Platelet-activating Factor Induced Platelet Aggregation Research Articles

Aqueous Extract of Malus domestica Inhibits Human Platelet Aggregation Induced by Multiple Agonists

Cardiovascular diseases such as hypertension, myocardial infarction, and atherosclerosis are the major cause of morbidity and mortality in humankind and not only afflict industrialized nations but also affect developing countries such as Pakistan. We attempted to screen one of the most common fruits, Malus domestica (MD or apple), used in Pakistan for its potential cardio-protective effects. The crude aqueous extract (MD-Aq) was prepared and investigated for inhibitory activity against several important cardiovascular drug targets. Activities of antioxidant enzymes such as glutathione peroxidase (GPx) and superoxide dismutase (SOD) were studied, as well as total antioxidant status (TAS). Calcium channel blocking activity was measured using guinea pig ileum in an isolated tissue bath setup. Platelet aggregation studies were carried out using a dual-channel aggregometer. MD-Aq showed dose-dependent improvement in the GPX activity. The highest SOD levels were observed with 10 mg/ml and were 185 U/L compared to 174 U/L for saline, which means no significant effect by MD-Aq. TAS levels were highest at 10 mg/ml of MD-Aq, reaching 1.75 mmol/L of plasma. At 10 mg/ml, close to 90% inhibition of KCl-induced opening of calcium channels was observed, compared with the effect of 1mM Verapamil. At each dose of 1, 5, and 10 mg/ml, a significantly higher arachidonic acid-induced inhibition of platelet aggregation was observed as compared to the saline effect. MD-Aq effects on platelet-activating factor-induced platelet aggregation were less potent. We concluded that MD-Aq could elevate GPX and TAS levels, is inactive at SOD, and is excellent as a calcium channel blocker, a more potent blocker of arachidonic acid-induced aggregation than platelet-activating factor-induced aggregation. These activities can help treat various cardiovascular diseases.

Cardioprotective Effects of Malus Domestica (Apple) May be Mediated Through Antiplatelet, Calcium Channel Blocking, and Antioxidant Properties

Research on the cardioprotective potential of Malus domestica (MD or apple) has been of considerable importance and efforts are being increasingly focused on investigating their effects on human health. However, relatively little work has been done to investigate the cardioprotective effects of crude extract of MD. In the present study, the antioxidant potential of crude methanolic extract of MD fruit part was investigated by measuring its effects on glutathione peroxidase (GPx), superoxide dismutase (SOD) enzymes, and total antioxidant status (TAS) in blood samples. Anti-platelet and calcium channel blocking activities were also investigated. Our results reveal that crude methanolic extract of MD showed a significant concentration-dependent increase in GPx, and SOD, but not in TAS compared with saline solution (0.9% NaCl) used as negative control and vitamin C, as a positive control. Furthermore, crude extract of MD inhibited calcium channels and showed strong antiplatelet effects against arachidonic acid and platelet-activating factor-induced platelet aggregation. These results suggest that the cardioprotective effects of MD may be mediated through multiple pathways including calcium antagonizing, antiplatelet, and antioxidant properties.

The in vitro antithrombotic properties of ale, lager, and stout beers

The in vitro antithrombotic properties of polar lipid microconstituents of three Irish beers (ale, larger, and stout) have been assessed against platelet-activating factor-induced platelet aggregation. Total lipid (TL) extracts were obtained using the Bligh and Dyer method and each beer was separated into the organic phase and hydromethanolic phase. The total polar lipid (TPL) and total neutral lipid (TNL) fractions of the organic phase and hydromethanolic phase of each beer were obtained using a counter-current distribution. The TL, TNL, and TPL extracts of each phase of each beer were measured for their capacity to inhibit platelet-activating factor-induced platelet aggregation. The low IC50 values obtained suggest that all three beers contain potent polar lipids that can inhibit platelet-activating factor-induced platelet aggregation. The fatty acid composition of the organic phase TPL extracts were measured using gas chromatography-mass spectrometry. The organic phase total polar lipid extracts were further fractionated using thin-layer chromatography (TLC) and the antithrombotic bioactivities of these TLC bands were measured. This data suggested that the TLC bands corresponding to phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, and cardiolipin were the most active phospholipids in the total polar lipids of the organic phase. These promising results indicate that beer consumption may provide antithrombotic effects.

