Rabbit Platelets Research Articles

The mediation of platelet quiescence by NO-releasing polymers via cGMP-induced serine 239 phosphorylation of vasodilator-stimulated phosphoprotein

Nitric oxide (NO) releasing (NORel) materials have been shown to create localized increases in NO concentration by the release of NO from a diazeniumdiolate-containing or S-nitrosothiol-containing polymer coating and the improvement of extracorporeal circulation (ECC) hemocompatibility. However, the mechanism and, in particular, the platelet upregulation of the NO/cGMP signaling protein, vasodilator-stimulated phosphoprotein phosphorylated at serine 239 (P-VASP (ser 239)), for the improved ECC hemocompatibility via NO release still needs elucidation. In this work, two NORel polymeric coatings were evaluated in a 4 h rabbit thrombogenicity model and the anti-thrombotic mechanism investigated for rabbit platelet P-VASP upregulation. Polymer films containing 25 wt% diazeniumdiolated dibutylhexanediamine (DBHD) or 5 wt% S-nitroso-N-acetylpenicillamine (SNAP) coated on the inner walls of ECC circuits yielded significantly reduced ECC thrombus formation and maintained normal platelet aggregation compared to polymer controls after 4 h of blood exposure. Platelet P-VASP (ser 239), a useful tool to monitor NO/cGMP signaling, was upregulated after 4 h on ECC and markedly increased after ex vivo sodium nitroprusside (SNP) stimulation. Interestingly, in the rabbit platelet, NO did not upregulate the cAMP P-VASP phosphoprotein P-VASP (ser 157) as previously shown in human platelets. These results suggest that NORel polymers preserve rabbit platelet quiescence by sustaining a level of cGMP signaling as monitored by P-VASP (ser 239) upregulation. The upregulation of this NO-mediated platelet signaling mechanism in this rabbit thrombogenicity model indicates the potential for improved thromboresistance of any NORel-coated medical device.

Anti-platelet-activating factor, antibacterial, and antiradical activities of lipids extract from silver carp brain

BackgroundEpidemiological studies have verified the protective role of fish lipids in cardiovascular diseases. However, the effects of fish lipids on health boost remain undefined. Large amounts of by-products, such as fish brain which contains high level of lipids, are produced with silver carp processing. Fish brain is rich in bioactive lipids which are overwhelmingly effective in preventing cardiovascular diseases. The aim of this study was to elucidate the pharmacological activities of silver carp brain lipids against diseases by inhibiting platelet-activating factor (PAF), suppressing bacterial growth and scavenging free radicals.MethodsTotal lipids (TL) were extracted from silver carp brain and separated into polar lipids (PL) and neutral lipids (NL). The capabilities of the lipid fractions in aggregating washed rabbit platelet or in inhibiting PAF-induced platelet aggregation were tested. Their antibacterial and antiradical activities were studied as well.ResultsThe lipid fractions exhibited strong inhibitory activities, and the activity of TL was mainly attributed to NL. TL exhibited antibacterial activity towards Staphylococcus aureus, while NL managed to fight against S. aureus and Escherichia coli. PL excelled TL and NL in simultaneously suppressing the growths of Shigella dysenteriae and Salmonella typhi besides those of S. aureus and E. coli. The scavenging effect of PL on 2,2-diphenyl-1-picrylhydrazyl radical was considerably higher than those of TL and NL.ConclusionThe present study may help to explain the protective role of fish lipids against diseases and may be responsible for the effectiveness of fish brain in benefiting health.

Blood Platelet Insulin sensitivity in Atherogenic Dieted Rabbits Fed Fish Oils with Comparable Eicosapentanoic Acid Content

