Platelet aggregation inhibitors from Agkistrodon acutus snake venom

Among all the purified components from A. acutus venom, including ADPase, 5'-nucleotidase, phospholipase A2 and fibrinogenases, only the venom ADPase (50-100 micrograms/ml) shows marked inhibitory action on ADP (10 microM)-, collagen (10 micrograms/ml)- and sodium arachidonate (100 microM)-induced platelet aggregations of rabbit platelet-rich plasma. The venom 5'-nucleotidase (100 micrograms/ml) inhibited ADP-induced platelet aggregation by 31 +/- 4% (n = 4, P less than 0.05). Fibrinogenolytic enzymes (fractions I and IX, 100 micrograms/ml) did not significantly inhibit platelet aggregation induced by ADP (10 microM), collagen (10 micrograms/ml) or sodium arachidonate (100 microM). However, when the fibrinogenase (fraction IX, 100 micrograms/ml) was preincubated with platelet-rich plasma for 30 min it inhibited collagen (20 micrograms/ml)- and ADP (10 microM)-induced platelet aggregations by 34 +/- 9% (n = 4, P less than 0.05) and 35 +/- 6% (n = 4, P less than 0.05), respectively. The phospholipase A2 (100 micrograms/ml) did not affect platelet aggregation. The venom ADPase is a single chain polypeptide with a molecular weight of 94,000. The specific ADPase activity is estimated to be 4.3 mu moles Pi/min/mg of protein. It also possesses phosphodiesterase and weak 5'-nucleotidase activities.     

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.     

Antiplatelet effects of the novel thromboxane A2 receptor antagonist sodium (E)-11-[2-(5,6-dimethyl-1-benzimidazolyl)-ethylidene]-6,11- dihydrodibenz[b,e] oxepine-2-carboxylate monohydrate.

The effects of KW-3635 (sodium (E)-11-[2-(5,6-dimethyl-1-benzimidazolyl)- ethylidene]-6,11-dihydrodibenz[b,e] oxepine-2-carboxylate monohydrate, CAS 127166-41-0) on platelet aggregation were examined. In human washed platelets, KW-3635 shifted the concentration-aggregation curves for U-46619, a thromboxane A2 (TxA2) mimetic, to the right. The pA2 value for KW-3635 was 8.8 +/- 0.10, while those for sulotroban and daltroban were 6.31 +/- 0.18 and 7.75 +/- 0.07, respectively. In human platelet rich plasma (PRP), KW-3635 at 10(-8) mol/l to 10(-6) mol/l inhibited the aggregations induced by U-46619 (1 mumol/l) or collagen (1.5 micrograms/ml). However, KW-3635 at up to 10(-5) mol/l did not affect the primary phase of platelet aggregation induced by adenosine diphosphate or epinephrine. KW-3635 at 10(-5) mol/l did not affect the antiaggregatory effects of the prostaglandins PGI2, PGE1 and PGD2. These results indicate that KW-3635 is a potent and selective TxA2 receptor antagonist. The TxA2 antagonistic effects of KW-3635 were compared with that of daltroban in PRP from various animals species. The effects of KW-3635 on platelet aggregation were species-dependent and KW-3635 exhibited the most prominent activity in human platelets. The activities of KW-3635 in mouse and rabbit PRP were much less potent. In PRP from guinea-pigs, dogs, cats and rats, KW-3635 exhibited moderate anti-aggregatory effects. In the guinea-pig PRP, KW-3635 at 10(-7) mol/l to 3 x 10(-6) mol/l inhibited both the platelet aggregation and the concomitant adenosine triphosphate secretion in a concentration-dependent manner, the effect being more potent than those of sulotroban and daltroban. In the experiments on the platelet aggregation ex vivo in guinea-pigs, KW-3635 at oral doses of 3 and 10 mg/kg inhibited the aggregations induced by U-46619 (1, 3 mumol/l), collagen (3, 6, 9 micrograms/ml) and arachidonate (50, 100 mumol/l). The effects lasted for longer than 7 h following oral administration. These results indicate that KW-3635 is a specific and orally active TxA2 receptor antagonist. KW-3635 is expected to be a drug useful for the treatment of patients with thrombotic disorders.     

