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Setting: The Eustachian tube plays a vital role in middle ear physiology. There has been evidence that Eustachian tube (ET) and angle are correlated with middle ear function. The measurements of these Eustachian tube features are now made possible with computed tomography and multiplanar reconstruction techniques. However, there has not been a standardised protocol devised to these measurements in limited window cone-beam CT scans of temporal bones., Objective: The primary object of the present study is to establish and validate a new landmark in closer proximity to the middle ear that is consistently captured, thereby allowing ET angle and length to be measured from the majority of cone-beam CT scans. Secondarily, the ET anatomies of patients with middle ear dysfunction manifesting as acquired cholesteatoma are analysed with this new method of measurement., Methods: This study undertook a step-by-step method to first validate the methods of ET measurement with Reid's standard plane, then identifying an alternative landmark, thus a new plane visible on limited window cone-beam CT scans of temporal bones and lastly, validating the application of this new plane in the measurements of ET angle and length. This new method of measurement was coined the Ku-Copson plane and was applied to 30 cochlear implant patients and 30 patients with acquired cholesteatomas. Their ET anatomies were analysed and compared., Results: It was found that the new Ku-Copson mandibular fossa plane was a reliable and accurate plane for the measurement of ET angle and length. Furthermore, it was found that patients with acquired cholesteatomas have statistically significant smaller ET angles and shorter ET lengths when compared with patients with cochlear implants, of normal middle ear function., Conclusion: The newly proposed method utilising the right mandibular fossa as an anatomical landmark for ET angles and lengths measurement appears to be viable. The close proximity of this landmark to the middle ear means that it is highly likely to be captured in most cone-beam CT scans of the petrous temporal bones. This enables the retrospective examination ET angles and lengths to be conducted on CB CT scans. This study reports statistically significant difference in ET anatomy in patients with middle ear dysfunction., (© 2022. The Author(s).)

Background: Developing since colonisation, Australia's healthcare system has dismissed an ongoing and successful First Nations health paradigm in place for 60,000 years. From Captain James Cook documenting 'very old' First Nations Peoples being 'far more happier than we Europeans' and Governor Arthur Phillip naming Manly in admiration of the physical health of Gadigal men of the Eora Nation, to anthropologist Daisy Bates' observation of First Nations Peoples living 'into their eighties' and having a higher life expectancy than Europeans; our healthcare system's shameful cultural safety deficit has allowed for an Aboriginal and Torres Strait Islander child born in Australia today to expect to live 9 years less than a non-Indigenous child. Disproportionately negative healthcare outcomes including early onset diabetes-related foot disease and high rates of lower limb amputation in Aboriginal and Torres Strait Islander Peoples contribute to this gross inequity., Main Body: In 2020, the Australian Health Practitioner Regulation Authority released the National Scheme's Aboriginal and Torres Strait Islander Health and Cultural Safety Strategy 2020-2025 - empowering all registered health practitioners within Australia to provide health care to Aboriginal and Torres Strait Islander Peoples that is inclusive, respectful and safe, as judged by the recipient of care. This recently released strategy is critically important to the podiatry profession in Australia. As clinicians, researchers and educators we have a collective responsibility to engage with this strategy of cultural safety. This commentary defines cultural safety for podiatry and outlines the components of the strategy in the context of our profession. Discussion considers the impact of the strategy on podiatry. It identifies mechanisms for podiatrists in all settings to facilitate safer practice, thereby advancing healthcare to produce more equitable outcomes., Conclusion: Aboriginal and Torres Strait Islander Peoples access health services more frequently and have better health outcomes where provision of care is culturally safe. By engaging with the National Scheme's Aboriginal and Torres Strait Islander Health and Cultural Safety Strategy, all registered podiatrists in Australia can contribute to achieving equity in health outcomes for Aboriginal and Torres Strait Islander Peoples.

Objectives: To compare the efficacy of in-shoe heel lifts to calf muscle eccentric exercise in reducing pain and improving function in mid-portion Achilles tendinopathy., Methods: This was a parallel-group randomised superiority trial at a single centre (La Trobe University Health Sciences Clinic, Discipline of Podiatry, Melbourne, Victoria, Australia). One hundred participants (52 women and 48 men, mean age 45.9, SD 9.4 years) with clinically diagnosed and ultrasonographically confirmed mid-portion Achilles tendinopathy were randomly allocated to either a (1) heel lifts (n=50) or (2) eccentric exercise (n=50) group. The primary outcome measure was the Victorian Institute of Sport Assessment-Achilles (VISA-A) questionnaire at 12 weeks. Differences between groups were analysed using intention to treat with analysis of covariance., Results: There was 80% follow-up of participants (n=40 per group) at 12 weeks. The mean VISA-A score improved by 26.0 points (95% CI 19.6 to 32.4) in the heel lifts group and by 17.4 points (95% CI 9.5 to 25.3) in the eccentric exercise group. On average, there was a between-group difference in favour of the heel lifts for the VISA-A (adjusted mean difference 9.6, 95% CI 1.8 to 17.4, p=0.016), which approximated, but did not meet our predetermined minimum important difference of 10 points., Conclusion: In adults with mid-portion Achilles tendinopathy, heel lifts were more effective than calf muscle eccentric exercise in reducing pain and improving function at 12 weeks. However, there is uncertainty in the estimate of effect for this outcome and patients may not experience a clinically worthwhile difference between interventions., Trial Registration Number: ACTRN12617001225303., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: The effect of different orthotic materials on plantar pressures has not been systematically investigated. This study aimed to review and summarise the findings from studies that have evaluated the effect of orthotic materials on plantar pressures., Methods: We conducted a systematic review of experimental studies that evaluated the effect of foot orthotic materials or shoe insole materials on plantar pressures using in-shoe testing during walking. The following databases were searched: MEDLINE, CINAHL, Embase and SPORTDiscus. Included studies were assessed for methodological quality using a modified Quality Index. Peak pressure, pressure-time integral, maximum force, force-time integral, contact area, and contact time were variables of interest. Data were synthesised descriptively as studies were not sufficiently homogeneous to conduct meta-analysis. Standardised mean differences (Cohen's d) were calculated to provide the size of the effect between materials found in each study., Results: Five studies were identified as meeting the eligibility criteria. All five studies were laboratory-based and used a repeated measures design. The quality of the studies varied with scores ranging between 20 and 23 on the modified Quality Index (maximum index score 28). The included studies investigated the effects of polyurethane (including PORON®), polyethylene (including Plastazote®), ethyl vinyl acetate (EVA) and carbon graphite on plantar pressures. Polyurethane (including PORON®), polyethylene (including Plastazote®) and EVA were all found to reduce peak pressure., Conclusion: Based on the limited evidence supplied from the five studies included in this review, some orthotic materials can reduce plantar pressures during walking. Polyurethane (including PORON®), polyethylene (including Plastazote®) and EVA reduce peak pressure beneath varying regions of the foot. Future well-designed studies will strengthen this evidence.

