were measured as well as platelet aggregation induced by ADP and collagen. At each time a dose response curve was studied for aliquots stored at each pH level. When two aliquots were maintained at different pH levels in the range 6.85-7.90, there was a significant increase in aggregation at the higher pH, even when the pH difference was as small as 0.2 units. In this range, pH did not influence the rate of deterioration of the aggregation response, but when pH was above 8.0, there was marked deterioration of the response. Increased pH was associated with an increase in plasma levels of beta-thromboglobulin and 14C-serotonin, which was more marked when pH was above 8.0. It appears that increases in pH are harmful to platelets and even small pH changes should be avoided during storage of platelet-rich plasma for tests of platelet function.     

Inhibitory mechanism of dipyridamole on platelet aggregation ex vivo

The effects of dipyridamole on platelet aggregation ex vivo and in vitro and on platelet cyclic AMP phosphodiesterase (PDE) were studied, and the mechanism of the ex vivo effects was assessed. Both the ADP- and collagen-induced aggregations ex vivo were inhibited dose-responsively by oral administration of dipyridamole. Maximum dipyridamole levels in the plasma were reached at 30 min after the administration. The inhibitory effects of dipyridamole on platelet aggregation ex vivo reached a maximum at between 1 and 2 hrs. On the other hand, the ADP-induced aggregation in vitro and cyclic AMP PDE activity were not inhibited until after 10 min of incubation at a low concentration of dipyridamole. This mode of inhibition of platelet aggregation in vitro and of cyclic AMP PDE activity agreed with the mode of inhibition in the case of platelet aggregation ex vivo. It is suggested therefore that the ex vivo effects, observed with only a low dipyridamole concentration in the plasma, may be due primarily to inhibition by dipyridamole of the cyclic AMP PDE in platelets.     

金纳多对急性脑梗死患者血小板聚集和血液流变学的影响

目的 :观察金纳多对脑梗死患者血小板聚集和血液流变学的影响。方法 :将 6 8例急性脑梗死患者随机分为两组 ,金纳多治疗组 38例 ,对照组 30例 ,对两组患者检测治疗前后血小板聚集和血液流变学各指标并进行神经功能缺损评分。结果 :金纳多治疗组显效率为 89 5 % ,对照组为 76 7% (P <0 0 5 )。金纳多治疗后血粘度、纤维蛋白原、红细胞电泳时间、血小板第一时相聚集率 (Ⅰ°M)、血小板第二时相聚集率 (Ⅱ°M)均降低 ,明显优于对照组 (P <0 0 5 )。结论 :金纳多通过拮抗血小板聚集和改善血液流变学而对急性脑梗死具有治疗作用     

A study of intravascular platelet aggregation by continuous platelet counting

Intravascular platelet aggregation has been studied by measuring the response to aggregating agents using continuous platelet counting. Dose-dependent falls in platelet count occurred after the injection of collagen, prostaglandin H₂ (PGH₂), arachidonic acid (AA) and four synthetic prostanoids in rats. Indomethacin partially inhibited collagen- and AA-induced aggregation in the rat and collagen-induced aggregation in the rabbit. Indomethacin and aspirin did not produce a dose-dependent inhibition of collagen-induced aggregation over the range of doses tested. 1-n-butylimidazole inhibited collagen- and PGH₂-induced aggregation in the rabbit but did not inhibit collagen- or AA-induced aggregation in the rat.     

effects of picotamide on human platelet aggregation, the release reaction and thromboxane B2 production