In vitro and in vivo studies of the effect of vitamin E on microsomal cytochrome P450 in rat liver

Administration of a diet supplemented with 0.06% vitamin E acetate to male rats over a 6-week period doubled hepatic microsomal stores of alpha-tocopherol over those in control (vitamin E adequate) rat liver. Total cytochrome P450 content and NADPH-cytochrome P450 reductase activity were significantly elevated in hepatic microsomes from vitamin E-supplemented rats to 111% and 123% of respective control values. Androstenedione 16 alpha-hydroxylase activity was increased in these fractions (2.57 +/- 0.31 nmol product/min/mg protein vs 1.81 +/- 0.38 in controls) whereas activities of the 6 beta-, 7 alpha- and 16 beta-hydroxylase pathways were unchanged. Immunoquantitation of the microsomal 16 alpha-hydroxylase, P450 IIC11, indicated a corresponding increase in the hepatic content of the enzyme. In view of the established antioxidant role of tocopherols, the effects of dietary vitamin E manipulation on the concentration of protein sulphydryl groups and the susceptibility of microsomes to ferric sulphate-ADP-NADPH-mediated lipid peroxidation were also assessed. Dietary supplementation did not influence microsomal protein sulphydryl content (68 +/- 10 nmol glutathione equivalents/mg protein) but decreased the extent of lipid peroxidation produced by the ferric sulphate-ADP-NADPH system in vitro. Further in vitro experiments demonstrated that vitamin E acetate (2 microM) protected protein sulphydryl groups and lipids against peroxidation in control microsomes and partially reduced the associated losses of P450-mediated steroid hydroxylase activities. Western immunoquantitation of P450 IIC11 revealed that exogenous vitamin E acetate protected completely against peroxidation-induced apoprotein loss. These studies establish that the in vitro protective effects of vitamin E acetate against sulphydryl and lipid peroxidation extend to protection of the P450 apoprotein but that enzyme activity is only partially protected. This finding suggests that peroxidation-dependent loss of P450 in vitro is mediated by haem degradation from the P450 holoenzyme and is not directly related to lipid/sulphydryl oxidation. In contrast, the in vivo effects of dietary vitamin E on drug metabolizing enzymes are regulatory in nature and are unrelated to effects on lipid peroxidation.     

Oral vitamin C reduces arterial stiffness and platelet aggregation in humans.

Atherosclerosis is associated with stiffening of conduit arteries and increased platelet activation, partly as a result of reduced bioavailability of nitric oxide (NO), a mediator that normally has a variety of protective effects on blood vessels and platelets. Increased levels of oxygen free radicals are a feature of atherosclerosis that contributes to reduced NO bioavailability and might lead to increased arterial stiffness and platelet activation. Vitamin C is a dietary antioxidant that inactivates oxygen free radicals. This placebo-controlled, double-blind, randomized study was designed to establish whether acute oral administration of vitamin C (2 g), would reduce arterial stiffness and in vitro platelet aggregation in healthy male volunteers. Plasma vitamin C concentrations increased from 42+/-8 to 104+/-8 microM at 6 h after oral administration, and were associated with a significant reduction in augmentation index, a measure of arterial stiffness (by 9.6+/-3.0%; p = 0.016), and ADP-induced platelet aggregation (by 35+/-13%; p = 0.046). There was no change in these parameters after placebo. Vitamin C, therefore, appears to have beneficial effects, even in healthy subjects. The mechanism responsible is likely to involve protection of NO from inactivation by oxygen free radicals, but this requires confirmation. If similar effects are observed in patients with atherosclerosis or risk factors, vitamin C supplementation might prove an effective therapy in cardiovascular disease.     

