Role of the 5-lipoxygenase-activating protein (FLAP) in murine acute inflammatory responses.

Leukotrienes are potent inflammatory mediators synthesized from arachidonic acid (AA) predominately by cells of myeloid origin. The synthesis of these lipids is believed to be dependent not only on the expression of the enzyme 5-lipoxygenase (5-LO), which catalyzes the first steps in the synthesis of leukotrienes, but also on expression of a nuclear membrane protein termed the 5-LO-activating protein (FLAP). To study the relationship of these two proteins in mediating the production of leukotrienes in vivo and to determine whether the membrane protein FLAP has additional functions in various inflammatory processes, we have generated a mouse line deficient in this protein. FLAP-deficient mice develop normally and are healthy. However, an array of assays comparing inflammatory reactions in FLAP-deficient mice and in normal controls revealed that FLAP plays a role in a subset of these reactions. Although examination of DTH and IgE-mediated passive anaphylaxis showed no difference between wild-type and FLAP-deficient animals, mice without FLAP possessed a blunted inflammatory response to topical AA and had increased resistance to platelet-activating factor-induced shock compared to controls. Also, edema associated with Zymosan A-induced peritonitis was markedly reduced in animals lacking FLAP. To determine whether these differences relate solely to a deficit in leukotriene production, or whether they reflect an additional role for FLAP in inflammation, we compared the FLAP-deficient mice to 5-LO-deficient animals. Evaluation of mice lacking FLAP and 5-LO indicated that production of leukotrienes during inflammatory responses is dependent upon the availability of FLAP and did not support additional functions for FLAP beyond its role in leukotriene production.

Collagen-induced arthritis is reduced in 5-lipoxygenase-activating protein-deficient mice.

Collagen-induced arthritis in the DBA/1 mouse is an experimental model of human rheumatoid arthritis. To examine the role of leukotrienes in the pathogenesis of this disease, we have developed embryonic stem (ES) cells from this mouse strain. Here, we report that DBA/1 mice made deficient in 5-lipoxygenase-activating protein (FLAP) by gene targeting in ES cells develop and grow normally. Zymosan-stimulated leukotriene production in the peritoneal cavity of these mice is undetectable, whereas they produce substantial amounts of prostaglandins. The inflammatory response to zymosan is reduced in FLAP-deficient mice. The severity of collagen-induced arthritis in the FLAP-deficient mice was substantially reduced when compared with wild-type or heterozygous animals. This was not due to an immunosuppressive effect, because anti-collagen antibody levels were similar in wild-type and FLAP-deficient mice. These data demonstrate that leukotrienes play an essential role in both the acute and chronic inflammatory response in mice.

Kinetics of single-layer graphite oxidation: evaluation by electron microscopy.

Etch-decoration reveals that the rate of removal of carbon atoms exposed at monolayer steps on graphite surfaces is very different from the rate of removal, under identical conditions, at multilayer steps. At 1113 degrees K and a pressure of 1.33 newtons per square meter of oxygen, the rate of oxidation (along the layer planes) is less by a factor of nearly 100 than that at multilayer steps.

Does the frequency of routine follow-up after curative treatment for head-and-neck cancer affect survival?

Background: Routine follow-up is a cornerstone of oncology practice, but evidence to support most aspects of follow-up is lacking. Our objective was to investigate the relationship between frequency of routine follow-up and survival. Methods: This population-based study used electronic health care data relating to 5310 patients from Ontario diagnosed with squamous-cell head-and-neck cancer during 2007-2012. Treatments included surgery (24.6%), radiotherapy with or without chemotherapy (52.4%), and combined surgery and radiotherapy (23%). We determined the oncologist who was following each patient after treatment; calculated the average follow-up visits to the oncologist during the subsequent 2.5 years for all patients who were doing well; and used Kaplan-Meier and multiple variable regression analysis to compare, by treatment, overall survival for patients in the high, typical, and low follow-up oncologist groups. Results: Many oncologists saw patients 40%-80% more often than other oncologists did. No relationship of appointment frequency with survival was observed for patients in any treatment group. Conclusions: The practice of routine follow-up varies and is costly both to a health care system and to patients. Without evidence about the effectiveness of current policies, further research is required to investigate new or optimal practices.

Variation in routine follow-up care after curative treatment for head-and-neck cancer: a population-based study in Ontario.

