Biomedical subjects
Publications and source records attributed to J M Gerrard.
Levels of prostaglandins in serum, plasma and middle ear effusions (MEE) in chinchilla were measured by radioimmunoassay. Higher levels of PGE2 and PGF2alpha were observed in the POM group than in the SOM group. Prostaglandins appear to play important roles as a mediator of the inflammatory response in experimentally induced purulent otitis media.
Vitamin E, an inhibitor of platelet aggregation, was evaluated for its effects on platelet intracellular calcium flux. These studies used a platelet membrane fraction containing membranes of the dense tubular system which actively sequesters calcium in the presence of ATP and magnesium. After these membrane vesicles have accumulated calcium, the cation can be released by addition of the calcium ionophore A23187. Vitamin E had no effect on uptake of calcium by the membrane vesicles, but showed a concentration dependent inhibition of the release of calcium induced by A23187. In similar or slightly higher concentrations than inhibited calcium release, vitamin E also inhibited platelet aggregation, internal contraction and secretion, but had no effect on prostaglandin and thromboxane synthesis and potentiated phospholipase A2 activity. It is suggested that vitamin E acts to inhibit platelet internal contraction and secretion by preventing efflux of calcium from the dense tubular system. The potentiation of phospholiplase A2 by vitamin E could be explained by a localized increase of calcium at the site of the phospholipase A2 on the inner side of the dense tubular system membrane proximal to the vitamin E block.
The molecular mechanism by which indomethacin exerts its inhibitory effects on the prostaglandin endoperoxide synthetase enzyme is unknown. In the present study we have explored the possibility that indomethacin might interact with Fe++ in the enzyme to produce its inhibitory effect. For this study we made use of the recent discovery that Fe++ alone can oxidize arachidonic acid, and the interaction of this fatty acid with the metal can be detected by following reduction of nitroblue tetrazolium (NBT) or by conversion of the Fe++ to Fe+++. Indomethacin markedly inhibited NBT reduction in the presence of arachidonic acid and Fe++ when the indomethacin had been preincubated with the Fe++. Indomethacin also inhibited the conversion of Fe++ to Fe+++ by arachidonic acid. Results obtained by varying the concentrations of indomethacin and arachidonic acid and measuring inhibition of the conversion of Fe++ to Fe+++ by the indomethacin are consistent with a one to one complex forming between indomethacin and Fe++. The complex between indomethacin and Fe++ separates on prolonged incubation of the complex with arachidonic acid. The nature of the binding is suggested by a molecular model. Our results suggest that indomethacin may act to inhibit the prostaglandin endoperoxide synthetase enzyme by complexing Fe++ in the enzyme. Ibuprofen and tolmetin, two other prostaglandin synthetase inhibitors, also inhibit the interaction of Fe++ with arachidonic acid suggesting this may be a general mechanism for this type of drug.
Previous studies have shown that NBT and VE together are potent inhibitors of platelet aggregation, secretion and PG synthesis. In this study, we evaluated the capacity of NBT to detect PG synthesis by SVGM. Aspirin pretreatment of SVGM decreased the amount of NBT reduced after addition of arachidonic acid, demonstrating that products generated by the cyclo-oxygenase were involved in NBT reduction. The influence of NBT and VE on PG synthesis by SVGM was then evaluated by measuring malondialdehyde (MDA) production. NBT or VE alone had no significant effect, but together these agents were as effective as aspirin in preventing MDA formation. The effect of NBT and VE on 14C-arachidonic acid conversion was followed by thin layer chromatography and radioscanning. Again, NBT or VE alone were ineffective, whereas the combination was as effective as aspirin in preventing conversion of arachidonic acid. We speculate NBT and VE together inhibit pg synthesis by scavenging a free radical species of arachidonic acid generated in the initial step of fatty acid peroxidation.
A model is proposed for the interaction of arachidonic acid with the heme associated with the cyclo-oxygenase enzyme which synthesizes prostaglandin endoperoxides. According to this concept, arachidonic acid attaches with its carboxylic acid residue to one ligand of the Fe++ in heme, then curls around the outside of the protoporphyrin to react with a molecule of oxygen associated with the ligand of the Fe++ on the other side of the protoporphyrin with addition of O2 at the C11 carbon, ring closure across C8-12, formation of the C9-11 endoperoxide and then addition of a second oxygen at C15 with an allylic shift of the double bond. This concept may resolve several experimental findings relating to the mechanism of prostaglandin synthesis and can account for much of the stereospecificity in the conversion of arachidonic acid to prostaglandin G2.
The synthesis of a prostaglandin endoperoxide analogue, 9,11-azo-13-oxa-15-hydroxyprostanoic acid (AOHP), is described. AOHP was found to block effectively both the thromboxane synthetase and the PGH2/TxA2 receptors in human platelets. It inhibits the platelet aggregation induced by arachidonic acid, 9.11-methanoepoxy-PGH2, PGH2, and TxA2 but does not affect the ADP-induced aggregation in aspirinated platelet-rich plasma. Some of the intermediates for the synthesis of AOHP also are effective in inhibiting platelet aggregation.