Effects of the new antiplatelet agent 2-methyl-3-(1,4,5,6-tetrahydronicotinoyl)pyrazolo[1,5-a]pyridine on platelet aggregation and thrombosis in experimental animals.

Antiplatelet and antithrombotic effects of KC-764 (2-methyl-3-(1,4,5,6-tetrahydronicotinoyl)pyrazolo[1,5-a]pyridine, CAS 94457-09-7) were studied. KC-764 inhibited arachidonic acid (AA)- and collagen-induced platelet aggregation with IC50s of 1.0 x 10(-8)-2.8 x 10(-7) mol/l for humans, rabbits, guinea pigs and dogs, and IC50s of 3.9 x 10(-6)-3.7 x 10(-5) mol/l for mice and rats in vitro. KC-764 inhibited AA- and collagen-induced aggregation with ID50s of 0.04-0.09 mg/kg p.o. in rabbits and dogs, and ID50 of 13.0 mg/kg p.o. in rats. These antiaggregatory activities of KC-764 were stronger than those of acetyl-salicylic acid (ASA), indometacin, cilostazol and ticlopidine. KC-764 inhibited the production of thromboxane B2 (TXB2) in rabbit platelet microsomes, washed platelets and reconstituted platelet rich plasma (RPRP) with IC50s of 2.9 x 10(-6) mol/l, 2.8 x 10(-7) mol/l and 4.3 x 10(-8) mol/l, respectively. The in vitro inhibitory activity of KC-764 on AA-induced platelet aggregation was more potent when RPRP was used rather than washed platelet suspension containing 30% rabbit plasma. ASA did not show such an augmentation. KC-764 prevented collagen- and AA-induced thrombosis at more than 1 mg/kg p.o. and more than 0.1 mg/kg i.v. in mice and rabbits. KC-764 showed the wider margin of dose between antiplatelet action and prolongation of bleeding time in rabbits than ASA and indometacin. These results indicated that KC-764 was a potent antithrombotic drug to prevent TXB2 production and less possible to induce untoward actions as compared with ASA or indometacin.     

Effect of stable fish oil on arterial thrombogenesis, platelet aggregation, and superoxide dismutase activity

We examined the influence of dietary stable fish oil on aortic thrombosis, platelet aggregation, and superoxide dismutase (SOD) activity in a rat model. Twenty-nine Sprague-Dawley rats were fed regular chow supplemented with stable fish oil preparation (for 1 or 3 weeks), and 37 rats fed regular chow served as controls. The abdominal cavity was opened, and the abdominal aorta isolated. Whatman paper impregnated with 35% FeCl3 was wrapped around the surface of the aorta, and aortic flow was continuously recorded. In control rats, an occlusive platelet-fibrin-rich thrombus was formed in 21 +/- 3 min. Dietary fish oil in a time-dependent fashion delayed time to thrombus formation (24 +/- 2 min in rats fed fish oil for 1 week and 31 +/- 2 min in rats fed fish oil for 3 weeks), inhibited platelet aggregation (21 +/- 5% vs. 45 +/- 6%; p < 0.01) and increased SOD activity (p < 0.01). We conclude that dietary supplementation with stable fish oil delays formation of arterial thrombus, probably by reducing platelet aggregation and oxidative stress-associated arterial injury.     