We studied the effects of picotamide (N,N′ bis 3 picolyl-4-methoxy-isophthalamide) on human platelet aggregation, the release reaction and the production of thromboxane B₂ (TxB₂) induced by several platelet agonists. The effects of picotamide were compared to those of acetylsalicylic acid (ASA). Picotamide (0.5 mmol/1) inhibited platelet aggregation, the release of ATP and TxB₂ production induced by ADP, arachidonic acid (AA), collagen or the prostaglandin endoperoxide (PE) analogue U46619. ASA (0.5 mmol/1) did not affect platelet aggregation and the release of ATP induced by U46619. Picotamide and ASA inhibited the AA-induced platelet TxB₂ production both under stirring and non-stirring conditions, whereas the pure thromboxane A₂ receptor antagonist BM13177 (0.5 mmol/1) was inhibitory only under stirring conditions. Since under non-stirring conditions platelet aggregation does not occur, picotamide directly inhibits TxB₂ production, whereas BM13177 inhibits the potentiation of TxB₂ production due to TxA₂/PE-dependent platelet aggregation. Malondialdehyde (MDA) production by unstirred platelets stimulated with AA was not significantly inhibited by picotamide. In conclusion, picotamide inhibits the TxA2/PE-dependent platelet responses to agonists by a double mechanism: (i), TxA₂/PE antagonism; (ii) inhibition of thromboxane synthase.     

ADP-induced second wave aggregation in platelet rich plasma from hypercholesterolemic rabbits

Platelet aggregation, ATP secretion and thromboxane formation were measured in platelet-rich plasma (PRP) of rabbits fed a cholesterol-rich (1%) diet for 3 months. Addition of ADP (0.6 – 2 μM) resulted in a clear biphasic aggregation in 7/8 cholesterol-fed rabbits. This was not seen in any of the 9 control animals kept on a standard diet for the same period of time (P < 0.01). The secondary ADP-induced aggregation was irreversible and was accompanied by enhanced ATP secretion and significant thromboxane generation. There was no ADP-induced thromboxane formation and only a minor ATP secretion at the highest ADP concentration studied (20 μM) in control rabbits. These data confirm platelet hyperreactivity in hypercholesterolemia and demonstrate for the first time the occurence of an ADP-induced second wave aggregation and granule secretion in citrated PRP of rabbits which may be triggered by thromboxane generation.     

Inhibition of platelet aggregation in whole blood by adrenoceptor antagonists

It has recently become possible to study platelet aggregation in whole blood which may more closely resemble the in-vivo situation as the platelets are left in their natural milieu with red and white cells present which themselves can influence aggregation. The effects of 4 adrenoceptor antagonists on platelet aggregation in whole blood were studied in-vitro using the Clay-Adams Ultra Flo 100 whole blood platelet counter. Labetalol, pindolol and propranolol inhibited aggregation to 0.5 microgram/ml collagen in a dose dependent manner, and were synergistic with prostacyclin in inhibiting collagen induced aggregation. These 3 drugs also promoted reversal of aggregation induced by 10 microM ADP, but only inhibited 0.5 mM arachidonic acid induced aggregation at high drug concentrations. Atenolol had no effect on either collagen, ADP or arachidonic acid induced aggregation. The anti-platelet effect of these drugs may be of value in the treatment of vascular disease.     

The effects of arm cranking exercise and training on platelet aggregation in male spinal cord individuals

Platelet aggregation at rest and in responses to exercise and training were compared between spinal cord injured (SCI) individuals (N=5) and able-bodied subjects (N=7). All participants performed arm cranking exercise at 60-65% VO(2peak) for 30 min. Venous blood samples were obtained before and after sub-maximal exercise and measured for platelet aggregation using ADP and collagen. To assess the effects of arm cranking training, platelet aggregation was re-measured in all subjects at rest and in response to the sub-maximal arm cranking exercise after 12 weeks of individually supervised training programme. Before training, the resting mean values of platelet aggregation induced by ADP and collagen were not different (P>0.05) between SCI and able-bodied. However the SCI individuals, but not the able-bodied subjects, exhibited a significantly (P<0.05) higher maximal platelet aggregation induced by ADP and collagen following sub-maximal arm cranking exercise. Although VO(2peak) after training was significantly increased (P<0.05) in both groups, the resting mean values of platelet aggregation induced with ADP and collagen were not significantly different (P>0.05) from those observed before training and were not different (P>0.05) between SCI and able-bodied. Post-training, the SCI individuals, but not able-bodied individuals, exhibited a significant decrease (P<0.05) in platelet aggregation following sub-maximal arm cranking exercise and this occurred with both ADP and collagen. These results suggest that SCI individuals, but not normal subjects increase their platelet aggregation following sub-maximal arm cranking exercise. Furthermore, arm cranking training in SCI individuals, appears to diminish the percentage of platelet aggregation ex vivo.     