Background: Corticosteroid injection is frequently used for plantar heel pain (plantar fasciitis), although there is limited high-quality evidence to support this treatment. Therefore, this study reviewed randomised trials to estimate the effectiveness of corticosteroid injection for plantar heel pain., Methods: A systematic review and meta-analysis of randomised trials that compared corticosteroid injection to any comparator. Primary outcomes were pain and function, categorised as short (0 to 6 weeks), medium (7 to 12 weeks) or longer term (13 to 52 weeks)., Results: A total of 47 trials (2989 participants) were included. For reducing pain in the short term, corticosteroid injection was more effective than autologous blood injection (SMD -0.56; 95% CI, - 0.86 to - 0.26) and foot orthoses (SMD -0.91; 95% CI, - 1.69 to - 0.13). There were no significant findings in the medium term. In the longer term, corticosteroid injection was less effective than dry needling (SMD 1.45; 95% CI, 0.70 to 2.19) and platelet-rich plasma injection (SMD 0.61; 95% CI, 0.16 to 1.06). Notably, corticosteroid injection was found to have similar effectiveness to placebo injection for reducing pain in the short (SMD -0.98; 95% CI, - 2.06, 0.11) and medium terms (SMD -0.86; 95% CI, - 1.90 to 0.19). For improving function, corticosteroid injection was more effective than physical therapy in the short term (SMD -0.69; 95% CI, - 1.31 to - 0.07). When trials considered to have high risk of bias were excluded, there were no significant findings., Conclusions: Based on the findings of this review, corticosteroid injection is more effective than some comparators for the reduction of pain and the improvement of function in people with plantar heel pain. However, corticosteroid injection is not more effective than placebo injection for reducing pain or improving function. Further trials that are of low risk of bias will strengthen this evidence., Registration: PROSPERO registration number CRD42016053216 .

Background: Plantar heel pain is a common foot complaint that causes significant disability and poorer health-related quality of life. Foot orthoses and corticosteroid injection are effective treatments for plantar heel pain; however, it is unclear whether one is more effective than the other., Objective: The aim of this trial was to compare the effectiveness of foot orthoses and corticosteroid injection for plantar heel pain., Methods: In this parallel-group, assessor-blinded, randomized clinical trial, participants received prefabricated, arch-contouring foot orthoses or a single ultrasound-guided corticosteroid injection. The primary outcome measure was the foot pain subscale of the Foot Health Status Questionnaire at 4 and 12 weeks., Results: One hundred three participants aged 21 to 72 years (63 female) with plantar heel pain were recruited from the community and received an intervention. For the primary outcome of foot pain, corticosteroid injection was more effective at week 4 (adjusted mean difference, 8.2 points; 95% confidence interval: 0.6, 15.8 points). However, foot orthoses were more effective at week 12 (adjusted mean difference, 8.5 points; 95% confidence interval: 0.2, 16.8 points). Although these findings were statistically significant, the differences between the interventions did not meet the previously calculated minimal important difference value of 12.5 points., Conclusion: Corticosteroid injection is more effective than foot orthoses at week 4, but this effect does not last; and appropriately contoured foot orthoses are more effective than corticosteroid injection at week 12. However, patients may not notice a clinically worthwhile difference between the interventions., Level of Evidence: Therapy, level 1b. J Orthop Sports Phys Ther 2019;49(7):491-500. Epub 26 May 2019. doi:10.2519/jospt.2019.8807 .

Increasing preferred step rate during running is a commonly used strategy in the management of running-related injuries. This study investigated the effect of different step rates on plantar pressures during running. Thirty-two healthy runners ran at a comfortable speed on a treadmill at five step rates (preferred, ±5%, and ±10%). For each step rate, plantar pressure data were collected using the pedar-X in-shoe system. Compared to running with a preferred step rate, a 10% increase in step rate significantly reduced peak pressure (144.5±46.5 vs 129.3±51 kPa; P=.033) and maximum force (382.3±157.6 vs 334.0±159.8 N; P=.021) at the rearfoot, and reduced maximum force (426.4±130.4 vs 400.0±116.6 N; P=.001) at the midfoot. In contrast, a 10% decrease in step rate significantly increased peak pressure (144.5±46.5 vs 161.5±49.3 kPa; P=.011) and maximum force (382.3±157.6 vs 425.4±155.3 N; P=.032) at the rearfoot. Changing step rate by 5% provided no effect on plantar pressures, and no differences in plantar pressures were observed at the medial forefoot, lateral forefoot or hallux between the step rates. This study's findings indicate that increasing preferred step rate by 10% during running will reduce plantar pressures at the rearfoot and midfoot, while decreasing step rate by 10% will increase plantar pressures at the rearfoot. However, changing preferred step rate by 5% will provide no effect on plantar pressures, and forefoot pressures are unaffected by changes in step rate., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Hyaluronan (HA) stimulates the motility of some but not all cell types. Here, we show that HA-promoted random motility of ras-transformed 10T1/2 (C3) fibroblasts requires activation of protein kinase C and is associated with rapid uptake of HA in a CD44 and RHAMM-dependent manner. The addition of HA to parental 10T1/2 fibroblasts (parental cells) does not stimulate random motility, but these cells can be 'primed' to respond to HA by treatment with the phorbol ester, PMA, for 4-6 h. This effect of PMA requires protein synthesis, PKC activity and is associated with enhanced uptake of HA. These results suggest that the ability of cells to respond to HA is regulated by a protein kinase C-dependent process that may promote uptake of HA.