The role of route of vitamin E administration on the plasma antioxidant activity and lipid peroxidation in newborn calves

The aim of our study was to evaluate plasma values of alpha-tocopherol, malondialdehyde (MDA) and antioxidant activity after a single-dose administration of vitamin E as intramuscular injection, oral supplementation and intramuscular injection plus oral supplementation at 4 hr after birth. Thirty calves were bled at birth and assigned to treatments as follows: control (n = 8), intramuscular injection (40 IU/kg, n = 7), oral supplementation (25 IU/kg, n = 7) and intramuscular injection (20 IU/kg) plus oral supplementation (12.5 IU/kg, n = 8). Blood was collected at 12 and 24 hr after birth and plasma alpha-tocopherol, MDA and antioxidant activity values were determined. Results showed that no changes in MDA values were observed after oral administration (P > 0.05). However, antioxidant activity values showed an increase at both 12 (9.57 +/- 0.65 mmol/l) and 24 hr (10.42 +/- 0.54 mmol/l) after birth when compared to control (3.73 +/- 0.75 mmol/l). Injection with or without oral supplementation increased serum antioxidant activity values at 12 (about 102%, 46%) and 24 hr (94%, 115%) after birth, when compared to control. In addition, MDA values were found to be lower in those animals receiving an injection of vitamin E or injection plus oral supplementation of vitamin E as compared to control at both time-points (P < 0.001). Injection of vitamin E provided beneficial effects to plasma antioxidant activity and MDA values. Therefore, injection may be the best method of vitamin E administration in newborn calves for protecting them in the stressful postnatal condition.     

Effect of the purified phospholipases A2 from snake and bee venoms on rabbit platelet function

Effects of seven purified phospholipases A2 from the venoms of snakes (Naja naja atra, Trimeresurus mucrosquamatus and T. gramineus) and honey bee (Apis mellifera) on rabbit washed platelet suspension in the absence of bovine serum albumin have been studied. Only phospholipases A2 from N. n. atra, T. mucrosquamatus and A. mellifera venoms induced platelet aggregation with small amounts of 14C-serotonin release. They showed tachyphylaxis and also cross-tachyphylaxis in inducing platelet aggregation. The former two phospholipases A2 exhibited biphasic responses in which irreversible aggregations appeared at concentrations of 1-10 micrograms/ml. At higher concentrations, they elicited the reversible aggregation. Exogenous Ca2+ was essential to their activity. Indomethacin and EDTA completely abolished both phospholipase A2 induced platelet shape change and aggregation, while mepacrine, prostaglandin E1, verapamil and nitroprusside inhibited only the aggregation response. p-Bromophenacyl bromide-modified phospholipases A2, which almost completely lost enzymatic activity, failed to induce platelet aggregation. Phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol inhibited the phospholipase A2-induced platelet aggregation. These phospholipases A2 induced thromboxane B2 formation which was inhibited by EDTA and indomethacin, but not by prostaglandin E1. Pre-treatment of platelet suspension with phospholipase A2 from N. n. atra or A. mellifera venom (50 micrograms/ml) inhibited platelet aggregation induced by sodium arachidonate or collagen, but not that induced by thrombin or ionophore A-23187. Exogenous sodium arachidonate or lysophosphatidylcholine also showed unaltered inhibitory spectrum on platelet aggregation. It is concluded that phospholipases A2 induce platelet aggregation by virtue of their enzymatic activity, cleaving the membrane phospholipids resulting in arachidonic acid release and formation of thromboxane A2. On the other hand, the cleaved products, lysophosphatidylcholine, arachidonic acid or arachidonate metabolites (via lipoxygenase pathway) may be responsible for anti-platelet activity.     

Acute effects of vitamin C on platelet responsiveness to nitric oxide donors and endothelial function in patients with chronic heart failure