The effects of two fish oils added to an atherogenic diet fed to New Zealand strain, male rabbits were studied for 9 weeks. Cod liver oil (CLO) containing 8–10% eicosapentaneoic acid (EPA) or MaxEPA, a concentrated mix of sardine and anchovie oil, with 18–20%EPA, was added to the atherogenic diet (AD) of 2% Cholesterol, 6% Corn Oil and 92% Purina Rabbit Chow and fed to the two experimental animal groups. There were 3 groups of 5 rabbits in the study: an ADC control group fed AD alone, an AD/CLO group fed AD with 8% CLO added and an AD/MaxEPA group fed AD with 4% MaxEPA. Blood Plasma Cholesterol assay mean values were 862mg/dl for ADC, 549mg/dl for AD/CLO and 781mg/dl in AD/MaxEPA animals and for blood platelets were 461mg/dl for ADC, 277mg/dl in AD/ CLO and 440mg/dl in AD/MaxEPA platelets. Plasma plasminogen activator activity (PAA) assays yielded mean %fibrinolysis values of 17.4% in ADC, 20.2% in AD/CLO and 18.2 %. in AD/ MaxEPA plasma . Platelet insulin sensitivity assays found glucose levels of 285.8mg% in ADC, 345.3 mg% in AD/CLO and 342.6 mg% in AD/MaxEPA platelets. Insulin sensitivity increased comparably in platelets of rabbits fed AD/CLO or AD/MaxEPA with the same EPA content. Both fish oils yielded moderately reduced blood plasma and platelet cholesterol and increased blood PAA compared to ADC tissues. At autopsy, aortae from rabbits fed AD/CLO or AD/ MaxEPA showed reduced atherosclerotic plaque development vs. ADC aortae. Funded by Louisiana Heart Association

Antithrombotic activity of HY023016, a novel Dabigatran prodrug evaluated in animal thrombosis models

IntroductionThrombin is a multifunctional trypsin-like serine protease that plays key roles in coagulation and thrombogenesis. HY023016, a novel Dabigatran prodrug, is an oral direct thrombin inhibitor. The purpose of this study was to compare the anti-thrombotic activities and haemorrhagic effects of HY023016 with Dabigatran etexilate and tetramethylpyrazine in several animal thrombosis models. MethodsTo investigate drug exposure, liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was used to determine the pharmacokinetic profile of HY023016. After single intragastric administrations of HY023016, Dabigatran etexilate or tetramethylpyrazine, the anti-thrombotic activities were evaluated through rabbit jugular vein thrombosis model, rat inferior vena cava thrombosis model, ex vivo rabbit platelet aggregation assay, in vivo rabbit coagulation assay, and direct thrombin binding assay. Meanwhile, we evaluated the effect of HY023016 on expression of tissue factor (TF) by RT-PCR. Rabbit cuticle bleeding assay and mouse tail bleeding assay were applied to evaluate the effects of HY023016 on haemorrhage. ResultsPharmacokinetic parameters indicated that HY023016 can convert to Dabigatran and tetramethylpyrazine. Our studies showed that HY023016 was able to significantly inhibit thrombus formation in a dose-dependent manner in rabbit and rat models (P<0.05). Similarly, it was able to dose-dependently inhibit thrombin- or ADP-induced platelet aggregation, prolonging the activated partial thromboplastin time (APTT) and prothrombin time (PT), inhibiting the activity of thrombin and inhibiting thrombin- or ADP-induced expression of TF (P<0.05 or 0.01). Dabigatran etexilate was also able to dose-dependently and significantly inhibit thrombus formation (P<0.01) but was unable to affect ADP-induced platelet aggregation and expression of TF. In contrast, tetramethylpyrazine could only exhibit mild antithrombotic activity compared with HY023016 and Dabigatran etexilate (P<0.05). HY023016 could prolong bleeding time (P<0.001), but the prolongations were significantly less than Dabigatran etexilate (P<0.05). ConclusionHY023016 showed thrombosis-inhibition activities comparable to those of Dabigatran etexilate, but better than those of tetramethylpyrazine. The attendant bleeding risk of HY023016 was lower than Dabigatran etexilate in rabbits and mice.