As part of the Keewatin Health Assessment Study, a comprehensive health interview and examination survey of Inuit and non-Inuit in the central Canadian Arctic during 1990-91, plasma samples were analyzed for phospholipid fatty acid composition. Compared to non-Inuit, the Inuit have reduced levels of dihomo-gamma-linoleic (DGLA) and arachidonic acid (ratios of 0.41 and 0.46) and the sum of all n-6 fatty acids (ratio of 0.65), but increased level of eicosapentaenoic (EPA) acid (ratio of 1.37). These trends are consistent with those reported from other circumpolar Inuit populations, especially the reduced arachidonic acid and increased EPA, although the Inuit excess in EPA is much less pronounced due to the greater importance of caribou rather than sea mammals in most of the Keewatin communities. The high linoleic/arachidonic acid ratio suggests increased inhibition of the metabolic pathway regulated by the enzyme delta-5 desaturase, which can be explained by the presence of high levels of highly unsaturated fatty acids of dietary origin, and/or a genetic deficiency. In multiple linear regression models with the independent variable list consisting of Inuit status, age, sex, education, physical activity, spending time on the land and consumption of wild meat and local fish, Inuit status is independently associated with lower levels of the n-6 acids but not the n-3 acids. This indicates that factors other than diet and lifestyle, perhaps genetic ones, may account for the observed "ethnic" differences. However, for those fatty acids in which Inuit differ from non-Inuit, there is no dose-response relationship in terms of self-reported degree ofnon-Inuit admixture. Dietary fatty acids play an important role in the risk of cardiovascular diseases and diabetes, diseases of increasing importance in the health transition experienced by the Inuit. Association studies of plasma fatty acids and DNA markers of candidate genes for atherosclerosis and insulin resistance may provide a clearer picture of the genetic basis for the observed differences in plasma fatty acid composition between Inuit and non-Inuit.

Zinc has been shown to inhibit β-receptor activation of adenylate cyclase at a post receptor site. We have postulated that the β-receptor is one of several receptors activated by reduction, followed by transmembrane elector transfer accelerated by GTP. GTP accelerates electron transfer in a model system and this accelerated electron transfer is inhibited by zinc. This could explain the mechanism of the post receptor inhibition by zinc of the adenylate cyclase stimulation which follows β-receptor activation.

Granulophysin, a protein described in platelet dense granule membranes, has been shown to be similar or identical to CD63, a lysosomal membrane protein. We have previously shown granulophysin to be present in neutrophils using immunofluorescence. We now localize granulophysin to the neutrophil azurophilic granules by fine structural immunocytochemistry. Granulophysin expression on the surface membrane of the neutrophil is increased following stimulation of the cells, demonstrated by flow cytometry and fine structural immunocytochemistry. A similar pattern is shown for an anti-CD63 antibody. Incubation of activated neutrophils with D545, a monoclonal antibody to granulophysin, blocks subsequent binding of anti-CD63 antibodies to the cell surface, and anti-CD63 antibodies prevent subsequent binding of D545 as assessed by flow cytometry and immunoblotting. Our results support the homology of CD63 and granulophysin previously demonstrated in platelets and confirm CD63 as an activation marker in neutrophils and the first azurophilic granule membrane marker of neutrophils.

Two sisters with lifelong bleeding tendencies were examined to determine whether their condition was associated with a platelet defect. Their platelet aggregation in response to epinephrine and collagen was abnormal, and the secretion of serotonin and ATP was markedly reduced. The platelet contents of serotonin, ADP, and ATP were all diminished and the ATP:ADP ratio was increased. Direct enumeration by whole-mount and quinacrine-fluorescence techniques demonstrated that the platelets from both sisters had significantly fewer dense granules than controls. These characteristics are similar to an individual with Hermansky-Pudlak syndrome and are consistent with a platelet dense granule deficiency. In contrast, immunofluorescence studies using an antibody against the dense granule membrane protein granulophysin suggested that both sisters had numbers of granules within the normal range. Evaluation by immunoblotting and ELISA indicated the presence of normal levels of granulophysin in the platelets from both sisters; FACS analysis demonstrated the surface expression of granulophysin under conditions of selective dense granule release. These results are consistent with these sisters having a form of dense granule storage pool deficiency where the granular membranes are present but the granules have reduced contents. This observation represents a novel form of storage pool disease which we have termed the empty sack syndrome.

We have evaluated platelet function in normal Simmental cattle and in those with a congenital, inherited bleeding disorder previously attributed to impaired platelet aggregation. Affected platelets failed to aggregate and secrete in response to ADP and the ionophore A23187, and showed impaired aggregation responses to collagen and ionomycin. Aggregation and secretion of normal and affected platelets was similar in response to thrombin and PMA. Resting cytosolic calcium levels and calcium mobilization in response to ADP and ionomycin were similar in control and four affected animals. Normal and affected bovine platelets phosphorylated myosin light chain and pleckstrin in response to ADP and A23187. Transmission electron microscopy of affected platelets following stimulation with ADP, showed shape change and some degree of centralization of the actomyosin gel. Affected platelets had comparable numbers of GPIIb/IIIa complexes and expressed comparable numbers of fibrinogen receptors as normal platelets in response to ADP. Cytoskeletal assembly in affected platelets was normal in response to PMA but incomplete in response to ADP and A23187. Failure of platelet aggregation in bleeding Simmental cattle is predicted to arise from abnormal cytoskeletal assembly following calcium mobilization and phosphorylation of myosin light chain in response to ADP.

