J.G. White

Phosphatidic acid releases calcium from a platelet membrane fraction in vitro

A platelet membrane fraction which actively sequesters calcium in the presence of ATP was prepared and the influence of phosphatidic acid evaluated. At 10--60 micrograms/ml phosphatidic acid caused a concentration dependent release of calcium from the membrane fraction. The calcium was released from inside the vesicles, since release occurred in the presence of EGTA used to bind calcium outside the membrane vesicles. Aspirin failed to inhibit release of calcium by phosphatidic acid. Our results may explain, in part, the prostaglandin and thromboxane independent calcium release which occurs in response to certain aggregating agents. Thus, phosphatidic acid, or a metabolite, may have an important role intracellularly in platelets in promoting calcium movement.

Platelet storage pool deficiency and prostaglandin synthesis in chronic granulocytic leukaemia.

Summary. Platelet function was evaluated in eight patients with chronic granulocytic leukaemia (CGL), seven Ph1 positive and one Ph1 negative. Seven of the eight patients’platelets had an absence of the second wave of adrenaline induced aggregation on at least one occasion, while five had impaired collagen aggregation. The platelets of all seven patients with abnormal responses to adrenaline, aggregated with arachidonic acid, thus ruling out a cyclo‐oxygenase deficiency. A marked decrease in the ADP, serotonin, and dense body content of platelets was found in all five patients evaluated. Mixtures of CGL patient platelets with platelets from normal donors who had ingested aspirin gave a normal biphasic response to adrenaline. Normal release of the storage pool contents from aspirin treated platelets was shown by stirring a mixture of CGL platelets and 14C‐serotonin labelled aspirin treated platelets with adrenaline. The CGL platelets alone or in the mixture produced malondialdehyde in response to adrenaline. These experimental results suggest that CGL platelets have a storage pool deficiency but can synthesize prostaglandins and thromboxanes in response to arachidonic acid and adrenaline.

The role of iron in prostaglandin synthesis: Ferrous iron mediated oxidation of arachidonic acid

Arachidonic acid (AA) is the essential substrate for production of platelet endoperoxides and thromboxanes. Iron or heme is an essential cofactor for the peroxidase, lipoxygenase and cyclo-oxygenase enzymes involved in formation of these products. The present study has examined the direct interactions between iron and arachidonic acid. Iron caused the oxidation of AA into more polar products which could be detected by UV absorbtion at 232 nM or the thiobarbituric acid (TBA) reaction. High pressure liquid chromatography, chem-ionization and electron-impact mass spectrometry and nuclear magnetic resonance spectroscopy suggest that the major product was a hydroperoxide of AA. Ferrous iron (Fe++) and oxygen were absolute requirements. Fe++ was converted to the ferric iron (Fe+++) state during oxidation of AA, but Fe+++ could not substitute for Fe++. No other enzymes, cofactors or ions were involved. Conversion of AA to a hydroperoxide by Fe++ was inhibited by the antioxidant, 2, (3)-Tert-butyl-4-hydroxyanisole, the radical scavenger, nitroblue tetrazolium, and iron chelating agents, including EDTA, imidazole and dihydroxybenzoic acid. The reaction was not affected by superoxide dismutase, catalase or aspirin. These findings and preliminary studies of the Fe++ induced oxidation product of AA as a substrate for prostaglandin synthesis and inhibitor of prostacyclin production indicate the critical role of Fe++ in AA activation.

Modification of human platelet response to sodium arachidonate by membrane modulation

Previous studies have shown that the majority of mongrel dogs have platelets that do not aggregate when stirred with sodium arachidonate, but respond normally to other aggregating agents. Here we have created a model in which human platelets mimic the unusual behavior of canine cells. Brief exposure of human platelets to low concentrations of prostacyclin will convert them to a physiologic state resembling that of canine platelets without causing elevation of intracellular levels of cAMP. Studies on the conversion of arachidonic acid showed that prostacyclin did not cause any inhibition of thromboxane B2 generation. Exposure of prostacyclin treated platelets to epinephrine restored their sensitivity to arachidonate without causing any detectable changes in levels of cAMP. The selective inhibition achieved by PGI2 could be mimicked by the endoperoxide analog. U44069, a thromboxane inhibitor, U-51605, and an endoperoxide/thromboxane receptor blocker, 13-APA. Inhibition induced by these compounds was also reversed by epinephrine. A calcium channel blocker, verapamil, and an alpha blocker, dihydroergocryptine, effectively blocked the corrective influence of epinephrine on prostacyclin-treated platelets. Results of these studies show that catecholamines and prostaglandin receptors share close sites on the membrane and exhibit a degree of cooperativity in calcium modulation. Dog platelets that do not respond to products of AA metabolism and the human platelets made to behave like canine platelets may have a defect in calcium mobilization and this defect is corrected by adrenaline through an alpha receptor modulation.