Ιn Vivo Effects of Tenofovir-DF/Emtricitabine and Abacavir/Lamivudine with Atazanavir-R on Platelet Activating Factor Metabolism in HIV Naive Patients

Antiretroviral therapy (ΑRT) has successfully decreased AIDS morbidity and mortality and increased the lifespan of HIV patients to several decades. However, numerous factors contribute with unknown mechanisms to chronic immune activation and inflammation leading to severe “non-AIDS morbidities’’. Platelet Activating Factor (PAF) is a potent lipid inflammatory mediator with important role in the ‘’non-AIDS morbidities’’. The purpose of this study was to investigate whether tenofovir-DF/emtricitabine and abacavir/lamivudine with atazanavir boosted ritonavir (ART_A and ART_B, respectively) affect in vitro PAF activity and in vivo PAF levels and metabolism. In this intent, the two ART regimens were examined in vitro against platelet aggregation induced by PAF. In addition, PAF levels and PAF metabolic enzymes were determined in HIV-1 infected volunteers before and after the initiation of antiretroviral therapy for a 12-month period. The in vitro results showed that ritonavir was the most potent inhibitor against PAF induced platelet aggregation while abacavir presented the less potent action. The in vivo results showed that tenofovir-DF/emtricitabine with atazanavir-r seems not to affect PAF levels and metabolism while abacavir/lamivudine with atazanavir-r increased bound and total PAF blood levels, PAF biosynthesis in platelets and also decreased Lp-PLA2 activity. In addition, ART_B revealed higher lyso-PAF-AT specific activity at 3rd, 6th and 9th month (p3=0.04, p6=0.04 and p9=0.03) compared to ART_A. In conclusion, there is a direct relation between in vitro and in vivo effect of antiretrovirals on PAF and abacavircontaining regimen activates PAF biosynthesis leading to elevated PAF levels.

Effect of safflower yellow on platelet activating factor mediated platelet activation in patients with coronary heart disease

The platelet aggregation and 5-HT release by washed platelet from coronary heart disease patients following platelet activating factor (PAF) treatment were detected by turbidimetry and O-phthalaldehyde assay. The free calcium concentration in the platelets was measured with the fura-2/AM probe fluorescent technique. Results showed safflower yellow could inhibit the PAF induced washed platelet aggregation and 5-HT release, which were in a safflor-yellow-dose dependent manner. When the PAF was 2.0×10 -9 mol/L, the inhibition rate of platelet aggregation was 26.2%, 41.3%, 58.1%, 81.2%, and the inhibition rate of 5-HT release was 3.7%, 11.9%, 29.9% and 54.4% after treatment with safflower yellow at 0.21, 0.42, 0.85 and 1.69 g/L, respectively. The study concludes safflower yellow can inhibit the PAF induced platelet aggregation, 5-HT release by platelets and elevation of free calcium in platelets.

Inhibitory effect of the Korean herbal medicine, Dae-Jo-Whan, on platelet-activating factor-induced platelet aggregation

The anti-thrombic properties of the Korean herbal medicine, Dae-Jo-Hwan (DJW) were investigated. Water extracts, a 70% methanol (MeOH) extract and an ethyl acetate (EtOAc) soluble fraction (III) from DJW inhibited platelet-activating factor (PAF)-induced platelet aggregation in vitro and in vivo assays. The extracts of DJW and eleven herbs from which it is derived, except for Panax ginseng Meyer, Angelica sinensis (OLIV.) DIELS and Schisandra chinensis Baill., inhibited AA-induced blood platelet aggregation to various extents. The effects observed with total DJW was synergistic over-additive rather that additive since the sum of single contributions was lower than the effect of the total extract. Fraction III was specially protected against the lethality of PAF, while verapamil did not afford any protection. Exogenously applied arachidonic acid (AA) (100 μM) led to a 89% platelet aggregation, the release of 14 pmol of ATP, and the formation of either 225 pg of thromboxane A2 (TXA2) or 45 pg of prostaglandin E2 (PGE2), each parameter being related to 10 6 platelets. An application of DJW 5 min before AA, dose-dependently diminished aggregation, ATP-re lease, and the synthesis of TXA2 and PGE2, with IC 50 values of 70, 87, 65 and 72 μg/ml, respectively. The similarity of the IC 50 values suggests the inhibition of cyclooxygenase (COX) by DJW as the primary target, thus suppressing the generation of TXA2, which induces platelet aggregation and the exocytosis of ATP by its binding on TXA2-receptors. These results indicate that DJW shows anti-thrombotic action on human platelets and inhibits the action of PAF in vivo by an antagonistic effect on PAF. Therefore, it may be useful in treating disorders caused by PAF.

Antiplatelet of vasorelaxing actions of some benzylisoquinoline and phenanthrene alkaloids.