Cholesterol enrichment of rabbit platelets enhances the Ca2 + entry pathway induced by platelet-derived secondary feedback agonists

AimsHypersensitivity of platelets due to increased platelet cholesterol levels has been reported in hypercholesterolemia. However, the signaling pathways linking increased platelet reactivity and cholesterol contents are not fully understood. This study aims to determine the direct effect of cholesterol enrichment of platelets on the pathways including Ca2+ mobilization and secondary feedback agonists such as adenosine diphosphate (ADP) and thromboxane A2 (TXA2). Main methodsIn vitro cholesterol enrichment of rabbit platelets was performed by incubation with cholesterol complexed with methyl-β-cyclodextrin. Ca2+ mobilization was monitored using platelets loaded with fura-PE3/AM, a fluorescent calcium indicator. Released ATP and TXB2 from platelets were measured by a luciferin–luciferase ATP assay system and a TXB2 ELISA Kit, respectively. Key findingsCholesterol enrichment of rabbit platelets significantly enhanced Ca2+ mobilization induced by thrombin, accompanying an augmented Ca2+ entry. The augmentation of Ca2+ entry by cholesterol enrichment was significantly suppressed by treatment with inhibitors for secondary feedback agonists. In cholesterol-enriched platelets, the amount of released ATP or TXB2 induced by thrombin was not significantly altered in comparison with control platelets, whereas an increase in [Ca2+]i induced by ADP or U46619, a TXA2 mimetic, was significantly enhanced. SignificanceThese results suggest that cholesterol enrichment of rabbit platelets results in enhanced Ca2+ mobilization via ADP/TXA2-dependent augmentation of the Ca2+ entry pathway. The results reveal a novel mechanism by which platelet hypersensitivity is regulated by cholesterol contents.

Effects of Imidazoline and Non-Imidazoline α-Adrenergic Agents on Rabbit Platelet Aggregation

Background/Aims: Imidazoline α₂-adrenergic agents exert complex effects on mammalian platelet aggregation. Although non-adrenergic, imidazoline (I) receptors have been revealed in human platelets, there is limited information about imidazoline’s action on platelet aggregation. This study aimed to investigate aggregatory and anti-aggregatory effects of various imidazoline or non-imidazoline α-adrenergic agents on rabbit platelets. Methods: Aggregatory responses of agents on rabbit platelets were examined by turbidimetric method. Radioligand binding assay to platelet I₁ and I₂ receptors was performed using [³H]-clonidine and [³H]-idazoxan, respectively. Results: Aggregation was not induced by α-adrenoceptor agonists alone. Adrenaline and noradrenaline produced dose-dependent potentiation of ADP- or collagen-induced aggregation. Imidazoline adrenoceptor agonists clonidine and p-aminoclonidine also potentiated ADP-induced platelet aggregation. The α₂-adrenoceptor antagonists and/or certain imidazoline adrenergic agents inhibited adrenaline-potentiated aggregation in a dose-dependent manner, whereas α₁-adrenoceptor antagonists and non-imidazoline α-adrenergic agents were either ineffective or less effective in inhibiting adrenaline-potentiated aggregation. Rabbit platelets did not have I₁ receptors, but had I₂ receptors, indicating that adrenaline-potentiated platelet aggregation was inhibited by idazoxan, but not by imidazoline compounds clonidine and oxymetazoline. Conclusions/Implications: These results demonstrated that α₂-adrenoceptor-blocking agents and/or imidazoline α-adrenergic agents effectively inhibit adrenaline-potentiated platelet aggregation. It is proposed that imidazoline structure in part plays a role in the inhibition of adrenaline-potentiated aggregation.

Inhibition by Hydrogen Sulfide of Rabbit Platelet Aggregation and Calcium Mobilization

Hydrogen sulfide (H2S), a gasotransmitter, plays a variety of roles in the mammalian body including the cardiovascular system. Given evidence that H2S donors including NaHS inhibit human platelet aggregation, we examined and characterized the effects of NaHS on rabbit platelet aggregation and cytosolic Ca(2+) mobilization. Rabbit platelet aggregation was determined in platelet-rich plasma (PRP) and washed platelets. Intracellular Ca(2+) levels were monitored in Fura2-loaded washed platelets. NaHS prevented rabbit platelet aggregation induced by collagen or ADP, and the effective concentration range of NaHS was 0.1-0.3 mM in PRP and 1-3 mM in washed platelets. In washed platelets, NaHS attenuated cytosolic Ca(2+) mobilization induced by collagen or ADP and also reduced platelet aggregation induced by ionomycin, a Ca(2+) ionophore. The anti-platelet effect of NaHS was blocked by an adenylyl cyclase inhibitor and enhanced by a phosphodiesterase inhibitor. H2S thus suppresses rabbit platelet aggregation by interfering with both upstream and downstream signals of cytosolic Ca(2+) mobilization in a cAMP-dependent manner.