Dynamics of platelet activation in diabetic and non-diabetic subjects during the course of an acute myocardial infarction

INTRODUCTION: The dynamics of platelet activation during the course of a myocardial infarction is unknown but of great importance in terms of risk assessment and anti-thrombotic therapy. The aim of the present study was sequentially to analyse platelet activation in diabetic and non-diabetic subjects with an acute myocardial infarction. MATERIALS AND METHODS: We used a sensitive laser light scattering technique to assess platelet aggregation as a measure of activation. Measurements were made on the first, second, third and fifth day in-hospital. Two hundred and forty-three patients with an acute myocardial infarction, of whom 48 had diabetes, were included. RESULTS: Platelet activation increased until the third in-hospital day in both diabetic and non-diabetic subjects, despite intense anti-thrombotic therapy. The activation was more pronounced in diabetic subjects from the time of hospital admission. Platelet activation tended to decrease after the third in-hospital day. CONCLUSIONS: We conclude that platelet activation increases rapidly at the onset of a myocardial infarction, despite aggressive anti-thrombotic treatment. The activation is more pronounced in diabetic subjects and tends to decrease within a few days. More targeted and effective anti-platelet therapy has the potential further to reduce cardiac and cerebral ischemic events following myocardial infarction and ongoing clinical trials are addressing this issue.     

Modified forms of low density lipoprotein affect platelet aggregation

Modified forms of low density lipoprotein (LDL) unlike native LDL can lead to macrophage cholesterol accumulation and foam cell formation. Since platelets interact with both lipoprotein and macrophages in the atherosclerotic plaque, the present study was designed to analyze the effect of modified LDL on washed human platelet composition and aggregation. Platelet aggregation was increased following 2 h of incubation with native LDL. Phospholipase C modified LDL and hepatic lipase modified LDL but not acetyl LDL further increased collagen induced platelet aggregation in a dose dependent manner by up to 15% (p less than 0.01). Oxidized LDL, however, demonstrated 25% reduction in both collagen and ADP induced platelet aggregation in comparison to the effect of native LDL. Platelet aggregation was found to be directly related to changes in platelet phospholipid content whereas platelet cholesterol content was similarly affected by all lipoproteins. Platelet cholesterol/phospholipid ratio was directly related to platelet aggregation. Our study thus demonstrates that modified forms of LDL significantly affect platelet lipid composition and function and if similar interactions occur in vivo it might also affect the atherogenic process.     

Lack of thromboxane A2 synthesis in platelet aggregation induced by factor VIII/von willebrand factor

Thromboxane A₂ (TXA₂) synthesis during factor VIII/von Willebrand factor (FVIII/vWF) induced-platelet aggregation was studied in human platelets prelabeled with (1-¹⁴C)-arachidonic acid (¹⁴C-AA). Although previous studies demonstrate that thrombin, collagen or epinephrine induced platelet aggregation is associated with TXA₂ synthesis, no TXA₂ synthesis was detected in FVIII/vWF induced aggregation. Nevertheless, an increased release of ¹⁴C-AA and its probable conversion into HETE was detected during platelet aggregation induced by FVIII/vWF in the presence of ristocetin. When thrombin was added to ¹⁴C-AA labeled platelet suspensions, similar amounts of TXA₂ were synthesized with or without stirring, despite the fact that no platelet aggregation occurs without stirring. These results indicate that TXA₂ is not synthesized during FVIII/vWF induced platelet aggregation, and that aggregation of platelets is not a pre-requisite for TXA₂ synthesis.     

