Molecular mechanisms involved in human platelet aggregation by synergistic interaction of platelet-activating factor and 5-hydroxytryptamine

Our recent studies have shown that co-activation of Gq and Gi proteins by 5-hydroxytryptamine (5-HT) and adrenaline show synergism in human platelet aggregation. This study was conducted to examine the mechanism(s) of synergistic interaction of 5-HT and platelet activating factor (PAF) in human platelets. We show that PAF, but not 5-HT, increased platelet aggregation in a concentration-dependent manner. However, low concentrations of 5-HT (2 microM) potentiated platelet aggregation induced by subthreshold concentration of PAF (40 nM) indicating a synergistic interaction between the two agonists and this synergism was blocked by receptor antagonists to either 5-HT or PAF. 5-HT also potentiated the effect of PAF on thromboxane A2 (TXA2) formation and phosphorylation of extracellularly regulated mitogen-activated protein kinases (ERK1/2). The synergism of 5-HT and PAF in platelet aggregation was inhibited by calcium (Ca2+) channel blockers, verapamil and diltiazem, phospholipase C (PLC) inhibitor, U73122, cyclooxygenase (COX) inhibitor, indomethacin, and MEK inhibitor, PD98059. These data suggest that synergistic effect of 5-HT and PAF on human platelet aggregation involves activation of PLC/Ca2+, COX and MAP kinase pathways.

Molecular mechanisms involved in human platelet aggregation by synergistic interaction of platelet-activating factor and 5-hydroxytryptamine

Our recent studies have shown that co-activation of Gq and Gi proteins by 5-hydroxytryptamine (5-HT) and adrenaline show synergism in human platelet aggregation. This study was conducted to examine the mechanism(s) of synergistic interaction of 5-HT and platelet activating factor (PAF) in human platelets. We show that PAF, but not 5-HT, increased platelet aggregation in a concentration-dependent manner. However, low concentrations of 5-HT (2 microM) potentiated platelet aggregation induced by subthreshold concentration of PAF (40 nM) indicating a synergistic interaction between the two agonists and this synergism was blocked by receptor antagonists to either 5-HT or PAF. 5-HT also potentiated the effect of PAF on thromboxane A2 (TXA2) formation and phosphorylation of extracellularly regulated mitogen-activated protein kinases (ERK1/2). The synergism of 5-HT and PAF in platelet aggregation was inhibited by calcium (Ca2+) channel blockers, verapamil and diltiazem, phospholipase C (PLC) inhibitor, U73122, cyclooxygenase (COX) inhibitor, indomethacin, and MEK inhibitor, PD98059. These data suggest that synergistic effect of 5-HT and PAF on human platelet aggregation involves activation of PLC/Ca2+, COX and MAP kinase pathways.

Evidence for the presence of a peptide inhibitor of arachidonic acid metabolism in human cerebrospinal fluid

Human cerebrospinal fluid [CSF], incubated with a homogenate of human platelets, inhibited the conversion of arachidonic acid into tri-hydroxy-eicosatrienoic acid, 12-hydroxy-eicosatetraenoic acid and thromboxane B[2], in a concentration-related manner. A preliminary investigation of physicochemical properties and the effect of proteolytic enzymes on CSF indicated that CSF contains a peptide of low molecular weight [about 1400 daltons] that inhibits arachidonic acid metabolism. The presence of such an inhibitor in CSF might represent a natural mechanism for controlling arachidonic acid metabolism in the subarachnoid space and the adjacent tissues

Ketotifen, an anti-asthmatic drugs protects platelet-activating factor-induced sudden death in rabbits

The effects of antiasthmatic drug; ketotifen on human platelet aggregation induced by platelet-activating factor [PAF], ADP, collagen and arachidonic acid [AA] have been investigated. Ketotifen selectively inhibited [at 0.04-4uM concentration] PAF-induced aggregation. Whereas other anti-asthma drugs such as sodium cromoglycate, theophylline and salbutamol had no effect. Similarly, ketotifen also inhibited the lethal effects of PAF in the rabbit. PAF [8-11ug/Kg i.v.] caused sudden death in rabbits due to platelet aggregation. Pretreatment of rabbits with ketotifen [1 mg/kg i.p.] protected conscious rabbits from PAF [11 ug/kg i.v.] -induced death. Since PAF is a powerful inducer of platelet aggregation via a mechanism mediated by specific PAF membrane receptors, our data is suggestive that ketotifen acts as a PAF antagonist in platelet aggregation

