The Inhibitory Effect of Human Plasma Albumin and Haptoglobin on Platelet Aggregation and 5-HT Release.

Platelet aggregation contributes to the pathogenesis of cardiovascular diseases. After activation it leads to dense granule secretion and 5-HT release. The question arises; how platelet aggregation is endogenously controlled during blood circulation. In preliminary studies, we observed that human platelets aggregate more rapidly when suspended in buffer as compared to those suspended in plasma (PRP). These observations point to the presence of an endogenous substance that may inhibit arachidonic acid- induced platelet aggregation. An analysis of plasma Cohn fractions demonstrated that most of the plasma inhibitory activity was associated with albumin-rich and α-globulin rich protein fractions. The identity of plasma endogenous inhibitors of platelet aggregation (EIPA) was established by affinity chromatography on Cibacron Blue F3G-A for specific removal of albumin. The association of α-globulins to EIPA activity was recognized as due to haptoglobin by affinity chromatography on a column of hemoglobin-sepharose. In addition, we also found that the distribution of EIPA activity varies according to sex and physiological state. These findings reveal that EIPA may act by modulation of arachidonic acid metabolism or sequestering the fatty acid substrate.

Nonsteroidal anti-inflammatory drugs as potential ecto-nucleotide phosphodiesterase inhibitors

Phosphodiesterases (PDE) are group of enzymes which catalyze the hydrolysis of cAMP and cGMP. Since these cyclic phosphate moieties worked as intracellular second messengers in numerous physiological processes, their inhibition can affect normal physiology of living system. NSAIDs are among the frequently prescribed medications, because of their efficacy as analgesic, antipyretic and anti-inflammatory agents. They are known to block cyclooxygenase pathway. In limited data NSAIDs has been shown anti-tumor potential, and phosphodiesterase inhibition has assumed to be one of the mechanism. To date no further evaluation being done. Further, NSAIDs are classified as cyclooxygenase inhibitors and phosphodiesterase inhibition can imprint its side effects. This study first time investigates the effects of NSAIDs on phosphodiesterase 1 inhibition. The activity against snake venom phosphodiesterase 1 was assayed on a microtitre plate reader spectrophotometer. Selective COX-2 inhibitor, celecoxib, exhibited a potent PDE1 inhibitory activity, at therapeutic doses, with an IC50 value of 29.4 µM. The findings of our study are indicative of new pharmacological actions of cyclooxygenase inhibitors. This article presents the PDE inhibitory properties as a new effects of already existing drugs. These additional effects could be potentially helpful for researchers to assess other physiological and pathological states.

Analgesic, anti-inflammatory and anti-platelet activities of the methanolic extract of Acacia modesta leaves.

The current study was aimed to evaluate Acacia modesta for analgesic, anti-inflammatory, and anti-platelet activities. The analgesic and anti-inflammatory effects were assessed in rodents using acetic acid and formalin-induced nociception, hot plate and carrageenan-induced rat paw oedema tests. The intraperitoneal (i.p.) administration of the methanolic extract (50 and 100 mg/kg) produced significant inhibition (P\0.01) of the acetic acid-induced writhing in mice and suppressed formalin-induced licking response of animals in both phases of the test. In the hot plate assay the plant extract (100 mg/kg) increased pain threshold of mice. Naloxone (5 mg/kg i.p.) partially reversed the analgesic effect of the extract in formalin and hot plate tests.A. modesta (100 and 200 mg/kg i.p.) exhibited sedative effect in barbiturate-induced hypnosis test similar to that produced by diazepam (10 mg/kg i.p.). The plant extract(50-200 mg/kg i.p.) produced marked anti-inflammatory effect in carrageenan-induced rat paw oedema assay comparable to diclofenac and produced a dose-dependent(0.5-2.5 mg/mL) inhibitory effect against arachidonic acid induced platelet aggregation. These data suggest that A. modesta possesses peripheral analgesic and antiinflammatory properties, with analgesic effects partially associated with the opioid system.

Investigating GABA and its function in platelets as compared to neurons.