We evaluated the effect of Quin-2 loading (> 20 microM) on platelet responses such as phosphoinositide turnover, elevation of cytosolic Ca2+, phosphorylation of myosin light chain (MLC) and a 47-kDa protein, and aggregation in human platelets stimulated with arachidonic acid (AA) and epinephrine. The formation of inositol phosphates (IP, IP2, and IP3) in platelets stimulated with AA was inhibited by 50.4, 59.5, and 61%, respectively, in the presence of Quin-2 (40 microM). A similar degree of inhibition was observed in platelets stimulated with epinephrine (50 microM) and thrombin (0.1 U/ml). Even though Quin-2-induced inhibition of aggregation in response to AA was reversed by epinephrine, its effect on phosphoinositide turnover remained unaffected. Monitoring of cytosolic Ca2+ changes further indicates that the ability of epinephrine to restore aggregation in Quin-2-loaded (40 microM) and AA-stimulated platelets is not coupled to an increase in cytosolic Ca2+. Quin-2 loading (40 microM) caused a significant inhibition of MLC phosphorylation (20 kDa) in platelets stimulated by AA. However, it had no effect on the phosphorylation of the 47-kDa protein induced by AA. Furthermore, Quin-2 loading (40 microM) exerted no significant effect on shape change, actin filament assembly, and spreading, but caused a significant inhibition of secretion and clot retraction. We conclude that the formation of inositol phosphates, increases in cytosolic Ca2+, and phosphorylation of MLC affected by Quin-2 are not coupled to the mechanisms by which platelets develop stickiness, undergo shape change, spreading, and aggregation in response to epinephrine and AA. It appears that the effect of epinephrine in restoring the aggregation response of refractory platelets is coupled to a calcium-mediated alpha-adrenergic receptor, and it may serve as a critical salvage pathway in platelets with compromised functions.

Thrombin is by far the most potent platelet agonist. Potentially this reflects multiple intracellular processes involved in transmitting the activation signal from the initial contact with a receptor, or binding site, to the final platelet response. Platelet membranes have two putative receptors: the high affinity glycoprotein Ib, whose function remains to be clarified, and the moderate affinity autoproteolytic receptor. The autoproteolytic receptor is a member of a family of receptors, with seven transmembrane domains, which interact with GTP-binding proteins. Distal to the membrane, several forms of phospholipase C are activated and roles for both heterotrimeric and low molecular mass GTP-binding proteins have been presented. Phospholipase C acts on inositol phospholipids to generate inositol trisphosphate and diacylglycerol, both of which function as second messengers in thrombin-induced platelet activation. Inositol trisphosphate mobilizes internal calcium stores and this is accompanied, and enhanced, by an influx of calcium from the external milieu. Diacylglycerol and calcium both serve to regulate the activity of multiple protein kinases which, in turn, mediate the phosphorylated state of numerous proteins. Phosphorylation can occur on serine, threonine or tyrosine residues of target proteins and the phosphorylated state of these proteins determines the final activation of the platelet.

The levels of glycoprotein (GP) Ib and GPV and phospholipase C activity were measured in platelets from three Bernard-Soulier syndrome patients. The patients' platelets had 46%, 46%, and 24% of control levels of GPIb alpha and 43%, trace, and 13% of control levels of GPV as determined by immunoblot analysis. Stimulation by thrombin, trypsin, the thromboxane analogue U46619, and the combination of U46619 and trypsin caused the formation of [32P]phosphatidic acid, an index of phospholipase C activity, in [32P]orthophosphate-prelabeled platelets. With all agonists, however, the formation of [32P]phosphatidic acid was markedly reduced in Bernard-Soulier syndrome platelets compared with control platelets. These data indicated a postreceptor defect in phospholipase C activation in Bernard-Soulier syndrome platelets and confirmed earlier observations of potential proteolytic and nonproteolytic mechanisms of platelet activation.

The effect of dietary alpha-linolenic acid (18:3n-3) and its ratio to linoleic acid (18:2n-6) on platelet and plasma phospholipid (PL) fatty acid patterns and prostanoid production were studied in normolipidemic men. The study consisted of two 42-d phases. Each was divided into a 6-d pre-experimental period, during which a mixed fat diet was fed, and two-18 d experimental periods, during which a mixture of sunflower and olive oil [low 18:3n-3 content, high 18:2/18:3 ratio (LO-HI diet)], soybean oil (intermediate 18:3n-3 content, intermediate 18:2/18:3 ratio), canola oil (intermediate 18:3n-3 content, low 18:2/18:3 ratio) and a mixture of sunflower, olive and flax oil [high 18:3n-3 content, low 18:2/18:3 ratio (HI-LO diet)] provided 77% of the fat (26% of the energy) in the diet. The 18:3n-3 content and the 18:2/18:3 ratio of the experimental diets were: 0.8%, 27.4; 6.5%, 6.9; 6.6%, 3.0; and 13.4%, 2.7, respectively. There were appreciable differences in the fatty acid composition of platelet and plasma PLs. Nevertheless, 18:1n-9, 18:2n-6 and 18:3n-3 levels in PL reflected the fatty acid composition of the diets, although very little 18:3n-3 was incorporated into PL. Both the level of 18:3n-3 in the diet and the 18:2/18:3 ratio were important in influencing the levels of longer chain n-3 fatty acid, especially 20:5n-3, in platelet and plasma PL. Production of 6-keto-PGF1 alpha was significantly (P < 0.05) higher following the HI-LO diet than the LO-HI diet although dietary fat source had no effect on bleeding time or thromboxane B2 production.(ABSTRACT TRUNCATED AT 250 WORDS)