Members of a series of benzylisoquinoline and phenanthrene alkaloids were tested for their antiplatelet and vasorelaxing actions. Atherosperminine HClO4 (15) and atherosperminium I (16) showed strong inhibition of adenosine 5'-diphosphate-induced platelet aggregation. dl-N-Methylcoclaurine (1), dl-coclaurine (6), 15, 16, xylopine hydroxylamine (18), and atherosperminine N-oxide (19) showed strong inhibition of arachidonic acid-induced platelet aggregation. Compounds 1, 15, 16, dicentrine methine (17), 18, and 19 showed strong inhibition of collagen-induced platelet aggregation. d-(-)-Magnocurarine I (8), 15, and 16 showed strong inhibition of platelet-activating factor-induced platelet aggregation. Compounds 15, 17, and 19 showed vasorelaxing action in rat thoracic aorta.

Stimulus-specific defect in platelet aggregation in polycythemia vera.

We have previously reported that polymorphonuclear granulocyte (PMN) and monocyte oxidative metabolism is reduced in polycythemia vera (PV) patients compared to healthy control subjects, after stimulation with cell surface receptor-dependent stimuli such as n-formyl-methionyl-leucyl-phenylalanine, leukotriene B4 and platelet-activating factor (PAF). In contrast, the oxidative response to phorbol myristate acetate (PMA) is normal. We now show that, in PV patients exhibiting significantly reduced PMN chemiluminescence after PAF stimulation, PAF induced platelet aggregation was also reduced--40 +/- 3% compared to 50 +/- 2% in controls (p < 0.01). The defective aggregatory response to PAF in PV remained over a wide range of stimuli concentrations. Platelet aggregation induced by PMA and ADP, however, was similar in PV and controls. In contrast, platelet aggregation induced by PAF (or by ADP and PMA) was not significantly reduced in patients with chronic myeloid leukemia, essential thrombocythemia and multiple myeloma. Furthermore, the release of beta-thromboglobulin was slightly but not significantly higher after PAF stimulation in PV and this argues against an abnormal PAF receptor as the cause of the defective function. Thus, not only PV neutrophils, but also PV platelets show a discrete defect of the stimulus response coupling for PAF, indicating a disease-specific abnormality that appears to be of clonal origin.

Effects of Low-Dose EPA-E on Glycemic Control, Lipid Profile, Lipoprotein(a), Platelet Aggregation, Viscosity, and Platelet and Vessel Wall Interaction in NIDDM

To assess the effects of low-dose eicosapentaenoic acid-ethyl-ester on diabetes regulation, lipid metabolism, blood rheology, and platelet reactivity. In a double-blind, randomized, placebo-controlled study, 24 NIDDM subjects received 1800 mg of EPA-E, 900 mg of EPA-E, or a placebo (1656 mg olive oil) daily for 8 wk. The EPA:arachidonic acid plasma ratio increased over an 8-wk period, then declined after a 4-wk wash-out period in the fish-oil groups in a dose-dependent way. Platelet-activating factor-induced platelet aggregation decreased from 75 +/- 7% at wk 0 to 35 +/- 21% at wk 8 in the 900-mg group (P = 0.016) and from 72 +/- 11 to 40 +/- 30% in the 1800-mg group (P = 0.039), but did not change in the placebo group. No effects on ADP- or collagen-induced aggregation could be attributed to EPA-E. In the 1800-mg group low-density-lipoprotein cholesterol increased significantly, without concomitant rise in apolipoprotein B. Triglycerides, glycemic control, lipoprotein (a), blood and plasma viscosity, erythrocyte deformability, and platelet adhesion to and aggregate formation on extracellular endothelial cell matrix were not significantly influenced. Purified EPA-E in doses of 900 and 1800 mg reduces Platelet-activating factor-induced platelet aggregation without negatively affecting glycemic control. Low-density-lipoprotein cholesterol was elevated in the 1800-mg group.

Synthesis and Study of the Anti-inflammatory Properties of Some Pyrazolo[1,5-a]pyrimidine Derivatives