Anti-platelet activity of diacetylated obovatol through regulating cyclooxygenase and lipoxygenase activities

Obovatol has been reported biological activities such as muscle relaxative, anti-gastric ulcer, anti-allergic and anti-bacterial activities. The present study was undertaken to investigate the effect of diacetylated obovatol, an obovatol derivative, on rabbit platelet aggregation, and their possible molecular mechanisms. Effects of diacetylated obovatol on platelet activation including aggregation and serotonin secretion were examined. In addition, we investigated the effect of diacetylated obovatol on archidonic acid and metabolites liberation and intracellular calcium mobilization. Diacetylated obovatol concentration-dependently inhibited the washed rabbit platelet aggregation induced by collagen and arachidonic acid, suggesting that diacetylated obovatol may selectively inhibits collagen- and arachidonic acid-mediated signal transduction. In accordance with these results, diacetylated obovatol showed a concentration-dependent decrease in cytosolic Ca(2+) mobilization and serotonin secretion. However, diacetylated obovatol did not inhibit arachidonic acid liberation; on the other hand, diacetylated obovatol inhibited the formation of arachidonic acid metabolites such as thromboxane A(2), prostaglandin D(2) and 12-HETE through interfering with cyclooxygenase (COX)-1 and lipoxygenase (LOX) activities. The results demonstrated that diacetylated obovatol has antiplatelet activities through inhibition of COX-1 and LOX activities.

JJK694, a Synthesized Obovatol Derivative, Inhibits Platelet Activation by Suppressing Cyclooxygenase and Lipoxygenase Activities

Obovatol has various biological activities, including anti-proliferative, neurotrophic, anti-fibrillogenic, anti-platelet, anti-fungal and anti-inflammatory activities. In this study, we investigated the effects of JJK694, a synthesized obovatol derivative, on rabbit platelet activation and its molecular mechanisms. JJK694 significantly inhibited washed rabbit platelet aggregation and serotonin secretion induced by collagen and arachidonic acid, but had little effect on thrombin- or U46619-induced aggregation. These results suggest that JJK694 selectively inhibits collagen- and arachidonic acid-mediated signaling. JJK694 also showed a concentration-dependent decrease in cytosolic Ca(2+) mobilization, but it had no effect on arachidonic acid liberation. On the other hand, it significantly inhibited the formation of arachidonic acid metabolites, including thromboxane A(2) (TXA(2)), prostaglandin D(2), and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), by suppression of cyclooxygenase (COX)-1 and lipoxygenase (LOX) activities. These results indicate that JJK694 hasanti-platelet activities through inhibition of arachidonic acid metabolite production by suppression of COX-1 and LOX activities.

Platelet-Activating Factor (PAF) Antagonistic Activity of a New Biflavonoid from Garcinia nervosa var. pubescens King

The methanol extract of the leaves of Garcinia nervosa var. pubescens King, which showed strong inhibitory effects on platelet-activating factor (PAF) receptor binding, was subjected to bioassay-guided isolation to obtain a new biflavonoid, II-3,I-5, II-5,II-7,I-4',II-4'-hexahydroxy-(I-3,II-8)-flavonylflavanonol together with two known flavonoids, 6-methyl-4'-methoxyflavone and acacetin. The structures of the compounds were elucidated by spectroscopic methods. The compounds were evaluated for their ability to inhibit PAF receptor binding to rabbit platelets using 3H-PAF as a ligand. The biflavonoid and acacetin showed strong inhibition with IC50 values of 28.0 and 20.4 µM, respectively. The results suggest that these compounds could be responsible for the strong PAF antagonistic activity of the plant.