We have recently suggested that platelets could be used as a model for neuronal receptors. In this paper we have investigated gamma-aminobutyric acid (GABA) metabolism and GABA receptors in platelets and in cultured neurons to see whether platelets' GABA mimics neuronal GABA receptor activities. We used the ELISA technique for detecting the GABA concentration in platelet rich plasma and cultured neurons. The functional effects of GABA and its receptor ligands on platelets were determined using an aggregometer. We found that the GABA concentration is 30% lower in platelets than in neurons and in both preparations GABA was metabolized by GABA transaminase (GABA-T). GABA potentiated calcium dependent platelet aggregation with a higher value in washed platelets suspension (WPS) then in platelet rich plasma (PRP). This effect was inhibited by benzodiazepines, calcium channel blockers and the selective phosphoinositide 3-kinase antagonist Wortmannin. GABA neurotransmission is involved in most aspects of normal brain function and can be perturbed in many neuropathologic conditions. We concluded that platelets could be further developed to be used as a peripheral model to study neuronal GABAergic function and its abnormality in diseases such as epilepsy and schizophrenia. Furthermore our results indicated that PI3-kinase is involved in calcium dependent GABA induced platelet aggregation as this synergistic effect is inhibited by Wortmannin in dose dependent manner.

Ethnopharmacological studies on antispasmodic and antiplatelet activities of Ficus carica.

ETHNOPHARMACOLOGICAL RELEVANCE: The ripe dried fruit of Ficus carica Linn. (Moraceae) commonly known as "Fig" has medicinal value in traditional system of medicine for its use in gastrointestinal and inflammatory disorders. AIM OF THE STUDY: To rationalize the medicinal use of Fig (Ficus carica) in gastrointestinal and inflammatory disorders. MATERIALS AND METHODS: The aqueous-ethanolic extract of Ficus carica (Fc.Cr) was studied for antispasmodic effect on the isolated rabbit jejunum preparations and for antiplatelet effect using ex vivo model of human platelets. RESULTS: Fc.Cr tested positive for alkaloids, flavonoids, coumarins, saponins, sterols and terpenes. When tested in isolated rabbit jejunum, Fc.Cr (0.1-3.0mg/mL) produced relaxation of spontaneous and low K(+) (25 mM)-induced contractions with negligible effect on high K(+) (80 mM) similar to that caused by cromakalim. Pretreatment of the tissue with glibenclamide caused rightward shift in the curves of low K(+)-induced contractions. Similarly, cromakalim inhibited the contractions induced by low K(+), but not of high K(+), while verapamil equally inhibited the contractions of K(+) at both concentrations. Fc.Cr (0.6 and 0.12 mg/mL) inhibited the adenosine 5'-diphosphate and adrenaline-induced human platelet aggregation. CONCLUSION: This study showed the presence of spasmolytic activity in the ripe dried fruit of Ficus carica possibly mediated through the activation of K(+)(ATP) channels along with antiplatelet activity which provides sound pharmacological basis for its medicinal use in the gut motility and inflammatory disorders.

Quantitative gait analysis as a method to assess mechanical hyperalgesia modulated by disease-modifying antirheumatoid drugs in the adjuvant-induced arthritic rat.

In the present study, azothioprine, chloroquine, D-penicillamine, methotrexate and sodium aurothiomalate (gold salt) were evaluated for possible disease-modifying effects in the adjuvant-induced arthritis model of human rheumatoid arthritis in rats. Gait analysis was used to examine the role of disease-modifying antirheumatic drugs in the development of pain. Body weights were also measured to monitor the progression of disease and the systemic antiarthritic effects of the test compounds used in this study, as well as their systemic toxicity. Our results showed that azothioprine (5 mg/kg/day), chloroquine (12.5 mg/kg/day), sodium aurothiomalate (2.5 mg/kg/day) and methotrexate (1 mg/kg/week) not only inhibited the macroscopic changes such as erythema and swelling of limbs, but also exhibited significant reversal of gait deficits seen in the untreated or saline-treated arthritic rats. No reduction in the body weights were observed in the arthritic rats treated with azothioprine, chloroquine, sodium aurothiomalate and methotrexate. D-Penicillamine (12.5 mg/kg/day), however, showed a significant reduction (P < 0.03) in the body weights of the arthritic rats over a period of 22 days; furthermore, it was unable to show any reduction in arthritic score (P < 0.1). In earlier experiments, chloroquine and methotrexate failed to suppress carageenan-induced edema, suggesting that the mode of antiarthritic action may be different from those of nonsteroidal anti-inflammatory agents. Since these disease-modifying antirheumatic drugs are reported to have an immunomodulatory role, especially the gold salt, which influences the monocyte-macrophage system, it is suggested that the observed antiarthritic effects of disease-modifying antirheumatic drugs may be partly attributed to their immunomodulatory activity.

Some new prospects in the understanding of the molecular basis of the pathogenesis of stroke.