Background: The actual practices of routine follow-up after curative treatment for head-and-neck cancer are unknown, and existing guidelines are not evidence-based. Methods: This retrospective population-based study used administrative data to describe 5 years of routine follow-up care in 3975 head-and-neck cancer patients diagnosed between 2007 and 2012 in Ontario. Results: The mean number of visits per year declined during the follow-up period (from 7.8 to 1.9, p < 0.001). The proportion of patients receiving visits in concordance with guidelines ranged from 80% to 45% depending on the follow-up year. In at least 50% of patients, 1 head, neck, or chest imaging test was performed in the first follow-up year; that proportion subsequently declined (p < 0.001). Factors associated with follow-up practices included comorbidity, tumour site, treatment, geographic region, and physician specialty (p < 0.05). Conclusions: Given current practice variation and the absence of an evidence-based standard, the challenge in identifying a single optimal follow-up strategy might be better addressed with a harmonized approach to providing individualized follow-up care.

Oxidative ß-C-H sulfonylation of cyclic amines.

A transition metal-free strategy for the dehydrogenative ß-sulfonylation of tertiary cyclic amines is described. N-Iodosuccinimide facilitates regioselective oxidative sulfonylation at C-H bonds positioned ß to the nitrogen atom of tertiary amines, installing enaminyl sulfone functionality in cyclic systems. Mild reaction conditions, broad functional group tolerance and a wide substrate scope are demonstrated. The nucleophilic character of the enaminyl sulfone is harnessed, demonstrating potential application for scaffold diversification.

C1-esterase inhibitor deficiency and elective caesarean section.

C1-esterase inhibitor deficiency is a rare disorder of the complement system characterised by episodes of cutaneous and mucosal oedema. Life-threatening airway oedema can follow airway instrumentation or minor trauma. We describe the successful management of a 37-year-old primiparous woman with inherited C1-esterase inhibitor deficiency who was admitted at 38 weeks' gestation for elective caesarean section. Whilst undergoing general anaesthesia 18 months previously she had experienced facial and pharyngeal oedema despite prophylaxis (one unit of fresh frozen plasma). On this occasion she underwent elective caesarean section following intrathecal anaesthesia with 0.5% hyperbaric bupivacaine 2 mL and diamorphine 300 microg. Cardiovascular stability was ensured using glycopyrolate and intravenous Hartmann's solution 2 L; a live female infant was delivered successfully. There were no peri- or postoperative complications. Regional anaesthesia is the safest method for providing surgical anaesthesia in the obstetric patient. We believe elective caesarean section under regional anaesthesia should be considered if there are predicted difficulties with vaginal delivery.

Formation of 6-keto prostaglandin E1 in mammalian kidneys.

1 The metabolism of prostacyclin (PGI2) and 6-keto prostaglandin F1 alpha (6-keto PGF1 alpha) was studied in cell-free homogenates of rat, rabbit and guinea-pig kidney. 2 Rabbit kidney converted both PGI2 and 6-keto PGF1 alpha to a stable metabolite with chromatographic and biological activity identical to that of authentic 6-keto PGE1. Activity was found in the kidney cortex but not medulla, was inhibited by NAD+ or NADP+ (5 mM) and showed an optimum temperature requirement of 37 degrees C. 3 Guinea-pig kidney converted PGI2 but not 6-keto PGF1 alpha to a labile, biologically active metabolite which was not 6-keto pge1. 4 No conversion of prostacyclin or 6-keto PGF1 alpha to biologically active metabolites occurred in cell-free homogenates of rat kidney, liver and colon or guinea-pig liver and colon. 5 6-keto PGE1 rapidly lost spasmogenic activity on the rat stomach strip following incubation with rabbit or guinea-pig kidney supernatant in the absence of added cofactors. No loss of activity occurred on incubation with rat kidney. 6 Rutin (50 microM) potently inhibited synthesis of 6-keto PGE1 from added PGI2 by rabbit kidney cortex. This reaction was potentiated by a similar concentration of sulphasalazine, carbenoxolone, imidazole, papaverine or indomethacin. 7 The relevance of these findings for the possible physiological and pathological roles of 6-keto PGE1 in the kidney is discussed.

Conversion of prostacyclin to 6 oxo prostaglandin E1 by rat, rabbit, guinea-pig and human platelets.