Thromboxane A2, produced from the arachidonic acid released from platelet phospholipids by phospholipase A2, stimulates platelet aggregation. It remains unresolved whether additional products of platelet phospholipase A2 might promote aggregation. To address this question, we have used aspirin-treated platelets to block thromboxane A2 formation and studied the influence of the phospholipase A2 inhibitor U10029A on platelet aggregation and secretion in response to thrombin. U10029A at 100 microM markedly inhibited platelet aggregation, but had no effect on platelet secretion. Since this concentration of U10029A effectively blocked lysophosphatidic acid (LPA) formation, LPA was added and found to substantially reverse the inhibitory effect of U10029A in these platelets. Furthermore, the action of U10029A was not due to inhibition of phosphatidate phosphohydrolase because U10029A, unlike propranolol, did not inhibit this enzyme. Although it is not possible to conclusively rule out an effect of U10029A in addition to its inhibition of phospholipase A2, our results reveal that a product of phospholipase A2 other than thromboxane A2 is important for platelet aggregation, but not for secretion in response to thrombin. Our data suggest that this product is LPA. Since the amount of phosphatidic acid (PA) increased dramatically concurrent with inhibition of platelet aggregation, it is safe to conclude that PA has no direct role to promote platelet aggregation in response to thrombin.

We compared the effect of different aspirin schedules, dosages, and formulations on various bleeding time parameters including bleeding time, plasma and total blood volume, and levels of the stable metabolites of thromboxane A2 (TXA2) and prostacyclin (PGI2) (respectively, TXB2 and 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha)) to determine the optimal dosage and formulation of aspirin to inhibit TXA2 production while sparing PGI2. In a randomized, parallel study, 52 healthy male volunteers (62 independent observations) with no history of bleeding disorders were given 80 mg or 325 mg of regular aspirin, or 325 mg of enteric-coated aspirin to ingest daily (14 pills) or every other day (7 pills) for a continuous 14 day period. Bleeding times were performed on day 1 before aspirin, 6 h after aspirin on day 1, and before aspirin on day 14. Bleeding times, plasma volume, and total volume increased significantly from before aspirin to after 6 h and 14 days (p < 0.0001 for all parameters) for all aspirin formulations. For day 1 before aspirin ingestion to 6 h later, both TX and PGI2 (p < 0.008) decreased significantly. 6 h after ingestion of aspirin on day 1 to day 14, both TX and PGI2 levels also significantly decreased (p < 0.0001). There was a highly significant decrease in PGI2 production on every other day aspirin schedules (p = 0.0001) particularly with 80 mg of aspirin, while the decrease in PGI2 production on daily aspirin was not significant (p = 0.10). The most favourable ratio of 6-keto-PGF1 alpha to TXB2 occurred with 80 mg daily.(ABSTRACT TRUNCATED AT 250 WORDS)

Human platelet activation is associated with, and regulated by, the phosphorylation of a number of proteins. Recently, attention has been focused on tyrosine phosphorylation of proteins and their function in platelet activation. Here vanadate, an inhibitor of tyrosine phosphohydrolase, was used to examine the role that tyrosine phosphorylation plays in platelet activation. Vanadate (7.5 to 100 mumol/L) stimulated the dose-dependent aggregation of saponin-permeabilized, but not intact, platelets. Electron-microscopic studies indicated small degranulated aggregates. Vanadate-induced aggregation was inhibited by pretreatment with prostacyclin (1 to 10 nmol/L), genistein (1 to 10 micrograms/mL), aspirin (100 mumol/L), or BW755C (80 mumol/L). Aggregation was associated with the aspirin-sensitive formation of [32P]phosphatidic acid and the phosphorylation of platelet proteins, notably pleckstrin and myosin light chain. Immunoblotting studies indicated that vanadate caused the tyrosine phosphorylation of proteins of approximate molecular weights 26, 29, 32, 40, 42, 80, and 90 Kd. Preincubation with BW755C abolished the phosphorylation of the 26-, 29-, 32-, 40-, and 42-Kd proteins but not the 80- and 90-Kd proteins. Vanadate stimulated the release of [3H]-arachidonic acid that was not affected by pretreatment with BW755C. The subsequent conversion of [3H]-arachidonic acid to [3H]-thromboxane A2 was significantly inhibited. These findings show that vanadate stimulates platelets by promoting arachidonic acid release from phospholipids. Tyrosine phosphorylation, potentially of the 80- or 90-Kd proteins, may regulate a platelet phospholipase A2. The release arachidonic acid was converted to thromboxane A2 that produced secondary effects such as phospholipase C activation, protein phosphorylation, and aggregation, and was associated with the tyrosine phosphorylation of the 26-, 29-, 32-, 40-, and 42-Kd proteins.

Background: The identification and characterization of granule membrane proteins are becoming increasingly important in understanding the packaging and secretory function of granules and characterizing diseases involving granules. A granule membrane protein, granulophysin, has recently been identified in the membranes of platelet dense granules, organelles that contain stored ADP, ATP, serotonin, and calcium. Antibodies that recognize granulophysin also stain granules of monocytes, neutrophils, and lymphokine activated killer cells., Experimental Design: In the present study, the distribution of epitopes recognized by antigranulophysin monoclonal antibodies in human tissues was investigated using immunohistochemistry on paraffin sections. Quantitation of the protein was also performed by enzyme-linked immunosorbent assay. The protein was also analyzed in various tissues using Western blotting., Results: Granulophysin was localized to the granules of skin melanocytes, neurons, endocrine gland cells, exocrine glands (except mucin producing cells), and surface lining cells. Analysis by Western blots revealed a typical staining pattern for granulophysin in lung, adrenal gland, liver, brain, prostate, and pituitary. Atypical bands were present in the pancreas head (47 kDa) and skeletal muscle (34 kDa). A clear distinction was demonstrated between granulophysin and synaptophysin through both immunochemistry and blotting, despite the known cross-reactivity of these two proteins., Conclusions: The findings demonstrate that granulophysin is a widely distributed protein that is frequently associated with granules. We speculate that it may be critical in granule function.