Chronic heart failure (CHF) is characterized by a prothrombotic state, which may relate to increased platelet aggregability, endothelial dysfunction, and increased oxidative stress. We investigated the effect of vitamin C in CHF on ex vivo platelet aggregation and platelet responsiveness to the anti-aggregatory effects of the nitric oxide (NO) donors glyceryl trinitrate (GTN) and sodium nitroprusside (SNP). We also examined parameters of oxidative stress and endothelial function in patients. In this double-blind, randomized, crossover study vitamin C (2 g) or placebo was given intravenously to 10 patients with CHF. We measured adenosine 5-diphosphate (ADP)-induced platelet aggregation, flow-mediated dilatation (FMD) in the brachial artery using ultrasonic wall-tracking, and plasma levels of lipid-derived free radicals using electron paramagnetic resonance spectroscopy. Vitamin C did not affect ex vivo platelet aggregability but enhanced the inhibition of platelet aggregation by SNP (62.7+/-10.2 to 82.7+/-4.8%, p = 0.03) and tended to increase responses to GTN (40.5+/-9.0 to 53.4+/-7.3, p = 0.06). The effect of vitamin C on platelet responsiveness to the antiaggregatory effects of SNP was inversely related to basal response to SNP (r = -0.9, p < 0.01); a similar trend was observed with GTN (r = -0.6, p = 0.1). Vitamin C also increased FMD (1.9+/-0.6 to 5.8+/-1.5%, p = 0.02) and reduced plasma lipid-derived free radicals by 49+/-19% (p < 0.05). In patients with CHF acute intravenous administration of vitamin C enhances platelet responsiveness to the anti-aggregatory effects of NO donors and improves endothelial function, suggesting a potential role for vitamin C as a therapeutic agent in CHF.     

维拉帕米对糖尿病病人体外血小板聚集的影响

糖尿病Ⅱ型病人２０例（男性１０例，女性１０例，年龄５８±ｓ７ａ）在０．５μｍｏｌ／ＬＡＤＰ作为血小板致聚条件下加入０．２５－１．０μｍｏｌ／Ｌ维拉帕米，观察对体外血小板聚集的影响。结果表明糖尿病Ⅱ型病人在二相聚集比正常者显著增强，维拉帕米在０．２５－１．０μｍｏｌ／Ｌ浓度范围内均可部分减缓糖尿病Ⅱ型病人所增高的血小板聚集。     

Differential modulation of bradykinin-induced relaxation of endothelin-1 and phenylephrine contractions of rat aorta by antioxidants

AIM: We tested the hypothesis that bradykinin (BK)-induced relaxation of phenylephrine (PE) and endothelin-1 (ET-1) contractions can be differentially modulated by reactive oxygen species (ROS). METHODS: Aortic rings isolated from Sprague-Dawley rats were used for the study. The contribution of ROS to PE (1 x 10(-9)-1 x 10(-5) mol/L)- and ET-1 (1 x 10(-10)-1 x 10(-8) mol/L)-induced contractions and the influence of ROS in BK (1 x 10(-9)-1 x 10(-5) mol/L) relaxation of PE (1 x 10(-7) mol/L) or ET-1 (1 x 10(-9) mol/L)-induced tension was evaluated in the aorta in the presence or absence of the following antioxidants: catalase (CAT, 300 U/mL), superoxide dismutase (SOD, 300 U/mL), and vitamin C (1 x 10(-4) mol/L). Results: Tension generated by ET-1 (1 x 10(-9) mol/L) or PE (1 x 10(-7) mol/L) was differentially relaxed by BK (1 x 10(-5) mol/L), producing a maximal relaxation of 75%+/-5% and 35+/-4%, respectively. The BK (1 x 10(-5) mol/L)-induced relaxation of PE (1 x 10(-7) mol/L) tension was significantly enhanced from 35%+/-4% (control) to 56%+/-9%, 60%+/-5%, and 49%+/-6% by SOD, CAT, and vitamin C, respectively (P<0.05, n=8). However, the relaxation of ET-1 (1 x 10(-9) mol/L) tension was significantly attenuated from 75%+/-5% (control) to 37%+/-9%, 63%+/-4%, and 39%+/-7% by SOD, CAT, and vitamin C, respectively (P<0.05, n=8). On the other hand, CAT had no effect on PE-induced tension, while SOD enhanced PE-induced tension (36%, P<0.05, n=10) and vitamin C attenuated (66%, P<0.05, n=8) the tension induced by PE. By contrast, SOD or vitamin C had no effect, but CAT attenuated (44%, P<0.05, n=9) the tension induced by ET-1. CONCLUSION: We have demonstrated that O2(-) and H2O2 differentially modulate BK relaxation in an agonist-specific manner. O2(-) attenuates BK-induced relaxation of PE contraction, but contributes to the relaxation of ET-1 contraction. O2(-) seems to inhibit PE contraction, while H2O2 contributes to ET-1-induced contraction. Thus, ROS differentially modulate vascular tone depending on the vasoactive agent that is used to generate the tone.     