Decanoyl-, palmitoyl-, and oleoyl-lysophosphatidic acid (LPA) were studied for their effects on platelet aggregation and intracellular calcium flux. Palmitoyl-LPA and oleoyl-LPA both caused a concentration-dependent aggregation of human blood platelets at concentrations of 12--300 microM. Aggregation by adenosine diphosphate (ADP) was enhanced at slightly lower concentrations. First-wave aggregation induced by these LPAs was not blocked by aspirin, indomethacin, or heparin, suggesting similarities to ADP aggregation. However, in washed platelets with a high calcium concentration, no serotonin secretion was observed, even though full aggregation occurred, suggesting that aggregation was not due to released ADP. This concept was supported by studies of platelets deficient in the storage pool of ADP and serotonin, which had a normal first-wave aggregation response to palmitoyl-LPA. Aggregation induced by palmitoyl LPA was inhibited by prostaglandin E1 (PGE1), theophylline, and ethylenediaminotetraacetate (EDTA), though in the presence of EDTA shape change occurred. Aggregation stimulated by palmitoyl-LPA or oleoyl-LPA was characterized by changes in the shape of the platelets with development of pseudopods and centralization of granules closely surrounded by contractile microfilaments and supporting microtubules. The addition of palmitoyl-LPA and oleoyl-LPA, but not decanoyl-LPA, caused the release of calcium from a platelet membrane fraction that contains elements of the intracellular calcium storage system and actively concentrates this cation in the presence of adenosine triphosphate (ATP) and magnesium. It is suggested that LPAs cause aggregation by stimulating the release of calcium intracellularly.
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Serum complement studies were carried out in five children with acute lymphocytic leukemia. During therapy with L-asparaginase, prednisone and vincristine, hypocomplementemia developed in all patients, and disappeared within 2 weeks after the discontinuation of L-asparaginase. Complement breakdown products were not present in plasma. The changes of serum complement levels paralleled those of plasma fibrinogen. These findings suggest that the hypocomplementemia observed in these patients may be related to impaired protein synthesis induced by L-asparaginase.
In order to explore a possible relationship between platelet aggregation induced by lysophosphatidic acid (LPA) and that induced by adenosine diphosphate (ADP), we have studied the influence of palmitoyl-LPA (P-LPA) on platelets from dogs and rabbits and on human platelets made refractory to LPA. Dog platelets did not aggregate with P-LPA alone, but P-LPA enhanced ADP aggregation, and after a small dose of ADP, P-LPA was itself effective in causing aggregation and internal contraction in dog platelets. Rabbit platelets showed no response to P-LPA alone, but, as with dog platelets, P-LPA enhanced ADP aggregation. In addition, when P-LPA was added during or immediately after ADP aggregation, it caused a contraction within the platelets and a small wave of aggregation by itself. P-LPA added to human platelets caused aggregation without the need for ADP. However, when a small dose of P-LPA was added to human platelets and the wave of aggregation was allowed to reverse, these platelets subsequently were unresponsive to P-LPA, although they showed an enhanced response to ADP. The addition of a small dose of ADP to the P-LPA refractory platelets partially reversed the refractory state, and the platelets then showed aggregation with P-LPA. The results demonstrate that ADP and P-LPA have significant interactions in their effects on platelets. These interactions are discussed in terms of a two-component mechanism for the ADP-induced intracellular calcium flux, LPA, or possibly phosphatidic acid, being one component.
We have evaluated the influence of NBT, vitamin E, and the combination of NBT and vitamin E on the fine structure and biochemistry of platelets during incubation, and the effects of these compounds on the aggregation and secretion of platelets stimulated by collagen, thrombin, epinephrine, and ADP. Results demonstrate that NBT and vitamin E, rather than injuring platelets, appear to protect them during incubation. Togheter NBT and vitamin E blockedaggregation by epinephrine, collagen, and thrombin, but permitted a small first wave stimulated by ADP. This pattern of response to aggregating agents was similar to reactions observed in platelets pretreated with aspirin and indomethacin, both potent inhibitors of platelet prostaglandin synthesis. The findings support the concept that conversion of arachidonic acid to an activated state is an important step in prostaglandin synthesis and that electron transfer or oxidation--reduction reactions are intimately involved in the development of platelet stickiness. Although vitamin E alone does not block prot to regulate formation of endoperoxides and thromboxanes.
Blood platelets contain a variety of contractile protein species, including the glycoprotein alpha-actinin, which is found at the Z disc in skeletal muscle cells. In the present study, we have considered the possibility that alpha-actinin might be one of several previously described platelet surface glycoproteins. Purified anti-alpha-actinin antibody was found to react strongly with partially purified platelet glycoprotein III, weakly with platelet glycoprotein IIb, and not at all with platelet glycoproteins Ib and IV. Platelets from three siblings with thrombasthenia, a disorder characterized by severe deficiency of platelet glycoproteins IIb and III, were found also to be equally deficient in alpha-actinin. These findings indicate that alpha-actinin and glycoprotein III are identical and suggest that this protein may be an anchor point for actin on the inside of the membrane. Combined with ultrastructural studies of normal and thrombasthenic platelets, the new findings provide a clearer understanding of contraction in single cells and small aggregates.