The levels and expression of the proteins CD63 and granulophysin in platelets from control and from a Hermansky-Pudlak syndrome subject (a condition characterized by dense granule and lysosomal deficiencies and the accumulation of ceroid-like material in reticuloendothelial cells) were examined. Immunofluorescence studies indicated that anti-CD63 and anti-granulophysin antibodies recognized similar numbers of granules; coapplication of antibodies did not identify more granules than the individual antibodies. Significantly fewer granules were recognized in Hermansky-Pudlak syndrome platelets than in control using either antibody. Immunoblotting studies demonstrated that anti-CD63 and anti-granulophysin antibodies apparently recognize the same protein, which was deficient in Hermansky-Pudlak platelets. Analysis by fluorescence-activated cell sorter (FACS) showed biphasic expression of CD63 and granulophysin after thrombin stimulation of control but not Hermansky-Pudlak platelets. Anti-CD63 effectively blocked detection of the protein by anti-granulophysin using immunofluorescence, ELISA, immunoblotting, and FACS analysis. Amino-terminal sequencing over the first 37 amino acids revealed that granulophysin was homologous to CD63, melanoma antigen ME491, and pltgp40. These results suggest that granulophysin and CD63 are possibly identical proteins. This is the first report of a protein present in platelet dense granules, lysosomes, and melanocytes, but deficient in a patient with Hermansky-Pudlak syndrome.

The vasoactive eicosanoids, prostacyclin and thromboxane, are thought to play an important role in the genesis of cardiovascular disease. Since an altered basal production of these eicosanoids among individuals exhibiting the Type A behavior pattern had previously been observed by the authors, the present study evaluated the extent to which the TABP-eicosanoid relationship would be altered by two lifestyle variables known to affect platelet activity: alcohol consumption and stressful physical activity. 55 male participants aged 18-25 years, participated in the study. They were classified as either Type A or Type B on the basis of the Structured Interview and as either moderate, heavy, or abstinent alcohol drinkers. Bleeding times were performed and bleeding time thromboxane and prostacyclin metabolites were measured in all subjects both before and following treadmill exercise. The results indicated that following exercise, Type A participants, who reported moderate alcohol intake, had decreased levels of thromboxane B2 formation relative to Type As reporting heavy consumption. Further, prostacyclin production, measured as the primary metabolite, 6-keto-prostaglandin F1 alpha, was significantly suppressed following exercise among drinkers as compared with participants reporting abstinence. These results were discussed in relation to the proposition that moderate alcohol consumption reduces coronary heart disease risk.

The results of investigations in platelets provide evidence for an intracellular messenger role for histamine. Studies of neutrophils and of cellular proliferation suggest that there may be a wider role for histamine as an intracellular messenger modulating activation processes in cells.

The involvement of intracellular histamine in thapsigargin (Tg)-induced platelet aggregation was studied. Platelet aggregation induced by 0.25 and 0.5 microM Tg was not accompanied by a rise in intracellular histamine but a significant (p < 0.01) increase in the level of intracellular histamine was observed at 1 microM Tg. Preincubation of platelets with inhibitors of histamine metabolizing enzymes had little effect on intracellular histamine levels in platelets stimulated by 0.5 microM Tg. In addition, the inhibitors of histidine decarboxylase (HDC), alpha-methyl histidine (alpha-MH) and alpha-fluoromethyl histidine (alpha-FMH) failed to inhibit Tg-induced aggregation. The intracellular histamine receptor antagonist, N,N-diethyl-2-[4-(phenylmethyl)phenoxy] ethanamine. HCl (DPPE), inhibited Tg-induced aggregation but with IC50 values dependent on the concentration of agonist used. The inhibitory effects of DPPE on Tg-induced aggregation were not reversed by the addition of histamine to saponin-permeabilized platelets suggesting non-histamine mediated effects of DPPE on Tg-induced aggregation. Tg stimulated an increase in the cytosolic free calcium concentration which was unaffected by DPPE indicating that the effects of DPPE are also not due to the inhibition of mobilization of cytosolic calcium. The ultrastructural studies suggest that the major Tg-induced changes (pseudopod formation and granule centralization) are consistent with a primary role for Tg to mobilize calcium; DPPE had very little effect on these ultrastructural changes. The results indicate that the effects of Tg on human platelets are mediated by an increase in cytosolic calcium but not by intracellular histamine.

Abnormalities in platelet dense granules, small intracellular organelles containing ATP, ADP, calcium, serotonin, and pyrophosphate, have frequently been reported in patients with leukemia and myeloproliferative disorders, particularly acute and chronic myelogenous leukemia. Recent studies of a family which includes several members with an autosomal dominant dense granule deficiency condition show an association between the presence of this form of dense granule deficiency and the development of acute myelogenous leukemia. Studies in two additional patients, one with the Monosomy 7 syndrome and the second with a myelodysplastic syndrome, revealed a defect in platelet dense granules. This defect appears to be due to an abnormality in the formation of these granules rather than the presence of empty vesicular structures or decreased contents due to activation associated secretion. The results suggest that the defect in platelet dense granules associated with leukemia or myelodysplastic syndromes may result from a chromosome alteration in the megakaryocyte cell line leading to decreased formation of dense granules. Studies in the family with an inherited bleeding disorder suggest that a gene coding for a protein important for the formation of dense granules is located adjacent to a gene which, when abnormal, may predispose to the development of leukemia.

The dense tubular system (DTS) functions to regulate platelet activation by sequestering or releasing calcium, similar to the sarcotubules of skeletal muscle. In resting platelets, the DTS exists as thin elongated membranes. Within 10 seconds of the addition of thrombin, platelets show a major ultrastructural change in their DTS: from the thin elongated form to a rounded vesicular form. These morphologic changes were demonstrated with two different stains and two different fixation methods. Platelets exposed to the calcium ionophore A23187 showed the same ultrastructural changes in the DTS. In contrast, the DTS remains in a thin elongated form when platelets are stimulated by the protein kinase C activators phorbol 12-myristate 13-acetate (PMA) and oleoylacetylglycerol (OAG). These morphologic changes may be related to the discharge of calcium from the DTS because this is stimulated by thrombin and A23187, but not by PMA. Preincubation of the platelets with the intracellular calcium chelator 5,5'-dimethyl-bis-(0-aminophenoxy)-ethane-N,N,N',N tetra acetic acid (BAPTA) largely prevented both the thrombin-induced rise in intracellular calcium and the changes in DTS morphology, suggesting that the changes in DTS morphology are secondary to the increase in cytosolic calcium. The results provide a morphologic correlate to existing biochemical evidence showing that the DTS is involved early during paltelet activation.