I4, a new synthetic sulfonylurea compound, inhibits the action of TXA2in vivo and in vitro on platelets and aorta vascular smooth muscle

Introduction1-[4-[2-(4-Bromobenzene-sulfonamino)ethyl]phenylsulfonyl]-3-(trans-4-methylcy-clohexyl)urea(I4, CAS865483-06-3); a totally synthetic new sulfonylurea compound, combining the hypoglycemic active structure of Glimepiride (CAS 93479-97-1) and anti-TXA2 receptor (TP) active structure of BM-531(CAS 284464-46-6), was designed and synthesized. Its effects on TXA2 synthesis and TP have not been reported yet. AimTo study the inhibitory effects of I4 and its mechanisms of action on TXA2 and TP. MethodsPlatelet aggregation studies were performed on human platelet, rat whole blood platelet and rabbit platelet, platelets aggregation was induced by TP agonist U-46619(stable analog of TXA2, CAS 56985-40-1). Plasma TXB2 and 6-keto-prostaglandin F1α (6-keto-PGF1α) were used as markers to determine the effect of I4 on thromboxane synthesis. Fluo-3-AM was used to measure the cytosolic Ca2+ concentrations ([Ca2+]i) in rabbit platelet. Aorta rings with and without endothelium were prepared and aorta contraction was induced by U-46619. A model of type 2 diabetes mellitus was established by intraperitoneal injection of low dose of streptozocin to rats fed a high-calorie diet. Both normal rats and type 2 diabetic rats were used to assay the inhibitory effect of I4 on platelet aggregation induced by U-46619. ResultsI4 exhibited a higher inhibitory potency than Glimepiride on U-46619 induced platelet aggregation in vitro and in vivo. I4 increased the ratio of plasma PGI2/TXA2 and decreased [Ca2+]i release from platelet internal stores. In addition, I4 presented a vasorelaxant activity on isolated rat aorta contraction induced by U-46619.Oral administration of I4 (1~10mg/kg) markedly and dose-dependently inhibited platelet aggregation in both normal rats and type 2 diabetic rats. ConclusionI4 significantly inhibited platelet aggregation induced by U-46619 in vitro and in vivo, and rat aorta contraction. It probably acts by partly blocking TXA2 action, decreasing the platelet intracellular Ca2+, and increasing the PGI2/TXA2 ratio.

Platelet activating factor levels and metabolism in tangier disease: a case study

BackgroundTangier disease (TD) is a phenotypic expression of rare familial syndrome with mutations in the ABCA1 transporter. The risk of coronary artery disease in patients with TD is variable. On the other hand the pivotal role of Platelet-Activating Factor (PAF) mediator in atheromatosis was found. Plasma lipoproteins are transporters of the PAF acetylhydrolase (PAF-AH) in cells and known as lipoprotein-phospholipase A2 (Lp-PLA2) in plasma and regulators of PAF levels in blood. In addition, PAF can be biosynthesized from the remodeling and the de novo pathways in which Lyso-platelet activating factor-acetyltransferase (Lyso-PAF-AT) and platelet activating factor-cholinephosphotransferase (PAF-CPT) are the regulatory enzymes. The aim of this study is to investigate in a TD patient with a unique mutation (C2033A), the concentration of PAF in blood, the Equivalent Concentration for 50% aggregation (EC50) values of platelet rich plasma (PRP) toward PAF, adenosine diphosphate (ADP) and thrombin, and the activities of PAF metabolic enzymes Lp-PLA2, PAF-AH, Lyso-PAF-AT and PAF-CPT.MethodsThe EC50 value of PRP was measured by an aggregometer. The determination of the specific activity of PAF-CPT and Lyso-PAF-AT was made after in vitro enzymatic assay, chromatographic separation and measurement of the produced PAF in a biological assay with washed rabbit platelets. The determination of PAF-AH and Lp-PLA2 was made after an in vitro enzymatic assay from the decay of radioactive PAF.ResultsThe TD patient had lower bound-PAF values in blood, decreased specific activity of PAF-CPT and Lyso-PAF-AT, increased specific activity of PAF-AH in platelets and leukocytes and Lp-PLA2 activity in plasma compared to healthy women. The EC50 of PAF and Thrombin were higher compared to healthy women.ConclusionThe increased Lp-PLA2 activity, as well as, the decreased activities of PAF-CPT and Lyso-PAF-AT, explain the decreased bound-PAF level in TD patient and the EC50 of PAF. However, total PAF is in a normal range and this probably can explain one of the reasons this TD patient has no CAD.