Desquamation of canine arterial endothelium was induced by stripping the luminal surface of arteries with an inflated embolectomy catheter. The dogs were killed by intravascular perfusion fixation at intervals varying from 2 hours to 12 weeks after the procedure, and the arteries were studied by light microscopy, transmission electron microscopy, and scanning electron microscopy. A heretofore unreported, apparently specific interaction of aggregated platelets and polymorphonuclear leukocytes (PMNs) was observed on the luminal surface of the denuded arteries. This interaction was abolished by the administration of a single dose of aspirin prior to the application of the catheter. These observations suggest that this platelet--PMN interaction is distinctly different from the classic inflammatory reaction, and that it is an integral part of the arterial response to injury that may affect endothelial regeneration.
Much of our current knowledge about the physiology of hemostasis has come from intensive study of platelets from patients with inherited and acquired bleeding disorders or an increased risk of thrombotic disease. Appreciation of the role of plasma proteins in platelet stickiness, of platelet surface membrane glyco-proteins in aggregation, of the substances stored in platelet organelles in cell-cell interaction, vascular injury and atherosclerosis, and of endoperoxides and thromboxanes in platelet intercellular communication have resulted largely from investigations on various types of defective platelets. While the techniques of physiology and biochemistry have generated critical details about abnormal platelets, electron microscopy and ultrastructural cytochemistry have provided an improved morphological framework in which to integrate the new discoveries. The present review has attempted to correlate physiological, biochemical and ultrastructural concepts as they relate to the current understanding of inherited platelet disorders.
The platelet dense tubular system, an internal smooth endoplasmic reticulum membrane system occupies a pivotal position in the initiation and modulation of platelet activation. The best available evidence suggests platelet prostaglandin and thromboxane synthesis and an internal calcium store critical to platelet activation are both found in the platelet dense tubular system. Studies of the structural, physiologic and chemical properties of thromboxane A2 support the concept that this product of platelet prostaglandin synthesis acts to carry the calcium from the dense tubular system into the cytoplasm where the calcium is released to initiate contraction of the platelet contractile protein. Evaluation of platelet phospholipase A2 suggests that calcium may be critical to activation of this enzyme. Two of the steps occurring during platelet activation which appear to require calcium 1) phospholipase A2 acting as the critical carrier of calcium from one location to the other.
A stress test has been designed which shows a consistent abnormality in platelets from carriers of the Wiskott-Aldrich-syndrome (W.A.S.) gene. 2-deoxy-D-glucose (D.D.G.), an inhibitor of glycolysis, completely inhibited second-wave adrenaline (epinephrine)-induced aggregation of platelets from 10 W.A.S. carriers, whereas it had no effect on the response of control platelets. Antimycin A (Ant A), an inhibitor of oxidative phosphorylation, had no effect on adrenaline-induced platelet aggregation of either carriers or controls. Incubation of control platelets with a combination of Ant A and D.D.G. inhibited aggregation in a way comparable to the effect of D.D.G. alone on carrier cells. Thus, W.A.S. carriers have a defect in platelet metabolism similar to that produced in normal platelets with Ant A. The D.D.G. stress test is a simple reproducible assay for detection of W.A.S. carriers.
The oxidation of arachidonic acid by ferrous sulfate provides a useful model to study the role of iron in lipid oxidation reactions. We have employed nitroblue tetrazolium (NBT) in the present investigation to evaluate the mechanism of this reaction. In the presence of arachidonic acid, Fe +++, and O2, the yellow dye NBT was rapidly reduced to the blue form, NBTH2. The molar ratio of arachidonic acid to Fe++ in this rapid reaction was 1:1, showing an interaction of one fatty acid molecule per iron molecule. Approximately one molecule of NBT was reduced per four molecules of arachidonic acid and Fe++. Reduction of NBT was accompanied by oxidation of Fe++ to Fe+++, suggesting the transfer of four electrons from the Fe++ to NBT to reduce the dye. Arachidonic acid was found to be unchanged when extracted at the end of the reaction, indicating formation of a complex that could dissociate leaving intact arachidonic acid. Evidence for the presence of such a complex which slowly dissociates during the reaction was obtained after longer incubations with small amounts of arachidonic acid. NBT reduction was not inhibited by agents which hydrolyze superoxide, by catalase or by agents which trap hydroxy radicals. We, therefore, propose a model in which NBT traps a radical generated on the arachidonic acid molecule. The proposed model suggests mechanisms for other fatty acid oxidation reactions such as prostaglandin and hydroperoxy fatty acid synthesis.
The ability of platelet microsomes to generate platelet aggregating activity on addition of arachidonic acid was evaluated in women taking oral contraceptives and in controls taking no medication but matched for age, sex, and family history. Oral contraceptive users generated significantly more platelet aggregating activity per 100 ug of platelet microsomal protein than controls. Variation in generation of platelet aggregating activity during the menstrual cycle was also observed with highest activity during the third week. These studies show an altered balance of platelet prostaglandin and thromboxane synthesis in oral contraceptive users which may contribute to their increased incidence of thromboembolic phenomena.