The relationship between bleeding and bruising and the production of prostacyclin and thromboxane was assessed in children who were to have a tonsillectomy and/or an adenoidectomy. Eicosanoids in the blood oozing from the bleeding time incision were measured and correlated with the reported frequency of bruising and epistaxis. A striking association (P = .0003) between prostacyclin production and the frequency of bruising was found; children reporting bleeding at least biweekly had the highest prostacyclin synthesis. Successively lower levels of the prostacyclin metabolite, 6-keto-prostaglandin F1 alpha, were found in children reporting less frequent bruising. Prostacyclin production in bleeding time blood was also correlated inversely with systolic blood pressure and hemoglobin level, although neither of these variables could explain the association between prostacyclin production and bruising. There was no correlation between thromboxane formation, systolic blood pressure, hemoglobin level, age, or bleeding time and the frequency of bruising. The ratio of thromboxane B2 to 6-keto-prostaglandin F1 alpha was correlated inversely with the length of the bleeding time (P = .016). It is concluded that vascular prostacyclin production may have a role in bruising symptomatology. It is suggested that prostacyclin formed at the injured vessel surface collects within the first few seconds after injury inside the tissue space at the site of the bruise and, by influencing the formation of the platelet/fibrin plug and/or the leakage of blood from the vessels, plays a significant role in modifying the development of bruising.

We recently reported the characterization of a platelet granule membrane protein of molecular weight (mol wt) 40,000 called granulophysin (Gerrard et al: Blood 77:101, 1991), identified by a monoclonal antibody (MoAb D545) raised to purified dense granule membranes. Using immunoelectron-microscopic techniques on frozen thin sections, this protein was localized in resting and thrombin-stimulated platelets. In resting platelets, labeled with antigranulophysin antibodies and immunogold probes, label was localized to the membranes of one or two clear granules per platelet thin section. D545 also labeled dense granules in permeabilized whole platelets and isolated dense granule preparations examined by whole-mount techniques. Expression of granulophysin on the platelet surface paralleled dense granule secretion as measured by 14C-serotonin release under conditions in which lysosomal granule release, as measured by beta-glucuronidase secretion, was less than 5%. After thrombin stimulation, both the surface-connected canalicular system and the plasma membrane were labeled, demonstrating redistribution of granulophysin associated with degranulation. Double labeling experiments with D545 and antibodies to the alpha-granule membrane protein, P-selectin, demonstrated labeling of both P-selectin and granulophysin on dense granule membranes. Distribution of both proteins on the plasma membrane after platelet stimulation was similar. The results demonstrate that granulophysin is localized to the dense granules of platelets and is redistributed to the plasma membrane after platelet activation.

The immunological crossreactivity between the two granule-specific membrane glycoproteins, synaptophysin and granulophysin, was studied using a series of site-specific monoclonal and polyclonal antibodies. The epitope relatedness of six monoclonal antibodies against granulophysin was examined by competitive ELISA. The antibodies are shown to recognize distinct, but overlapping epitopes within a compact region that is constructed by the three-dimensional configuration of the molecule. All these antibody clones also recognize rat neuronal synaptophysin. Two monoclonal antibodies against synaptophysin, of which one is the well-characterized SY38 antibody, directed against the carboxy terminal of the molecule, are also shown to react with granulophysin. Characterized polyclonal antibodies against different peptide antigens of synaptophysin failed to recognize granulophysin. Synaptophysin and granulophysin are distinctly recognized in brain cell (white matter) and the pituitary both qualitatively and quantitatively. Based on these and other observations, it is suggested that the repeat motif in the cytoplasmic tail of synaptophysin represents an immunodominant construct that is the target for the observed crossreactive antibodies and that a similar tertiary construct has been preserved in granulophysin and in other transmembrane proteins.

The authors evaluated the interaction between physical fitness and Type A behavior on vascular production of prostacyclin and platelet thromboxane in response to a standard vessel injury. Ninety-seven male university students were classified as Type A or B on the basis of the Structured Interview of Rosenman. Reactivity, as measured by changes in systolic blood pressure (SBP), and heart rate (HR), measured before and after a stressful Stroop task 1 week later, showed that Type A individuals were more reactive to the stressor than Type Bs, providing independent confirmation of the behavioral interview assessment procedure. Fitness level was determined by a graded treadmill test and was not different for Type As and Type Bs. Basal thromboxane production, measured as the primary metabolite, thromboxane B2, in blood oozing from the bleeding-time site, was highest among unfit Type A subjects. Fit Type As and Type Bs showed significantly lower thromboxane production. No significant differences in prostacyclin production were seen. This study marks the first time that behavior pattern has been linked to an adverse aspect of a thrombosis-related parameter likely to be involved in the genesis of cardiovascular disease. The present results also indicate that physical fitness may, in some fashion, ameliorate the "toxic" effects of Type A behavior.

Fatty acid compositions were determined of phospholipids isolated from venous cord plasma and from the walls of umbilical arteries and veins, collected from healthy, a terme, Inuit and Caucasian (Dutch) neonates. The Inuit fatty acid profiles were characterized by a lower essential fatty acid (EFA) status, with higher levels of monounsaturated fatty acids, of Mead acid [20:3(n-9)] and its direct elongation product, and with lower amounts of the longer chain (greater than or equal to 20 carbon atoms), highly unsaturated (greater than or equal to 4 double bonds) fatty acids of both the (n-3) and (n-6) families. Levels of linoleic- and dihomo-gamma-linolenic acids were higher in Inuit as compared to Caucasian neonates, which suggests a low activity of the delta-5-desaturase in the Inuit. Within the Inuit group, a higher intake of marine food was associated with a better neonatal (n-3) status. Although the differences between Inuit and Caucasian neonates may be of genetic rather than of dietary origin, the results imply that dietary long-chain (n-3) or (n-6) fatty acids may be particularly important during pregnancy in Inuit mothers. Further studies are indicated with respect to the EFA content of the habitual Inuit diet and levels of delta-5-desaturase activity in the Inuit.