The enzymatic catabolism of prostacyclin (PGI2) to 6 oxo prostaglandin E1 (6 oxo PGE1) was studied in platelet-rich and platelet-poor-plasma of rat, rabbit, guinea-pig and man. Rat, rabbit and human platelets convert PGI2 to a product with biological activity and thin layer chromatographic mobility identical to that of authentic 6 oxo PGE1. Platelets from these species also converted 9 beta-[3H]-PGI2 to non-radioactive 6 oxo PGE1 as shown by the progressive loss of extracted radioactivity following incubation. Formation of 6 oxo PGE1 was inhibited by the flavonoid drugs, rutin and naringenin. Guinea-pig platelets did not convert PGI2 to 6 oxo PGE1. Rat, rabbit and guinea-pig platelets do not spontaneously release a 6 oxo PGE1-like substance when incubated at 37 degrees C in the absence of added PGI2 or aggregating agents. The relevance of these findings to the possible physiological and pathophysiological roles of 6 oxo PGE1 in the regulation of platelet function is discussed.

A modified mouse air pouch model for evaluating the effects of compounds on granuloma induced cartilage degradation.

1. Employing rat femoral head cartilage implanted in a 6 day old mouse air pouch, the effects of inflammatory stimuli (i.e. cotton pellets, carrageenan, zymosan) on the loss of proteoglycan and collagen and granuloma formation have been studied. 2. Wrapping of the cartilage in cotton resulted in granuloma formation with accelerated loss of proteoglycan and collagen over the 14 day implantation period. The amount of loss increased with increasing weight of cotton. 3. The effects of different classes of anti-rheumatic drugs on granuloma formation and proteoglycan and collagen loss from cotton wrapped femoral head cartilage in the mouse air pouch have been studied. 4. Non-steroidal anti-inflammatory drugs (NSAIDs) had no influence on granuloma formation, but in general accelerated the rates of proteoglycan and collagen loss. 5. Dexamethasone and prednisolone significantly reduced granuloma formation and had a marked protective effect on cartilage breakdown. 6. Of the slow acting anti-rheumatic drugs examined, only gold sodium thiomalate (GSTM) and dapsone significantly decreased cartilage loss, with an accompanying modest decrease in granuloma formation. 7. The immunosuppressants cyclophosphamide and methotrexate, but not azathioprine, reduced cartilage degradation, but had no effect on granuloma formation. 8. The results for the different classes of anti-inflammatory and anti-rheumatic drugs are discussed in relation to their effects in other animal models and their reported therapeutic activities in man. It is concluded that the mouse air pouch method as described offers advantages as an animal model over existing procedures to predict therapeutic efficacy in man.

Identification of 6-oxo-prostaglandin E1 as a naturally occurring prostanoid generated by rat lung.

The spontaneous release of prostanoids from rat isolated perfused lungs was studied after acid/organic extraction of perfusates by bioassay, radioimmunoassay, thin layer and high performance liquid chromatographic methods and by gas chromatography-negative ion mass spectroscopy (g.c.n.i.m.s.). An acid/organic extractable anti-aggregatory vasodilator prostaglandin which inhibited the twitch response of the field-stimulated guinea-pig vas deferens was released from the Krebs-perfused rat lung in nanogram amounts similar to those of other detected prostanoids. Parallel biological assay suggested that this prostaglandin had very closely similar pharmacological activity to authentic 6-oxo-prostaglandin E1 (6-oxo-PGE1), a metabolite of prostacyclin (PGI2) generated by the action of the enzyme 9-hydroxyprostaglandin dehydrogenase (9-PGDH). 6-oxo-PGE1 was identified conclusively in extracts of rat lung perfusate by thin layer chromatography, high performance liquid chromatography and g.c./m.s. combined with bioassay (inhibition of platelet aggregation), and its covalent structure was defined by g.c. negative ion chemical ionization mass spectroscopy. The rank order of spontaneous release of prostanoids (measured by radioimmunoassay) from the perfused rat lung was 6-oxo-PGF1 alpha greater than thromboxane B2 (TXB2) greater than PGE2 greater than 6-oxo-PGE1 (measured biologically) greater than PGF2 alpha. Release of all five prostanoids was inhibited by indomethacin, but only that of 6-oxo-PGE1 was inhibited by naringenin. Rat lung 100,000 g cytosolic supernatants contained 9-PGDH activity capable of removing 9 beta-tritium from labelled prostacyclin and forming an acid/organic extractable 6-oxo-PGE1-like anti-aggregatory substance. This 9-PGDH activity was inhibited by naringenin (IC50 10.3 microM). 6 The relevance of these findings to the possible physiological role of 6-oxo-PGE1 in the lung is discussed, and we propose that 6-oxo-PGE, should be accorded the status ofa physiologically relevant, naturally occurring metabolite of arachidonic acid.