Prostaglandin endoperoxides promote calcium release from a platelet membrane fraction in vitro

A calcium sequestering platelet membrane fraction was prepared and the effect of arachidonic acid, PGG2 and PGH2 on calcium content evaluated. At 4 degrees C, 6.7--16.7 micrometers arachidonic acid caused significant release of calcium from preloaded vesicles. Such release was completely inhibited by aspirin pretreating the platelets from which the membrane fraction was prepared. Gamma-linolenic acid, not a substrate for prostaglandin synthesis, did not cause calcium release. At 37 degrees C, after a 5 minute calcium loading of the membrane vesicles, arachidonic acid, PGG2, and PGH2 caused release of calcium. Calcium release by the PGG2 and PGH2 was only slightly inhibited by aspirin. Imidazole, which prevented conversion of the prostaglandin endoperoxides to thromboxanes, also only slightly inhibited calcium release. Other prostaglandins including PGD2, PGE1, PGE2 and PGD2 had no effect on the calcium content of the vesicles. These studies suggest that PGG2 and PGH2 may exert their effects on platelets by mobilizing calcium from an internal membrane store to make it available to promote platelet activation.

Vitamin E inhibits the release of calcium from a platelet membrane fraction in vitro

Abstract 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 phospholipase 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.

Influence of trifluoperazine on platelet aggregation and disaggregation

Calmodulin has been shown to activate platelet phospholipase A2 and initiate the metabolism of arachidonic acid. Trifluoperazine (stelazine), an agent which selectively binds calmodulin, inhibits phospholipase activity induced by calcium. We have evaluated the effect of this agent on the aggregation, disaggregation and reaggregation of human platelets. Results of our study have shown that stelazine is a selective inhibitor of the second wave response of platelets to the stimulation of agonists. The compound causes dissociation of aggregated platelet clumps and induces a refractory state in dispersed cells. Epinephrine reverses the refractory state and potentiates the response of dissociated cells to the action of thrombin, arachidonate and ionophore A23187. Stelazine elevates cAMP levels to some extent and inhibits release of labeled arachidonic acid from platelet membranes in response to thrombin induced stimulation. Epinephrine may correct stelazine induced inhibition by acting through alpha-adrenergic mechanism, by inducing changes in membrane permeability to calcium, by releasing membrane associated calcium or by directly promoting deacylation of phospholipids.

Platelet aggregation independent of adp release or prostaglandin synthesis in patients with Hermansky-Pudlak syndrome

Platelets from patients with the Hermansky-Pudlak (HPS) syndrome are deficient in the storage pool of adenine nucleotides and serotonin. As a result, the storage pool deficient (SPD) platelets develop only single waves of clumping when stimulated by threshold concentrations of aggregating agents which cause irreversible, biphasic aggregation of normal platelets. Yet, patients with HPS either have no bleeding problems or only mild symptoms. In the present study we have evaluated the importance of prostaglandin synthesis and secretion to the irreversible aggregation of HPS platelets. Results of the study demonstrate that aspirin-treated SPD platelets, which cannot form thromboxane or undergo the release reaction on stimulation by arachidonate, can still undergo irreversible aggregation in response to thrombin and ADP if treated first with epinephrine. A mechanism of membrane modulation mediated by alpha-adrenergic receptors cooperatively linked to the endoperoxide and thromboxane receptor can secure irreversible aggregation of normal or abnormal platelets despite absence of secretion and prostaglandin synthesis.

Labile aggregation stimulating substance (LASS): the factor from storage pool deficient platelets correcting defective aggregation and release of aspirin treated normal platelets.

Summary Mixtures of equal volumes of Hermansky‐Pudlak (HP) syndrome (adenine nucleotide storage pool deficient) and aspirin treated (AT) platelets (inhibited release of adenine nucleotides) undergo irreversible aggregation when exposed to collagen or adrenaline, whereas neither alone will do so. The present investigation has explored the basis for the mutual correction. Correction in the mixed system was accompanied by release of significant amounts of [14C]5‐hydroxy‐tryptamine from the AT platelets, whereas very little isotope was released when AT platelets alone were exposed to collagen. The correction of aggregation and the release of isotope could both be suppressed by pre‐treating HP platelets with aspirin. Labile aggregation stimulating substance (LASS) composed of two closely linked intermediates of prostaglandin (PG) E2 and PGF2a biosynthesis, produced using a microsomal fraction of HP platelets, could correct the aggregation and secretion defect of AT platelets exposed to collagen. These findings indicate that HP platelets when mixed with AT platelets and exposed to collagen secrete a substance which is responsible for the correction. LASS, identified as the factor involved, acted as an intercellular messenger which mediated the correction by overcoming the influence of aspirin on normal cells.

Ferrous iron mediated oxidation of arachidonic acid: studies employing nitroblue tetrazolium (NBT).

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.