A series of pyrazolo[1,5-a]pyrimidin-7-ones (1c–17c) were synthesized to evaluate in vivo and in vitro effects induced by structural modifications at the 2 position of 4,7-dihydro-4-ethyl-2-phenylpyrazolo[1,5-a]pyrimidin-7-one (FPP028). This substance, which has been previously studied, is a weak inhibitor of prostaglandin biosynthesis and a nonacid analgesic and anti-inflammatory agent devoid of ulcerogenic properties. To gain more insight into the mechanism of action of this class of compounds, several in vivo tests were carried out, such as carrageenan-induced rat paw edema and pleurisy. In vitro tests include some studies of leukocyte functions, such as superoxide production and myeloperoxidase release. In vitro effects on arachidonic acid-, adenosine 5’-diphosphate-, and platelet-activating factor-induced platelet aggregation were also studied. Different anti-inflammatory activities were observed, depending on the nature of substituents at the 2 position; these differences are probably linked to the capacity of these compounds to inhibit leukotrienes and/or prostaglandin biosynthesis with different selectivity. 4,7-Dihydro-4-ethyl-2(2-thienyl)pyrazolo[1,5-a]pyrimidin-7-one (7c) proved to be the most interesting compound of the novel synthesized series, showing powerful pharmacological activity in vivo as well as in vitro, together with very weak acute toxicity.

Effect of gliclazide on platelet-activating factor-induced platelet aggregation in patients with non-insulin-dependent diabetes mellitus

Effect of gliclazide on platelet-activating factor-induced platelet aggregation in patients with non-insulin-dependent diabetes mellitus

Platelet‐activating factor type activity in plasma from patients with septicemia and other diseases

The purpose of the present study was to determine whether increased levels of platelet-activating factor (PAF) type activity can be detected in plasma from patients with septicemia and other diseases. A level of PAF below 0.5 ng/mL of plasma was considered normal. We found that plasma from a patient with adverse anaphylactoidic reaction to intravenous analgetics contained 2.1 ng PAF/mL. In seven patients with septicemia, including urosepsis, endocarditis and peritonitis, and with positive blood culture, increased plasma PAF levels (1-20 ng PAF/mL) were observed. Other patients with clinical indications of septicemia had negative blood cultures and/or increased levels of C-reactive protein (CRP). Yet, in the plasma from these patients, no increased PAF levels were detected under the assay conditions used. Two patients with allergic asthma, requiring treatment with steroids, had no measurable plasma PAF. In the plasma from a patient with idiopathic thrombocytopenic purpura (ITP) only an "endogenous" inhibitor of PAF induced platelet aggregation was initially observed. In spite of this, the patient responded to treatment with the PAF antagonist WEB 2086 with a dramatic increase in platelet count (Lohmann et al., Lancet ii, 1147, 1988). Thereafter, also increased PAF levels (3.3 ng PAF/mL) were detected in plasma, although some "endogenous" inhibitor of PAF was still present. In conclusion, increased PAF levels in plasma from patients support a role of PAF in certain human disease states, such as in anaphylactoid reaction, sepsis and septic shock. The type, relevance and specificity of endogenous inhibitors of PAF deserve further study.

Effect of dipyridamole alone and in combination with aspirin on whole blood platelet aggregation, PGI2 generation, and red cell deformability ex vivo in man

The aim was to investigate the effects of dipyridamole, aspirin, and a combination of dipyridamole plus aspirin on platelet aggregation in whole blood, PGI2 generation, and red cell deformability ex vivo. were 16 male volunteers, aged 22-39 years, mean age, 26.6 years. This was a randomised, double blind, placebo controlled trial. The volunteer received each of the following treatments 10 days apart: dipyridamole 200 mg; aspirin 300 mg; dipyridamole 200 mg plus aspirin 300 mg; matched placebos. Blood was taken for platelet function tests, PGI2 metabolite assay, and red cell deformability before and 2 h after the trial dose was taken. Platelet aggregation was quantified by measuring the fall in single platelet count after stimulation with 2 micrograms.ml-1 collagen or 50 nM platelet activating factor (PAF), or by rollermixing aliquots of blood to initiate spontaneous aggregation. The platelet function tests were completed at 37 degrees C within 10 min of venepuncture. The stable metabolite of PGI2, 6-keto PGF1 alpha, was measured in serum. There was inhibition of spontaneous platelet aggregation by dipyridamole (p less than 0.004), aspirin (p less than 0.005), and the combination of dipyridamole plus aspirin (p less than 0.0001) as compared with placebo. PAF induced platelet aggregation was inhibited by dipyridamole (p less than 0.002) and the combination of dipyridamole plus aspirin (p less than 0.0001) but aspirin alone had no inhibitory effect. Collagen induced platelet aggregation was inhibited by all three treatments: dipyridamole (p less than 0.06), aspirin (p less than 0.0001), and the combination of dipyridamole plus aspirin (p less than 0.0001). PGI2 generation was markedly inhibited by aspirin (p less than 0.0001) and the combination doses (p less than 0.0001) but was unaffected by dipyridamole alone. Of the three active treatments, only dipyridamole alone significantly (p less than 0.001) increased red cell deformability; there was a modest decrease in red cell deformability with aspirin. The results with PAF support the view that dipyridamole inhibits platelet activation by more than one mechanism; the effect on collagen induced and spontaneous platelet aggregation suggests that the effect of the combination doses is additive and that on red cell deformability the synergy is negative.