Regulation of tumour necrosis factor production by adrenal hormones in vivo: insights into the antiinflammatory activity of rolipram.

1. The role of adrenal hormones in the regulation of the systemic and local production of tumour necrosis factor (TNF alpha) was examined in male Balb/c mice. 2. Intraperitoneal injection of 0.3 mg E. coli lipopolysaccharide (LPS, 0111:B4) led to high levels of circulating TNF alpha without stimulating TNF alpha production in the peritoneal cavity. Systemic production of TNF alpha in response to LPS was increased in adrenalectomized animals and in normal animals treated with the beta-adrenoceptor antagonist, propranolol. The glucocorticoid antagonist, RU 486, did not modify systemic TNF alpha production. These results indicate that systemic TNF alpha production is regulated by adrenaline but not by corticosterone. 3. When mice were primed with thioglycollate, TNF alpha was produced in the peritoneal cavity in response to low dose LPS (1 micrograms). The levels of TNF alpha in the peritoneal cavity were not enhanced by adrenalectomy or by treatment with either propranolol or RU 486, indicating local production of TNF alpha in the peritoneal cavity is not regulated by adrenaline or corticosterone. 4. The phosphodiesterase type IV (PDE-IV) inhibitor, rolipram, inhibited both the systemic production of TNF alpha in response to high dose endotoxin (ED50 = 1.3 mg kg-1) and the local production of TNF alpha in the peritoneal cavity in response to low dose endotoxin (ED50 = 9.1 mg kg-1). In adrenalectomized mice there was a slight reduction in the ability of rolipram to inhibit the systemic production of TNF alpha (ED50 = 3.3 mg kg-1) while the ability of rolipram to inhibit the local production of TNF alpha in the peritoneal cavity was virtually abolished (24% inhibition at 30 mg kg-1). The glucocorticoid antagonist, RU 486, also reduced the ability of rolipram to inhibit local TNF alpha production while propranolol was without effect. 5. Systemic treatment with rolipram increased the plasma concentrations of corticosterone in normal mice but not in adrenalectomized mice indicating that rolipram can cause adrenal stimulation in vivo. 6. In summary, these data indicate that systemic production of TNF alpha in response to high dose endotoxin is controlled differently from the local production of TNF alpha in response to low dose endotoxin. The systemic production of TNF alpha is regulated by catecholamines, but not by corticosterone, while the local production of TNF alpha in the peritoneal cavity is not regulated by basal levels of either catecholamines or corticosterone. 7. These data also show that the ability of rolipram to inhibit the local production of TNF alpha is dependent on the release of corticosterone from the adrenal glands.

The effect of some flavone drugs on the conversion of prostacyclin to 6-oxoprostaglandin E1.

Flavonoid drugs (rutin, naringenin and quercetin) were compared with indomethacin and sulphasalazine as inhibitors of rat and rabbit renal 9-hydroxyprostaglandin dehydrogenase, prostaglandin synthesis (bovine seminal vesicle microsomes) and inactivation (rabbit colon 100,000 g supernatant), vascular PGI2 formation (rat aortic rings) and for effects on platelet aggregation and on the isolated rat stomach strip. Rutin and naringenin potently inhibited rabbit renal conversion of PGI2 and PGF2 alpha to 6-oxoPGE1 and PGE2, respectively, but had no effect on rat renal 9-hydroxyprostaglandin dehydrogenase activity, prostaglandin breakdown, vascular PGI2 synthesis, platelet aggregation or the anti-aggregatory effect of PGI2 and 6-oxoPGE1. High concentrations (100 microM) of both drugs inhibited the spasmogenic effect of PGI2 on the rat stomach strip. Naringenin and quercetin (1 mM) inhibited whilst rutin (1 mM) stimulated microsomal prostaglandin synthesis. These results suggest that rutin and naringenin may be useful experimental tools to study the biological roles of 6-oxoPGE1.