Stroke is one of the leading causes of mortality and morbidity in advanced countries of the world. Despite the fact that reactive oxygen and nitrogen species (ROS and RNS) are the by-products of normal metabolic processes and mediate important physiological processes, they can inflict damage to the cell if produced in excess due to oxidative stress. In the present review, we focus on the cellular and molecular aspects of ROS and RNS generation and its role in the pathogenesis of stroke produced by hypoxia-reperfusion (H-R) phenomena that elicit oxidative stress. We outline the reasons for the vulnerability of the brain to ischaemic insult, chronic infection and inflammation as well as the natural defence mechanisms against radical mediated injury. We deal with the effect of ROS and RNS on intracellular signaling pathways together with the phenomena of apoptosis, mitochondrial injury and survival associated with these pathways. The intracellular signaling mechanisms influenced by reactive species can have significant effects on the outcome of the condition. Future studies should focus on understanding the molecular mechanisms involved in the action of anti-radicals agents, and their mode of action.

Inhibitors of phosphatidylinositide 3-kinase: effects on reactive oxygen species and platelet aggregation.

Phosphoinositide 3-kinase (PI 3-kinase) exists in cells as a family of isoforms. The enzymes are important regulators of fundamental metabolic processes, such as energy utilization, growth, cell proliferation and survival. They are activated by cell surface receptors for hormones, and by G-protein coupled receptors. Enzyme p110 gamma, in particular, catalyzes production of second messengers from inositol phospholipids, including phosphoinositide (3,4,5) triphosphate or PtdIns (3,4,5) P3, PtdIns (3,4) P2 and Ptdins (3) P. The objective of this study was to corroborate the role of PI 3 kinase in ROS generation and in platelet aggregation through the use of four chemically unrelated inhibitors of PI 3 kinase: wortmannin, LY-294002, resveratrol and quercetin. In this study, we describe the effects of four PI 3-kinase inhibitors on the production of reactive oxygen species (ROS) and platelet aggregation induced by a diversity of agonists. Neutrophils and platelets were obtained from human blood and macrophages from mouse peritoneal cavity. ROS production was measured by a luminol-enhanced chemiluminescence assay; aggregation was measured in platelet-rich plasma (PRP) with a Chronolog Dual Channel Lumi-Aggregometer. Effects of graded concentrations of four enzyme inhibitors (wortmannin, LY-294002, resveratrol and quercetin) were evaluated. All inhibitors caused concentration-dependent depression of ROS generation and human platelet aggregation. They differed only in their potencies as revealed by concentration-response data. Moreover, inhibitors blocked activity of three chemically unrelated stimulants of aggregation: ADP, collagen and epinephrine. We conclude that inhibition of PI 3-kinase would appear to be a useful therapeutic goal in those conditions where the activities of platelets and/or phagocytes become aberrant.

Dual inhibition of cyclooxygenase and lipoxygenase enzymes by human cerebrospinal fluid.

Thromboxane A2 (TXA2) formed in damaged brain tissue and after thromboembolism and subarachnoid haemorrhage is responsible for cerebral vasospasm. In the present study, we examined the effect of human cerebrospinal fluid (CSF) on the production of thromboxane-A2 (TXA2) and 12-hydroxy-eicosatetraenoic acid (12-HETE) by human blood platelets. CSF was drawn by lumbar puncture from normal healthy volunteers (n = 17) and samples judged to be normal after routine examination in the clinical laboratories and were used fresh. We found that CSF inhibited the production of TXA2 and 12-HETE by blood platelets incubated with C14 labelled arachidonic acid (AA) in a concentration-related manner. Further biochemical analysis using proteolytic enzymes, gel filtration and membrane partition chromatography showed that the inhibitory activity was peptidic in nature and associated with a peptide of low molecular weight (1,400 Da). This study is the first to demonstrate that human CSF contains a dual inhibitor of cyclooxygenase (COX) and lipoxygenase enzymes in CSF.

Dual inhibition of cyclooxygenase and lipoxygenase by human haptoglobin: its polymorphism and relation to hemoglobin binding.

Haptoglobin (Hp) binds hemoglobin (Hb) specifically and stoichiometrically. Since Hb stimulates prostaglandin (PG biosynthesis), we investigated if Hp effects arachidonic acid (AA) metabolism. The results showed that Hp (50-250 microg protein) inhibited the biosynthesis of PGs via cyclooxygenase (COX) and 12-HETE via lipoxygenase pathway in human platelets. Additional evidence was obtained by the loss of Hp inhibitory activity upon removal of Hp by affinity chromatography on hemoglobin sepharose and by inhibition of AA or bradykinin-induced bronchoconstriction in the guinea pig. Hb reduced the inhibitory effect of Hp in a concentration-related manner such that all its inhibitory activity was lost when completely bound by Hb. Of the three Hp phenotypes, Hp 1-1 showed maximum binding capacity to Hb indicating its greater protective role. These findings implicate Hp in the regulation of COX and lipoxygenase pathways and show Hp involvement in the body's endogenous defense system against inflammation. This indicates that mammals have dual defense system, i.e., a specific immune system and non-specific Hp defense system.