Prostaglandins, menstruation, and menstrual disorders

Prostaglandins play an important role in the mechanism of human menstruation. Furthermore aberrant prostaglandin production may be an intimate component of the pathogenesis of menstrual disorders such as dysmenorrhoea, menorrhagia and perhaps the premenstrual syndrome. Inhibitors of prostaglandin biosynthesis available today have proven universally useful in the treatment of dysmenorrhoea and menorrhagia. The development of such drugs and endogenous inhibitors of prostaglandin synthase [EIPS] with greater specificity and also perhaps antagonists of the actions of specific prostaglandins is currently a high priority. It may be anticipated that the development of these newer ranges of natural and synthetic drugs will allow treatments of menstrual disorders with greater efficacy, and reduced sideeffects

Evaluation of drug effects on rabbit intestinal alkaline phosphatase activity

Alkaline phosphatase [AP] plays an important role in the availability and absorption of inorganic phosphate [Pi] through the intestine. In this study we have investigated the effects of various orally administered drugs on AP prepared from rabbit intestinal mucosa. The enzyme had a Km of 0.5 X 10-3 M at pH 10.5 and produced linear reaction rates both with respect to enzyme and substrate concentration. The AP was totally inhibited by 2.68 mM EDTA. An analysis of the effects of various drugs on AP showed that cysteine, aminophylline, neomycin, theophylline, pilocarpine, caffeine, aspirin and threonine, in descending order of potency, inhibited intestinal AP in a concentration-dependent manner. Acetylcholine, carbachol, alropine, hyoscine, ampicillin, isoniazide, polymyxin B, sulphamethoxazole, trimethoprim, indomethacin, p-acetamidophenol, papaverine, cyclazine, diphenhydramine, hydrocortisone, tyramine, streptomycin, and promethazine had no effect on intestinal AP. Our data showed that a number of drugs inhibited AP. It is tempting to speculate that the inhibition produced by various drugs may involve interference with absorption processes of Pi or the availability of Pi in the gastrointestinal tract

Decreased ability of human plasma to inhibit lipid peroxidation in uremia

Blood samples from normal healthy volunteers and uremic patients were obtained and plasma were prepared. The effects of these plasma samples on lipid peroxidation [LP] in rabbit homogenate were determined. Both plasma samples inhibited LP activity in our experiments. The percent inhibition of LP produced by normal plasma, concentration 1 and 10%, v/v, was 21.78 +/- 4.15 and 75.5 +/- 2.39 [Mean +/- S.E.M. n=20] respectively. The corresponding values of LP inhibition obtained using the uremic plasmas, concentrations 1 and 10% v/v, were 6.85 +/- 7.04 and 57.12 +/- 13.71 respectively. These data show that there was a significant reduction in LP inhibitory activity in plasma of uremic patients. This suggests that there may be an increased production of associative LP products in uremic patients. Since LP products profoundly affect blood clotting it can be inferred that aberrant levels of LP inhibitor in uremic plasma could contribute to increased bleeding in uremia

Endogenous inhibitor [S] of platelet aggregation

Human blood plasma contains an endogenous inhibitor [s] of platelet aggregation [EIPA]. The EIPA activity was found to be associated with alpha - globulin-rich and albumin-rich protein fractions of blood plasma. Data presented in this paper suggests that EIPA may regulate platelet aggregation associated with excessive production of arachidonic acid metabolites

Activity of endogenous inhibitor [s] of platelet aggregation in plasma of normal males, females and pregnant women

The EIPA activity in human plasmas obtained from adult males, females, pregnant females and foetal umbilical cord was determined. EIPA activity was highest in the following order of potency; adult females > adult males > pregnant females > foetal umbilical cord plasma. The data suggest the EIPA activity, is highest in females than in males and may vary according to sex or pregnancy in females

Inhibition of lipid peroxidation by anti-inflammatory drugs, human plasma and serum

Serveral non-steroidal anti-inflammatory drugs known to inhibit prostaglandin [PG] biosynthesis were also found to inhibit lipid peroxidation. Similarly, endogenous inhibitors of PG biosynthesis, such as human blood plasma, serum and plasma - globulin fraction, also inhibited lipid peroxidation. These results throw new light on the mechanism of action of anti-inflammatory drugs and also open up the interesting possibility that the inhibition of lipid peroxidation may be a component of anti-inflammatory response to these drugs