Measurement of vitamin E in serum and plasma by high performance liquid chromatography with electrochemical detection

A deficiency of vitamin E has been associated with a wide variety of pathological conditions, many of them involving neonates. Previously published methods for the quantitation of vitamin E require large sample volumes as well as extensive extraction and concentration procedures. The present work describes a method for the quantitation of vitamin E in small volumes (less than or equal to 50 microliters) of serum or plasma without the need for extraction or concentration of the sample. With further optimization, even smaller volumes (less than or equal to 10 microliters) can be employed. Sample preparation consisted of precipitation of plasma proteins with absolute ethanol. After centrifugation, a small volume (10 to 50 microliters) of the supernatant was injected directly onto the high performance liquid chromatography (HPLC) column. A dual channel electrochemical detector was used for quantitation of the vitamin E. Pooled serum or plasma was used to determine within-day (8.43 +/- 0.19 mg/L) and between-day (8.49 +/- 0.34 mg/L) precision. Analytical recovery of added vitamin E (4-16 mg/L) averaged 102 +/- 7.0%. No interfering peaks were observed. Linearity was demonstrated over 0.5 to 800 ng. Vitamin E acetate is not detected by this method. HPLC with electrochemical detection is a highly sensitive and specific method for the quantitation of vitamin E in plasma or serum. This procedure is rapid and simple, and can be performed with very small sample volumes.     

Specific inhibition of ADP-induced platelet aggregation by clopidogrel in vitro

1. The thienopyridine clopidogrel is a specific inhibitor of ADP-induced platelet aggregation ex vivo. No direct effects of clopidogrel (< or = 100 microM) on platelet aggregation in vitro have been described so far. 2. Possible in vitro antiaggregatory effects (turbidimetry) of clopidogrel were studied in human platelet-rich plasma and in washed platelets. 3. Incubation of platelet-rich plasma with clopidogrel (< or = 100 microM) for up to 8 h did not result in any inhibition of ADP (6 microM)-induced platelet aggregation. 4. Incubation of washed platelets with clopidogrel resulted in a time- (maximum effects after 30 min) and concentration-dependent (IC50 1.9+/-0.3 microM) inhibition of ADP (6 microM)-induced platelet aggregation. Clopidogrel (30 microM) did not inhibit collagen (2.5 microg ml(-1))-, U46619 (1 microM)- or thrombin (0.1 u ml(-1))-induced platelet aggregation. The inhibition of ADP-induced aggregation by clopidogrel (30 microM) was insurmountable indicating a non-equilibrium antagonism of ADP actions. The R enantiomer SR 25989 C (30 microM) was significantly less active than clopidogrel (30 microM) in inhibiting platelet aggregation (32+/-5% vs 70+/-1% inhibition, P < 0.05, n = 5). 5. In washed platelets, clopidogrel (< or = 30 microM) did not significantly reverse the inhibition of prostaglandin E1 (1 microM)-induced platelet cyclic AMP formation by ADP (6 microM). 6. The antiaggregatory effects of clopidogrel were unchanged when the compound was removed from the platelet suspension. However, platelet inhibition by clopidogrel was completely abolished when albumin (350 mg ml(-1)) was present in the test buffer. 7. It is concluded that clopidogrel specifically inhibits ADP-induced aggregation of washed platelets in vitro without hepatic bioactivation. Inhibition of ADP-induced platelet aggregation by clopidogrel in vitro occurs in the absence of measurable effects on the reversal of PGE1-stimulated cyclic AMP by ADP.     