Anti-ischemic effects of nimesulide, a cyclooxygenase-2 inhibitor on the ischemic model of rabbit induced by isoproterenol.

The objective was to devise an animal model of myocardial infarction (MI) against which cardioprotective drugs might be tested. We describe the effects of nimesulide, a COX experience with development and validation of such a model. The rabbit was chosen in preference to rodents because its heart and cardiac circulation more closely resemble those of human. Thus, the cardiovascular system of anaesthetized male rabbits, 1 to 1.5 kg (n=11), was stressed by a single bolus intravenous injection of isoprenaline (ISP), 65 mg/kg. The effects of the injection were followed for sixteen days and were evaluated in four ways: 1) measurements of creatinine kinase isozyme and troponin-I (TPI) in serum 2) Electrocardiographic (ECG) changes (ST elevation and Q wave development) 3) Cardiac histopathology observed in tissue sections of the isolated of the heart. The histopathological analysis showed that rabbit heart on 2nd day after ISP injection showed changes of coagulation necrosis. Day 4 total coagulation with the loss of nuclear and striation associated with heavy interstitial infiltrate of neutrophils was found. Day 8 after infarction showed collagen deposition with capillary channels in between the remaining islands of myocytes in the infarcted area. On the 16th day scarring was complete. Coronary perfusion rates (CPR) and heart rate (HR) of the infarcted and nimesulide (a COX-2 inhibitor) treated rabbits displayed significant improvement (n=11) on each corresponding day after infarction as compared to the infarcted and saline treated rabbits (P<0.05). All four indices revealed similarities with effects commonly associated with MI in humans.

Role of cyclooxygenase-2 in myocardial infarction and ischaemia.

OBJECTIVE: To determine the role of cyclooxygenase-2 (COX-2), on coronary perfusion rate (CPR) in a rabbit heart model showing ischaemic damage and reperfusion injury to the myocardium. DESIGN: An experimental study. PLACE AND DURATION OF STUDY: The animal unit of the Dr. Panjwani Center for Molecular Medicine and Drug Research, ICCS, University of Karachi during July 2004 to June 2005. MATERIALS AND METHODS: Rabbits were divided into three groups of six animals each. Group I had normal rabbits, group II consisted of infarcted rabbits (infarction induced by 65 mg /kg isoproterenol (ISP) and group III consisted of infarcted rabbits that received nimesulide, a COX-2 inhibitor (25 mg/kg). MI was induced in rabbits by ISP, a beta-agonist drug given by the intraperitoneal route. In this model, the acute phases of myocardial necrosis and repair mimicked those which occurred in humans. Induction of MI was confirmed by measuring changes in serum creatinine phosphokinase (CPK) and troponin I (TPI) levels, electrophysiological (ECG) changes and histochemical changes in the left ventricle before and after ISP injection. Coronary perfusion rate (CPR) was monitored using Langendorff isolated heart preparation at day 2, 4, 8 and 16 of post-infarction. RESULTS: CPR of infarcted and nimesulide treated rabbits displayed significant improvement on each corresponding day post-infarction as compared to the infarcted and control rabbits (p <0.01). CONCLUSION: Using this model, it was found that nimesulide, a COX-2 inhibitor, exerted a cardioprotective effects in MI.

Latest approaches to the diagnosis and management of food allergies in children.

Adverse food reactions are a challenge for physicians. As the prevalence of this condition rises, it is important that paediatricians and other health care professionals adeptly diagnose this condition. We begin by discussing the relevant points in history and physical examination, then we discuss the recent effective diagnostic tests and techniques available for doctors and patients, along with several management options. Over the last decade, there have been major advancements in this field and novel mechanisms have been proposed which efficiently modulate immune mechanisms involved. Although results are only preliminary, they do however, indicate a promising future for patients with food allergies.

Myocardial ischaemia and reperfusion injury: reactive oxygen species and the role of neutrophil.