Inhibitory Effects of Acetylmelodorinol, Chrysin and Polycarpol from Mitrella kentii on Prostaglandin E2 and Thromboxane B2 Production and Platelet Activating Factor Receptor Binding

Acetylmelodorinol, chrysin and polycarpol, together with benzoic acid, benzoquinone and stigmasterol were isolated from the leaves of Mitrella kentii (Bl.) Miq. The compounds were evaluated for their ability to inhibit prostaglandin E2 (PGE2) and thromboxane B2 (TXB2) production in human whole blood using a radioimmunoassay technique. Their inhibitory effect on platelet activating factor (PAF) receptor binding to rabbit platelet was determined using 3H-PAF as a ligand. Among the compounds tested, chrysin showed a strong dose-dependent inhibitory activity on PGE2 production (IC50 value of 25.5 µM), which might be due to direct inhibition of cyclooxygenase-2 (COX-2) enzymatic activity. Polycarpol, acetylmelodorinol and stigmasterol exhibited significant and concentration-dependent inhibitory effects on TXB2 production with IC50 values of 15.6, 19.1 and 19.4 µM, respectively, suggesting that they strongly inhibited COX-1 activity. Polycarpol and acetylmelodorinol showed strong dose-dependent inhibitory effects on PAF receptor binding with IC50 values of 24.3 and 24.5 µM, respectively.

Effects of SOD/PEG and soy flour on insulin sensitivity of blood platelets of atherogenic dieted rabbits

The effects of antioxidant superoxide dismutase (SOD) injections and of SOD rich soybean flour were studied in New Zealand strain, male rabbits. The animals were fed an atherogenic diet (AD) of 2% Cholesterol, 6% Corn Oil and 92% Purina Rabbit Chow, for 5 weeks prior to starting the experiments using 3 experimental groups of 5 animals. Group I AD Controls –theADC group‐fed AD, Group II fed AD and injected with 2,000units SOD‐PEG −2 x/week‐IP‐ (approximately 11,000 units/gm hemoglobin) ‐ the AD‐SOD group, and Group III was fed AD with 5% SOD rich soybean flour added‐the SF+AD group. The treatments lasted for 5 additional weeks before the animals were sacrificed. Sensitivity to insulin was measured in blood platelets in each group by assaying their glucose uptake following treatment with insulin. Blood plasma and platelets were also assayed for cholesterol and phospholipid. levels. Mean glucose assays of insulin treated platelets from AD‐SOD rabbits measured 8.2 mg/dl of glucose vs 4.5 mg/dl from ADC .platelets‐an 82.2% increase. SF+AD platelets registered a 10.1% increase in insulin sensitivity vs ADC samples. Mean cholesterol levels ( mg/dl ) in blood platelets diminished by 18.8% in the AD‐SOD group and 10.7% in the SF+AD platelets compared to the ADC values. Mean plasma cholesterol levels fell by 16.8% in the AD‐SOD and 19.1% in SF+AD vs ADC plasma. Mean phospholipid levels (mg%) showed a decrease of 12.5% in AD‐SOD platelets and 9.2% in SF+AD platelets vs those in ADC assays. Mean phospholipid levels also fell in AD‐SOD by 17.3% and by 15.8% in SF+AD vs the ADC control values.

Effects of Danhong mixture on platelet aggregation of rabbits

Objective To study the effects of Danhong mixture on platelet aggregation of rabbits.Methods 40 rabbits were randomly divided into 5 groups,8 rabbits in each group,of blank control group (treated with 0.5％ sodium carboxy methyl cellulose of the same volume),Danhong mixture of 5,2.5,1.25 g/kg group,aspirin group (treated with 0.025 g/kg aspirin).Each group was performed gastric lavage according to the ration of lml/kg,once daily,for continuous 8 days.Blood was obtained from heart 1 hour after last administration,then adenosine diphosphate (ADP),collagen,arachidonic acid (AA) were used for platelet aggregation test.Results Inhibition rate of Danhong mixture (5,2.5,1.25 g/kg) on ADP induced rabbit platelet aggregation were 9.4％,5.1％,and 0.6％ respectively,on collagen induced platelet aggregation were 34.5％,11.1％,and 5.6％; on AA induced platelet aggregation were 21.4％,11.6％,and 2.3％ respectively.Conclusion Danhong mixture had inhibitive action on platelet aggregation. Key words: Danhongmixture; Plateletaggregation; ADP; Collagen; AA