Enzymatic inactivation of 6-keto-prostaglandin E1 in vitro: comparison with prostaglandin E1.

The inactivation of 6-keto PGE1, a biologically active and stable metabolite of prostacyclin, was studied in 100,000 g cytosolic supernatants by bioassay on rat stomach strip (contraction) and human platelets (inhibition of ADP-induced aggregation). PGE1 was used as a reference compound. Both PGs were inactivated in supernatants from colon, kidney and liver of rat, rabbit and guinea-pig. Inactivation was time- and NAD+ -dependent and was generally greater for PGE1 than 6-keto-PGE1. The enzyme responsible for 6-keto-PGE1 inactivation in cytosolic supernatants is distinct from prostaglandin 15-hydroxydehydrogenase and 9-keto reductase, is not inhibitable by sulphasalazine-like drugs and its activity is recoverable after precipitation by ammonium sulphate. We conclude that 6-keto-PGE1 can be inactivated by enzymes with wide tissue distribution, but further studies are needed for identification of these novel enzymes and the products formed as well as to assess their significance in the intact animal.

Age-dependent changes in the synthesis and catabolism of 6 oxo PGE1 and other prostanoids by the rat kidney in vitro.

Synthesis and catabolism of 6 oxo PGE1 was assessed in 100,000 g cell-free supernatant fractions of kidneys obtained from rats aged 20, 34 and 70 days. In addition the release of PGI2, TxA2 (measured as 6 oxo PGF1 alpha and TxB2, respectively), PGE2 and PGF2 alpha from kidney slices prepared from these three groups of rats was determined using specific radioimmunoassays. The conversion of PGI2 to 6 oxo PGE1 (but not 13,14 dihydro 15 oxo PGF2 alpha to 13,14 dihydro 15 oxo PGE2) was detected in supernatant fractions of kidneys from 20 day rats. Slices prepared from the kidneys of these animals spontaneously released significant amounts of three prostanoids (6 oxo PGF1 alpha greater than PGE2 greater than PGF2 alpha greater than TxB2 = 0). No formation of 6 oxo PGE1 from exogenous PGI2 was demonstrated in renal 100,000 g supernates from 34 and 70 day rats even though these supernates avidly oxidised 13,14 dihydro 15 oxo PGF2 alpha to 13,14 dihydro 15 oxo PGE2. In these animals the rank order of prostanoid release from kidney slices was PGE2 greater than 6 oxo PGF1 alpha greater than PGF2 alpha greater than TxB2 = 0. The catabolism of 6 oxo PGE1 is also age-dependent. In 20 and 34 day old rats 6 oxo PGE1 and PGE1 incubated with renal 100,000 g supernates undergo loss of biological activity as determined by the ability to inhibit ADP induced human platelet aggregation. In contrast, kidney 100,000 g supernates prepared from 70 day rats convert 6 oxo PGE1 to an unidentified metabolite with more potent anti-aggregatory activity. The possibility that 6 oxo PGE1 has a biological role in the developing rat kidney is discussed.

The effect of sepsis rates of closing and cleaning hospital wards.

Sepsis rates were studied in five hospital wards before and after closure for cleaning. Each ward was closed because of an outbreak of Staph. aureus infection caused by a cloxacillin-resistant strain. The study shows that sepsis rates, especially sepsis caused by hospital strains of Staph. aureus, were greatly reduced in the three-month period following re-opening of the ward, provided that patients infected with such organisms were not readmitted to or allowed to remain in the ward.

Induction of hepatic microsomal drug-metabolizing enzymes by inhibitors of 5-lipoxygenase (5-LO): studies in rats and 5-LO knockout mice.