A growing body of evidence suggests that oxygen radicals can mediate myocardial tissue injury during ischaemia and, in particular, during reperfusion. This review focuses on the role of neutrophil as a mediator of myocardial damage. Upon reperfusion, neutrophils accumulate and produce an inflammatory response in the myocardium that is responsible, in part, for the extension of tissue injury associated with reperfusion. It has shown that the inhibition of neutrophil accumulation and adhesion is associated with decreased infarct size. This strongly suggests that myocardial cells at risk region undergo irreversible changes upon reperfusion and accumulation of neutrophils. Several pharmacological agents (ibuprofen, allopurinol, prostacyclin, and prostaglandin E analogues) protect the myocardium from reperfusion injury. In addition, the mechanisms by which these agents act and directions of research that may lead to therapeutically useful approaches are also discussed in this review.

Synergistic interaction of epinephrine and calcium ionophore in platelet aggregation.

BACKGROUND: Platelets play a key role in haemostasis. Human Platelets contain alpha2 adrenergic receptors, which are coupled with guanine nucleotide proteins (G proteins). The platelet activation involves a number of receptors for agonists. It has also been shown that most of the agonists act in synergy and potentiate the effects of each other. The present experimental study was designed to study the potentiation of epinephrine on human platelets by calcium ionophore A23187 and the possible role of calcium in platelet aggregation as a second messenger. METHODS: Study was carried out at Department of Biological Sciences Aga Khan University, Karachi. Blood samples from healthy volunteers were collected; Platelet aggregation was measured using Dual channel Lumi Aggregometer. The chemicals used include epinephrine, calcium ionophore A23187, yohimbine, diltiazem, verapamil and S Nitrosoacetylpenicillamin (SNAP). RESULTS: Epinephrine at low concentrations (0.01-0.2 microM) and/or A23187 (0.1-0.5 microM) itself did not produce platelet aggregation. However, when added together, a marked potentiation of platelet aggregation was observed. This synergistic effect was inhibited by alpha2-receptor blocker yohimbine; (IC50 = 0.05 microM) showing that the response is receptor mediated. To find out the molecular basis of this potentiation, we used SNAP, a nitric oxide donor and Ca++ channel blockers, i.e. diltiazem and verapamil. The SNAP, diltiazem and verapamil inhibited the platelet aggregation induced by A23187 and epinephrine with IC50 value of 0.5 microM, 50 microM and 22 microM respectively. CONCLUSION: The results of the study suggest that epinephrine and calcium ionophore act synergistically and Ca++ plays an important role in this synergistic interaction. While calcium channels blocking drugs diltiazem and verapamil inhibit this synergism.

Involvement of thromboxane A2 and tyrosine kinase in the synergistic interaction of platelet activating factor and calcium ionophore A23187 in human platelet aggregation.

The present study was carried out to examine the mechanisms of the synergistic interaction of PAF and A23187 mediated platelet aggregation. We found that platelet aggregation mediated by subthreshold concentrations of PAF (5 nM) and A23187 (1 mM) was inhibited by PAF receptor blocker (WEB 2086, IC50 = 0.65 mM) and calcium channel blockers, diltiazem (IC50 = 13 mM) and verapamil (IC50 = 18 mM). Pretreatment of platelets with PAF and A23187 induced rise in intracellular calcium and this effect was also blocked by verapamil. While examining the role of the down stream signaling pathways, we found that platelet aggregation induced by the co-addition of PAF and A23187 was also inhibited by low concentrations of phospholipase C (PLC) inhibitor (U73122; IC50 = 10 mM), a cyclooxygenase inhibitor (indomethacin; IC50 = 0.2 mM) and inhibitor of TLCK, herbimycin A with IC50 value of 5 mM. The effect was also inhibited by a specific TXA2 receptor antagonist, SQ 29548 with very low IC50 value of 0.05 mM. However, the inhibitors of MAP kinase, PD98059 and protein kinase C, chelerythrine had no effect on PAF and A23187-induced platelet aggregation. These data suggest that the synergism between PAF and A23187 in platelet aggregation involves activation of thromboxane and tyrosine kinase pathways.

A closer look at food allergy and intolerance.

Food allergy is a condition prevalent in over 2 percent of the world's population. The topic has been subject to research from ancient Greek times and continues to attract modern scientific and medical communities. In susceptible individuals certain foods produce a wide spectrum of unwanted effects like eczema, asthma, and urticaria. The management of food allergy and intolerance chiefly involves elimination diets, accurate diagnosis and detecting the causative mechanism providing us with ample food for thought. In this article, we have attempted to summarize and simplify the research on the various aspects of food allergy and intolerance, and discuss the natural history, manifestations, mechanisms, diagnosis, and management of this condition affecting countless worldwide.