Inhibition of platelet aggregation and the release of P-selectin from platelets by cilostazol

To evaluate the in vitro effects of cilostazol, a phosphodiesterase III inhibitor, on platelet responses, we measured platelet aggregation and the levels of soluble P-selectin, a glycoprotein present on the -granule membrane in resting platelets, and cAMP. Platelet-rich plasma and washed platelets from healthy human volunteers were treated with cilostazol (5, 25 and 50 μM). Platelet-rich plasma was stimulated by ADP (1 and 5 μM) or collagen (5 μg/ml). Washed platelets were stimulated by thrombin (4 U/ml) in the presence or absence of 1 μM forskolin. In vehicle-treated samples, soluble P-selectin levels in response to 1 μM ADP-induced primary aggregation were similar to those of circulating levels of healthy volunteers but the levels in response to 5 μM ADP-induced secondary aggregation and collagen-induced aggregation increased markedly compared to those in response to primary aggregation. This result suggests that P-selectin is released from platelets according to the extent of platelet aggregation. Cilostazol inhibited platelet aggregation as well as P-selectin release in a concentration-dependent manner. Cilostazol inhibited completely thrombin-induced aggregation in the presence of 1 μM forskolin, when cAMP levels were two-fold higher than those in the absence of forskolin. Cilostazol, which increases intracellular cAMP in platelets, may be useful in the treatment of arterial occlusive diseases.     

Inhibition of rabbit platelet phosphodiesterase activity and aggregation by calcium channel blockers

Rabbit platelet cyclic AMP phosphodiesterase is inhibited by the three calcium channel blockers nifedipine, diltiazem, and verapamil with IC50's of 100 microM, 100 microM and 420 microM, respectively. Also, platelet aggregation induced by 4 microM ADP is inhibited by those compounds. Verapamil is the most potent aggregation inhibitor with an IC50 of 260 microM while diltiazem and nifedipine have IC50's of 630 microM and 840 microM, respectively. All three compounds display a maximum inhibition of 80-85%. Diltiazem and PGD2 potentiate the antiaggregatory activity of each other in that the inhibitions occurring in the presence of the combination of the two (at varying concentrations) exceed the calculated sums of the inhibitions produced by each alone. On the other hand, the antiaggregatory activities of verapamil or nifedipine, are additive with that of PGD2 in that no significant differences exist between the observed inhibitions produced by the combinations and the calculated summed values of the individual inhibitions. Our data suggest, therefore, that in addition to lowering intracellular calcium ions, which are required for platelet aggregation, the three calcium channel blockers inhibit the breakdown of cyclic AMP thereby promoting antiaggregation.     

Protease activated receptor-1 (PAR-1) mediated platelet aggregation is dependent on clopidogrel response

Introduction

Clopidogrel inhibits ADP mediated platelet aggregation through inhibition of the P2Y12 receptor by its active metabolite. Thrombin induces platelet aggregation by binding to protease activated receptor-1 (PAR-1), and inhibition of PAR-1 has been evaluated in patients treated with clopidogrel to reduce ischemic events after acute coronary syndromes. Residual PAR-1 mediated platelet aggregation may be dependent on extent of clopidogrel response.

Material and Methods

Platelet aggregation was measured in 55 patients undergoing elective PCI at 16-24 hours after 600 mg clopidogrel loading dose by light transmittance aggregometry using ADP 20 μM and thrombin receptor agonist peptide (TRAP) at 15 μM and 25 μM as agonists. Genomic DNA was genotyped for common CYP2C19 variants.

Results

Increasing quartiles of 20 μM ADP induced platelet aggregation after clopidogrel loading were associated with increasing levels of TRAP mediated platelet aggregation. Patients in the highest quartile (clopidogrel non-responders) of post treatment ADP aggregation had significantly higher TRAP mediated aggregation than the patients in the lowest quartile (clopidogrel responders) [TRAP 15 μM: 79.6 ± 5% vs. 69.5 ± 8%, p < 0.001].

Conclusions

Non-responders to clopidogrel show increased residual platelet aggregation induced by TRAP, whereas clopidogrel responders exhibit attenuated response to TRAP. Addition of PAR-1 antiplatelet drugs may be most effective in patients with reduced clopidogrel response and high residual TRAP mediated platelet aggregation.