哒嗪酮类新化合物9612的抗血小板聚集作用

研究了新型哒嗪酮类化合物 96 12对家兔和大鼠血小板聚集的影响 .结果发现 ,96 12在体外能明显抑制花生四烯酸 (AA) ,腺苷二磷酸 (ADP)和凝血酶 (thrombin)诱导的家兔血小板聚集 ,其IC50 分别为 3 2 0 ,9 44和 7 10 μmol/L .96 12 .灌胃给药 (ig) ,能显著抑制AA和ADP诱导的大鼠血小板聚集 ,其作用与同等剂量 (15 0mg/kg)的阿斯匹林 (ASA)相比无明显差异 (P >0 0 5 ) .     

Development of a small RGD peptide fibrinogen receptor antagonist with potent antiaggregatory activity in vitro

The development of potent antithrombotic agents from the fibrinogen platelet receptor binding sequences Fg-alpha 572-575 -Arg-Gly-Asp-Ser- and Fg-gamma 400-411 -HHLGGAKQAGDV, believed to be a cryptic RGD-type sequence, is described. The tetrapeptide Ac-RGDS-NH2 itself is capable of inhibiting platelet aggregation in vitro at high concentrations, IC50 91.3 +/- 0.1 microM [in vitro antiaggregatory activity employing dog platelet rich plasma (PRP)/ADP], due to low platelet fibrinogen receptor affinity, Ki 2.9 +/- 1.9 microM (purified, reconstituted human platelet GPIIb/IIIa), relative to fibrinogen, Ki 38.0 +/- 6.0 nM. The peptide is also unstable to plasma, suffering total loss of in vitro activity upon incubation in PRP for 3 h (T1/2 90 min). Only modest improvements in potency were achieved with linear analogues of Ac-RGDS-NH2, while dramatic results were achieved with cyclic analogues, culminating in the cyclic disulfide Ac-cyclo-S,S-[Cys-(N alpha-Me)Arg-Gly-Asp-Pen]-NH2 (SK&F 106760) with improved plasma stability (100% activity after 3 h), affinity (Ki 58 +/- 20 nM purified human receptor), and potency (IC50 0.36 +/- 0.4 microM dog PRP/ADP). The affinity of this peptide is 2 orders of magnitude greater than that of Ac-RGDS-NH2. The affinity of the analogue is also comparable to fibrinogen. This peptide constitutes a first potent small peptide entry into the class of novel antithrombotic agents called fibrinogen receptor antagonists.     

Inhibition of phospholipase A2 by tiaramide in rabbit platelets

The mechanism by which tiaramide inhibited platelet aggregation was investigated using phospholipid labelling techniques by 14C-arachidonic acid (AA) and thin-layer chromatography. Tiaramide did not affect cyclo-oxygenase nor thromboxane synthetase, because TXB2 was detected in tiaramide-treated platelets, unlike aspirin-treated ones, and PGE2 and PGD2 did not increase, unlike in platelets treated with OKY-1581 (an inhibitor of thromboxane synthetase). Total phospholipid radioactivity was 82.5% of radioactivity recovered before aggregation, and this decreased to 49.0% (n = 5, P less than 0.05) after aggregation by collagen (30 micrograms/ml). AA radioactivity was 9.6% before aggregation and 40.0% after. Tiaramide (100 microM) restored total phospholipid and AA levels to those before aggregation. Tiaramide decreased the amount of AA liberated from 2-(3H-arachidonyl)phosphatidylcholine by whole platelet phospholipase A2 (PLA2). Tiaramide at 10 microM inhibited collagen-induced aggregation, but not that by AA. Tiaramide did not affect 45Ca-uptake by itself nor collagen-induced 45Ca-uptake from the external medium. Tiaramide did not inhibit intracellular Ca mobilization, and it did not affect the calmodulin-dependent cyclic nucleotide phosphodiesterase of rabbit brain. These facts suggest that tiaramide inhibits platelet PLA2 through mechanisms other than the blockade of Ca-influx and intracellular Ca mobilization or antagonism to calmodulin.     