Endogenous inhibitors of cyclic nucleotide phosphodiesterase: isolation and partial characterization

We have developed an extraction procedure to isolate from mammalian organs and cells endogenous factors that inhibit cyclic nucleotide phosphodiesterase [PDE]. The procedure involves homogenization of an organ or cell type in HC1 followed by extraction with chloroform methanol. The aqueous-methanol phase is separated and lyophilised. This material containing PDE inhibitor activity is further purified by gel filtration chromatography on Sephadex G-25 and G-75 columns. In rat and bovine brain, calf thymus, guinea pig lungs and ileum, human stomach, rat peritoneal mast cells and mouse splenic lymphocytes, cAMP and cGMP dependent PDE activity was inhibited in dose dependent fashion by the lyophilised material derived from these tissues. In other experiments the material from calf thymus selectively inhibited the PDE of calf thymus and not of any other tissue. Preliminary investigation of the physico-chemical properties of rat and bovine extracts indicated that brain contained inhibitory peptides of low molecular weight whereas calf thymus extracts contained inhibitory peptides of both low and high molecular weights. This method therefore reveals the presence of endogenous inhibitors of PDE [EIPODS] in various mammalian organs and cells

Drugs affecting wound healing and alkaline phosphatase activity

Alkaline phosphatase [AP] plays an important role in absorption, transport processes, and the availability of inorganic phosphates in normal tissues. In this study we found that human wound tissue contains AP activity which displayed typical enzymic properties. The enzyme had a Km of 9.3 x 10-5M and Vmax of1.098 moles/ml/min. The effect of various antimicrobial and anti-inflammatory drugs o the activity of AP in burn wound tissue was also examined. Low concentrations of oxytetracycline [OT, doxycycline hyalite [DH] and doxycycline polyphosphate sodium complex [DMSC] in increasing order of potency, inhibited AP activity in a dose-related manner. Other drugs, ampicillin, bacitracin, Zn, erythromycin, hydrocortisone, neomycine, polymeric B, sulphamethoxazole and trimethoprim did not inhibit AP activity at concentrations up to 5mM. in addition, isoenzymes of AP from different rabbits and human burn wound tissues were inhibited to varying extents by DH. On the other hand, DMSC selectively inhibited the human burn wound tissue AP activity and had no effect on AP derived from other tissues

Altered regulation of platelet aggregation in renal disorders

The activity of endogenous inhibitors of platelet aggregation [EIPA] was measured in the plasma of 19 patients with renal disorder [RD]. Similar studies were conducted in 16 healthy subjects. The plasma of 12 RD patients stimulated platelet aggregation [P<0.005] and had negative EIPA activity. Plasma of 7 out of 19 RD patients exhibited EIPA activity which was lower compared to healthy subject [P<0.001]. All RD patients had abnormally, low levels of plasma albumin [a part of EIPA activity] as compared to normal [P<0.01]. The patients who had low or negative EIPA activity had platelets which were highly sensitive to induction of aggregation by adenosine-5' diphosphate [P<0.001] or adrenaline [P<0.05]. Our data suggests that low plasma EIPA activity in patients with RD may be an important factor in their hypersensitivity to platelet aggregating agents and could contribute to the thrombotic tendency

Differential inhibition of arachidonate cyclo-oxygenase and lipoxygenase enzymes by drugs and new approaches to anti-inflammatory therapy

We have compared the effects of various types of drugs on arachidonate cyclo-oxygenase and lipoxygenase enzymes. Several non-steroidal anti-inflammatory drugs [NSAID] were found to be predominant inhibitors of cyclo-oxygenase and had little or no effect on the lipoxygenase activity. On the other hand, anti-malarial drugs and various anti-oxidants inhibited both enzymes with varying potencies whereas anti-asthmatic drugs and anti-inflammatory steroids showed no effect. These results are demonstrative that metabolism of arachidonic acid by way of cyclo-oxygenase and lipoxygenase pathways are involved in inflammation, and that dual inhibition of cyclo- oxygenase and lipoxygenase may lead to a more selective type of anti-inflammatory action without the complicating side-effects of corticosteroids

Potential new uses of aspirin

Aspirin, the old standby, appears to have new potential in therapeutics as an inhibitor of prostaglandin biosynthesis. In the present review, we have identified new vistas on the use of aspirin in the treatment of various prostaglandin mediated disorders such as cardiac ischaemia, arrhythmias, shock, migraine and various ophthalmic conditions