Effect of freezing on quality of sea bass and gilthead sea bream

AbstractFreezing is an efficient method of fish preservation. The aim of the present work was to examine the impact of freezing in fatty acid composition and in the in vitro inhibitory activity of sea bass (Dicentrarchus labrax) and gilthead sea bream (Sparus aurata) fillet lipids against platelet activating factor (PAF). The Bligh and Dyer extraction method and the counter‐current distribution method were used to obtain total, polar and neutral lipids. The fatty acid analysis conducted using the internal standard method and the biological assay on washed rabbit platelets took place calculating the in vitro inhibitory activity of fish lipids against 2.5 × 10−11 M of PAF. No statistical changes (p&lt;0.05) occurred in fatty acid content of fresh and thawed gilthead sea bream, while fatty acid amount in thawed sea bass was significantly higher (p&lt;0.05) compared to fresh fish. Total lipids of both thawed fish species exhibited stronger anti‐thrombotic activity compared to fresh fish. Freezing preserved fish quality and reinforced the anti‐thrombotic properties of fish oils, since even after 6 months of freezing, fish oils preserve their nutritional value in terms of protecting against cardiovascular diseases.Practical applications: Fish fillets contain high amount of unsaturated lipids that may easily undergo lipid oxidation. Freezing and frozen storage prevent such oxidative changes so fish quality is retained. Fatty acids and PAF‐antagonists in fish are of major importance since they contribute to the nutritional value of fish. The practical application of this work lies on the evaluation of the nutritional value of fish in terms of cardio protection by examining the impact of freezing on the levels of fatty acids and PAF‐antagonists in aquacultured fish fillets.

Отримання та характеристика інгібітора агрегації тромбоцитів із отрути Gloydius blomhoffii brevicaudus

Gloydius venom is a rich natural source of proteins possessing platelet aggregation inhibition activity. These proteins or their recombinant analogs are universal instruments for the investigation of proteins and receptors interaction in hemostatic system. At the same time previously studied and potentially new pharmacological properties of platelet aggregation inhibitors from snake venoms create a background for the development of the manufacturing technologies. This study presents a part of technological development of several target proteins parallel obtaining from G. blomhoffii brevicaudus snake venom, in particular deve­lop­ment of manufacturing steps for platelet aggregation inhibitor.The target protein was purified using three different chromatographic approaches. It was shown that it is a single chain protein with molecular weight 12001.55 Da, isoelectrical point 8.03, which inhibited platelet aggregation in the rabbit platelet rich plasma with ID 50 = 3.26×10 -6 g or 542×10 -9 М and revealing no enzyme activity. Keywords: snake venom, G. blomhoffii brevicaudus , platelet aggregation inhibitor, protein chromatography.

Morusinol Extracted from &lt;i&gt;Morus Alba&lt;/i&gt; Inhibits Arterial Thrombosis and Modulates Platelet Activation for the Treatment of Cardiovascular Disease

Morus alba (white mulberry) has been used in traditional Chinese medicine as an anti-headache, diuretic, expectorant, and anti-diabetic agent. In previous studies, extracts of Morus alba demonstrated favorable biological properties, such as antioxidant activity, suppression of lipoxygenase (LOX)-1, cytotoxicity against cancer cells, and inhibition of the invasion and migration of cancer cells. This study further evaluated the effects of morusinol, a flavonoid derived from Morus alba root bark, on platelet aggregation and thromboxane B(2) (TXB(2) formation in vitro and thrombus formation in vivo. The antiplatelet potential of morusinol was measured using in vitro rabbit platelet aggregation and TXB(2) formation assays. Arterial thrombus formation was investigated using an in vivo ferric chloride (FeCl(3)-induced thrombosis model. Morusinol significantly inhibited collagen- and arachidonic acid-induced platelet aggregation and TXB(2) formation in cultured platelets in a concentration-dependent manner. Thrombus formation was reduced by 32.1, 42.0, and 99.0% for collagen-induced TXB(2) formation, and 8.0, 24.1, and 29.2% for arachadonic acid-induced TXB(2) formation, with 5, 10, and 30 µg/mL morusinol, respectively. Moreover, oral morusinol (20 mg/kg) or aspirin (20 mg/kg) for three days significantly increased the time to occlusion in vivo by 20.3±5.0 or 6.8±2.9 min, respectively, compared with the control (1% CMC, carboxymethyl cellulose). Taken together, these results indicate that morusinol may significantly inhibit arterial thrombosis in vivo due to antiplatelet activity. Thus, morusinol may exert beneficial effects on transient ischemic attacks or stroke via the modulation of platelet activation.