Mechanism of action of the platelet aggregation inhibitor purified from Agkistrodon halys (mamushi) snake venom

The platelet aggregation inhibitor purified from Agkistrodon halys snake venom inhibited rabbit platelet aggregations induced by thrombin, sodium arachidonate, collagen or ionophore A-23187. The ic₅₀ was about 11 μg/ml in platelet aggregation regardless of which aggregation inducer was used. β-Mercaptoethanol abolished both the phospholipase A enzymatic and platelet aggregation inhibitory activities of this venom inhibitor. p-Bromophenacyl bromide-treated venom inhibitor lost almost completely its phosphilipase A enzymatic activity, but retained its platelet aggregation inhibitory effect. In the presence of EGTA, the venom inhibitor still showed the same inhibitory activity on thrombin-, sodium arachidonate-, collagen- or ionophore A23187-induced platelet aggregations triggered by successive addition of Ca²⁺. The activation of platelet phospholipase A and the serotonin release reaction triggered by Ca²⁺ influx were unaffected by this venom inhibitor. It also inhibited the clot retraction of platelet-rich plasma. It is concluded that the inhibitory effect of the venom inhibitor on platelet aggregation is independent of its phospholipase A enzymatic activity. Its mode of action is different from those of other known platelet inhibitory drugs. This venom inhibitor possibly acts on a common step subsequent to platelet shape change, leading to inhibition of platelet aggregation.     

Effect of 7-aza- and 13-aza-prostanoids on platelet aggregation

The effects of 11 new analogues of 7-aza- and 13-azaprostanoic acid on platelet aggregation of the rat were studied in vitro and ex vivo. The analogues of 13-azaprostanoic acid in vitro (10(-7)-10(-8) mol/l) were found to exhibit the antiaggregation activity in the blood. The compounds containing five-member cycle are more active antiaggregants. They decrease by 70-80% the degree of platelet aggregation stimulated by thrombin and ADP. The modification of omega-chain of these analogues (alkyl, phenyl) exerts no effect on the antiaggregation activity. The antiaggregation action of these compounds in ex vivo condition (5-10 micrograms/kg) does not preserve. 7-azaprostanoids possess the proaggregation activity in vitro.     

Effects of the calcium antagonist nifedipine on thromboxane B2 level and platelet aggregation in hypertensive patients

The effects of a calcium antagonist nifedipine (Adalat) on platelet aggregation was examined in vitro and in vivo. In vitro examination: Platelet aggregation induced by adenosine disphosphate (ADP), epinephrine, collagen, arachidonate, and thrombin was all inhibited dose-dependently in the platelet-rich plasma prepared from the blood of healthy individuals by the addition of nifedipine to a final concentration of 5 or 10 micrograms/ml. In vivo examination: 20 patients with essential hypertension were treated with 30 mg/d of nifedipine for 8 weeks. Significant decreases in both systolic and diastolic pressures were observed 2 weeks after the beginning of the administration, and continued throughout the administration period. ADP-induced platelet aggregation decreased by 25% after 2 weeks, 36% after 4 weeks, and 44% after 8 weeks (p less than 0.05 for all decreases). Plasma thromboxane B2 level also decreased markedly from 217.3 +/- 91.7 pg/ml before the administration to 119.0 +/- 29.7 pg/ml (p less than 0.01) 2 weeks after, and 99.1 +/- 25.4 pg/ml (p less than 0.01) 8 weeks after the beginning of the administration, suggesting suppressed thromboxane A2 production.