The effect of 5-lipoxygenase (5-LO) inhibitors on the hepatic microsomal mixed-function oxidase (MFO) system of rodents was investigated. After establishing the relative in vitro and in vivo potencies of the 3 test compounds, male Crl:CD (SD) BR rats received CJ-11,802 (0, 10, 50, or 200 mg/kg/day), zileuton (0, 10, 60, or 300 mg/kg/day) or ZD2138 (0 or 200 mg/kg/day) once daily by oral gavage for 14 (zileuton and ZD2138) or 30 (CJ-11,802) consecutive days. Controls were given an equivalent volume of 0.5% methylcellulose vehicle. At necropsy, all livers were weighed, and sections from representative animals (control and highest dose for each compound) were utilized to prepare hepatic microsomal fractions, which were assayed for cytochrome P-450 (CYP) content and the activities of cytochrome c reductase (CRed), para-nitroanisole O-demethylase (p-NOD), ethoxyresorufin O-deethylase (EROD), and pentoxyresorufin O-dealkylase (PROD). A dose-related increase in liver weight occurred in rats given CJ-11,802 and zileuton, while animals administered ZD2138 were unaffected. Rats given CJ-11,802 (200 mg/kg/day) and zileuton (300 mg/kg/day) had increases in CYP, EROD, PROD, CRed and p-NOD compared to corresponding controls, while only the latter two activities were elevated in animals administered ZD2138. To determine if induction of the hepatic microsomal MFO system was related to 5-LO inhibition, male DBA wild-type and 5-LO knockout mice were administered either CJ-11,802 (200 mg/kg/day) or vehicle by oral gavage for 14 consecutive days. At necropsy, liver weight, CYP content, and CRed activity were measured and all were increased similarly in the treated wild-type and knockout mice compared to corresponding controls, indicating that induction was not related to inhibiting 5-LO.

Catabolism of 6 keto PGE1: biological activation by the rat kidney in vitro.

An increase in anti-aggregatory but not spasmogenic activity was observed when 6 keto prostaglandin E1 (but not PGE1) was incubated at 37 degrees C with rat kidney 100 000 X g supernatant. No such biological activation was observed in boiled rat kidney supernatant. After high-pressure liquid chromatography two absorbance peaks with anti-aggregatory activity were detected. One peak had a retention time identical to authentic 6 keto prostaglandin E1 whilst the second peak did not coincide with known anti-aggregatory prostaglandins.

Interaction of prostaglandins and clonidine in the rat vas deferens.

Stimulation of presynaptic alpha 2-adrenoceptors by clonidine may lead to local synthesis of prostaglandins which contribute to the inhibition of noradrenaline release observed with this drug. The present investigation was undertaken to determine the role of prostaglandins in the effect of clonidine and xylazine on the rat vas deferens. Both drugs inhibited the twitch response to field stimulation in this preparation. Inhibition was reversed by yohimbine. This effect of clonidine (but not xylazine) was reduced by preincubating vasa deferentia in Krebs containing indomethacin for 1h. Clonidine (but not xylazine) stimulated the synthesis of prostaglandin-like activity in pieces of intact vas deferens incubated in Krebs containing arachidonic acid. Such stimulation was prevented by inclusion of yohimbine (but not prazosin) in the incubation medium. Clonidine did not stimulate prostaglandin synthesis in a cell-free preparation of sheep seminal vesicle microsomes incubated with arachidonic acid or inhibit PGE2 catabolism by purified swine lung 15-PGDH. We conclude that clonidine (but not xylazine) stimulates prostaglandin synthesis possibly by activating phospholipase activity and releasing arachidonic acid from membrane phospholipids. This effect on prostaglandin production is secondary to activation of alpha 2-adrenoceptors.

The effect of 6-oxo-prostaglandin E1 on human platelet aggregation in whole blood in-vitro.

The effect of PGI2, 6-oxo-PGE1 and PGE1 on ADP-induced human platelet aggregation has been assessed in whole blood and in blood centrifuged to prepare platelet-rich plasma (PRP). PGI2 was the most potent anti-aggregatory agent in both media. The concentration of PGI2 required to produce 50% inhibition of platelet aggregation was approximately 0.3 ng ml-1 in each case. In contrast both E series prostaglandins exhibited significantly greater (400-700%) anti-aggregatory activity when tested in whole blood than when tested in PRP. Since whole blood presumably represents a truer reflection of platelet reactivity in-vivo, we believe that the potency of 6-oxo-PGE1 (and PGE1) as inhibitors of platelet aggregation has been underestimated in previous experiments using PRP. In human whole blood 6-oxo-PGE1 has approximately 40% the anti-aggregatory activity of PGI2. The reasons for the increased anti-aggregatory potency of E series prostaglandins in whole blood is not known. We suggest that 6-oxo-PGE1 and PGE1 (but not PGI2) may prevent the release of pro-aggregatory ADP from red blood cells thereby enhancing their ability to inhibit platelet aggregation.