Platelet‐activating factor detection, metabolism, and inhibitors in the ethanologenic bacterium Zymomonas mobilis

AbstractPlatelet‐activating factor (PAF) is a signaling phospholipid with a significant physiological role in multicellular and unicellular organisms, including fermentative organisms such as yeast. Zymomonas mobilis is an ethanologenic α‐proteobacterium currently studied for bioethanol production. In order to examine the presence of PAF and/or PAF inhibitors in Z. mobilis, a new one‐step high performance liquid chromatography (HPLC) separation procedure of total lipids was performed, using a C8 reversed‐phase semi‐preparative column. According to this method and to bioassays based on washed rabbit platelet aggregation, two lipid molecules with PAF‐like activity and same retention times as those of standard PAF were detected; electron‐spray ionization MS and MS/MS analysis revealed that they share similar structure with 16:0 and 18:0 PAF. Furthermore, other lipids extracted from Z. mobilis were found to exhibit a potent anti‐PAF activity. Enzyme activities indicative of key PAF biosynthetic enzymes, such as dithiothreitol‐insensitive cholinephosphotransferase (PAF‐CPT) and lyso‐PAF acetyltransferase were detected in Z. mobilis homogenates. As for PAF degradation, activity similar to that of PAF acetylhydrolase was also discovered. Overall, the presence of PAF, PAF‐specific inhibitors, and enzyme activities relating to PAF metabolism, suggests that PAF may play an intrinsic role in this biotechnological organism.Practical applications: Z. mobilis is a platform microorganism for bioethanol production and a potential source of high‐value chemicals of interest to the food and healthcare industries. Further investigation of PAF's role is bound to affect applications involving this and other biotechnological organisms. The finding that Z. mobilis lipids exhibit potent anti‐PAF activity opens up prospects for their identification, overproduction and pharmaceutical use. The presented HPLC method for lipid fractionation accomplishes a one‐step separation of lipids from dense samples, which may be successfully employed to other lipid‐rich sources such as blood.

Platelet-activating factor (PAF) receptor binding activity of the roots of Enicosanthellum pulchrum

Context: Enicosanthellum pulchrum (King) Heusden (Annonaceae) is a coniferous tree that is confined to mountain forests. The chemical constituents of this species have been studied previously; however, its biological activity has never been investigated before and is reported here for the first time.Objective: The extracts, fractions and compounds from the roots of E. pulchrum were investigated for their inhibitory effects on platelet-activating factor (PAF) receptor binding to rabbit platelets using 3H-PAF as a ligand.Materials and methods: The PAF receptor binding inhibitory effect using rabbit platelets was determined in vitro by measuring the difference between total amount of bound 3H-PAF in the presence and the absence of excess unlabelled PAF. The compounds were isolated by bioassay-guided fractionation and their structures were elucidated by spectroscopic techniques.Results and discussion: Among the extracts tested, the ethyl acetate extract was the most active with 85.6% inhibition, while hexane and methanol extracts showed 40.2 and 42.5% inhibition, respectively. Fractionation of the ethyl acetate extract using vacuum liquid chromatography (VLC) yielded six fractions AEA(I-–VI). Chromatography fraction AEA(VI) yielded a new compound, 1-(2′,3′,4′-trimethoxyphenyl)hexan-1-ol, while fraction AEA(III) afforded three compounds, namely liriodenine, cleistopholine and dehydroanonaine. 1-(2′,3′,4′-Trimethoxyphenyl)hexan-1-ol, cleistopholine and dehydroanonaine showed relatively strong inhibition with IC50 values of 26.6, 50.2 and 45.4 µM, respectively.Conclusion: The results suggest that these compounds could be responsible for the PAF antagonistic activity of the ethyl acetate extract of this plant.