Platelet aggregation induced by platelet-activating factor is suppressed by cystine protease inhibitor

The effect of NCO-700, a cystine protease inhibitor, on platelet-activating factor-induced platelet aggregation was determined. A newly synthesized cystine protease inhibitor (calcium-activated neutral protease and cathepsin B inhibitor), NCO-700 (bis[ethyl (2R, 3R)-3-[(S)-methyl-1-[4-(2,3,4- trimethoxyphenylmethyl) piperazine-1-ylcarbonyl]butylcarbonyl]oxiran-2-carboxy late]sulfate), inhibited platelet-activating factor-induced platelet aggregation. The inhibition was dependent on the preincubation time with NCO-700 and on the concentration of the inhibitor. The release of serotonin was also inhibited almost completely by the 20-min preincubation with 10(-4) M NCO-700. Leupeptin also inhibited platelet-activating factor-induced platelet aggregation. But calcium-activated neutral protease inhibitor did not inhibit it. These observations suggest that NCO-700-sensitive protease(s) such as cystine protease may contribute to platelet aggregation induced by platelet-activating factor.

Suppression by tetrandrine of human platelet aggregation induced by platelet-activating factor and other stimulants.

Tetrandrine was found to have inhibitory effects on platelet-activating factor induced platelet aggregation in a dose-dependent manner. There was preferential inhibition of platelet aggregation induced by agents such as collagen, thrombin, adrenaline, and adenosine diphosphate. No inhibitory effect of tetrandrine was observed on platelet aggregation induced by adenosine diphosphate, arachidonic acid, and the calcium ionophore A23187. These results support our previous findings of interference with the phosphatidylinositol second-messenger system as one of the sites of action of tetrandrine. Since platelets may have an important role in the pathogenesis of asthma and other allergic diseases, these findings suggest that tetrandrine may have clinical application as a non-steroidal broad-spectrum anti-allergic drug.

Triazolobenzodiazepines competitively inhibit the binding of platelet activating factor (PAF) to human platelets

PAF causes dose dependent platelet aggregation of human platelet rich plasma or gel filtered platelets (GFP). The benzodiazepines alprazolam and triazolam, but not diazepam (1-10 microM), inhibit PAF induced aggregation but have no effect on aggregation induced by other platelet agonists such as ADP, epinephrine and collagen. The IC50 for aggregation by PAF (4 nM) in GFP is 1 microM for both alprazolam and triazolam. The mechanism for this inhibition was explored by studying the binding of 3H-PAF(0.08 nM) to GFP in Tyrodes buffer containing albumin (0.35%), Mg++ (1mM) and Ca++ (0.5mM). GFP was incubated with different doses of the drug for 5 min prior to addition of 3H-PAF. Incubation was then carried out for 60 min at 25 degrees C to achieve binding equilibrium, as previously established. Alprazolam and triazolam, but not diazepam, caused competitive displacement of 3H-PAF from specific binding sites of GFP. The IC50 of alprazolam was 3.8 microM while that of triazolam was 0.82 microM. Lineweaver-Burk plots of 3H-PAF binding in the presence of inhibitor were also consistent with competitive inhibition. These results are consistent with the interpretation that the specific inhibition of PAF induced platelet aggregation by alprazolam and triazolam, respectively, is due to competitive inhibition of binding of PAF to its receptor.

Effect of C-reactive protein on platelet-activating factor-induced platelet aggregation and membrane stabilization.

C-reactive protein (CRP) is an acute phase reactant which humoral concentration rises drastically following tissue injury or inflammation. CRP of all species binds to phosphorylcholine residues. In the present studies CRP was found to inhibit platelet-activating factor-induced platelet aggregation, and to stabilize platelet membranes against the lytic effect of lysophosphatidylcholine. Inhibition of platelet aggregation by CRP is accompanied by an inhibition of arachidonic acid release from both phosphatidylcholine and phosphatidylinositol. This suggests that phospholipases are inhibited. Hydrolysis of multilamellar dipalmitoylphosphatidylcholine liposomes by purified phospholipase A2, was also inhibited by CRP. These results suggest that CRP can stabilize membranes from the detergent-like effects of lysolipids and from potentially toxic materials such as platelet-activating factor. By inhibition of phospholipases, production of inflammatory mediators would be blocked. CRP might thus act as an early protective recognition mechanism in acute inflammatory states.