A series of T24-H-ras-transformed 10T1/2 fibroblasts with varying metastatic potential was tested for the ability to aggregate platelets. Results indicate that although platelet activation was always detected in the highly metastatic cells, some non-metastatic cells also have the ability to cause platelet aggregation, suggesting that this is a necessary but not sufficient characteristic of the metastatic phenotype. Apyrase, an ADP scavenger, effectively inhibited platelet aggregation by metastatic cells, however, there was no significant increase in ADP secretion or relation to the ability of the tumor cells to activate platelets. Hirudin, a thrombin inhibitor, did not affect aggregation, suggesting that the pathway of activation is thrombin-independent. The glycoprotein processing inhibitor, castanospermine, which reduces glycosidase I activity and metastatic capability, inhibited the ability of metastatic cells to cause platelet aggregation. However, another inhibitor of oligosaccharide processing, swainsonine, which inhibits mannosidase II activity and does not reduce metastasis, had no effect on platelet aggregation. These results show that the integrity of N-linked oligosaccharide structure of glycoproteins is an important feature of the ability of ras-transformed fibroblasts to activate platelets.

A membrane glycoprotein of human platelet dense granules, called granulophysin, with serologic homology to synaptophysin has recently been identified. To determine if this protein was present in granulated leukocytes, we examined several cell types for the presence of the protein by indirect immunofluorescence. Antigranulophysin mAb staining was detected in a granular pattern in the cytoplasm of permeabilized IL-2-stimulated CD3+ peripheral lymphocytes, neutrophils, U937 monocytes, and mast cells. Immunohistochemistry of human lymph nodes showed cytoplasmic staining of macrophages, neutrophils, and some dendritic cells. Induction of granule exocytosis in granulated CD3+ lymphocytes after stimulation with PMA and calcium ionophore A23187 resulted in a redistribution of the reactive epitope from the cytoplasm to the plasma membrane. Subcellular fractions contained two peaks of reactivity; the first peak coincided with N-benzyloxycarbonyl-L-lysine thiobenzyl ester-esterase activity in dense granules whereas the second peak was present in lighter fractions. The affinity purified protein from both peaks was identical in Western blot analysis and had a molecular mass of 28 kDa under reducing conditions. The protein could only be solubilized in detergent suggesting that it was an integral membrane protein. We have named this protein leukophysin to differentiate it from the 40-kDa granulophysin of platelets. Monocytes contained a protein with identical m.w. to leukophysin, whereas a protein of a slightly higher m.w. was detected in neutrophils. We propose that leukophysin is a common granule membrane protein of leukocytes.

A family with an inherited bleeding disorder extending over four generations, and multiple cases of myeloblastic and myelomonoblastic leukaemia was studied. Ten members of the family had, by history, a haemorrhagic diathesis. There were three documented cases of myeloblastic leukaemia, two documented cases of myelomonoblastic leukaemia and two more cases of leukaemia by history. In four of the cases the bleeding diathesis clearly antedated the leukaemia, in two by many years. The bleeding disorder is characterized by a long bleeding time, abnormal platelet aggregation, low platelet ADP and decreased numbers of platelet dense bodies consistent with a dense granule storage pool deficiency. The number of dense granules was decreased by immunofluorescence employing quinacrine or using an antibody to the dense granule membrane protein, granulophysin, confirming an absolute decrease in dense granule numbers rather than the presence of empty granule sacs. This congenital storage pool deficiency is associated with a high incidence of acute myeloid leukaemia in this family.

Objective: To assess a possible relation between the incidence of hypertension during pregnancy and the consumption of fatty acids found in fish and sea mammals., Design: Retrospective survey of pregnancy-induced hypertension; prospective diet survey., Setting: Inuit women from seven communities in the Keewatin region of the Northwest Territories., Patients: All women from Arviat (formerly Eskimo Point), Baker Lake, Chesterfield Inlet, Coral Harbour, Repulse Bay, Sanikiluaq and Whale Cove who gave birth between Sept. 1, 1984, and Aug. 31, 1987., Main Outcome Measures: All blood pressure measurements recorded during the pregnancy, incidence of pregnancy-induced hypertension in the seven communities, harvest of country food (food obtained from the land or sea rather than bought in a store) for six of the communities, self-reported consumption of fish, sea mammals and terrestrial mammals by a subgroup of the subjects and levels of phospholipid fatty acids in cord serum samples from a subgroup of the infants., Main Results: Significantly lower mean diastolic blood pressure values during the last 6 hours of pregnancy were noted for the women from the three communities with a higher consumption of fish and sea mammals (78.2 [95% confidence limits (CL) 76.6 and 79.9] mm Hg) than for those from the four communities with a lower consumption of such food (81.5 [95% CL 80.1 and 82.9] mm Hg) (p less than 0.005). The relation between community diet type and blood pressure was independent of other factors. Correspondingly, the women from communities with a lower consumption of marine food were 2.6 times more likely to be hypertensive during the pregnancy than those from communities with a higher consumption of marine food (p less than 0.007). Parity (p less than 0.05) and prepregnancy weight (p less than 0.005) were also significantly associated with pregnancy-induced hypertension; however, the relation between hypertension and community diet type remained significant in logistic regression analysis (odds ratio 2.56, p = 0.03). The differences between the community groups were substantiated by the results of the diet survey, the levels of eicosapentaenoic acid (EPA) in the cord serum phospholipids and the harvest data., Conclusions: Increased consumption of fish may be beneficial for women at risk for hypertension during pregnancy. A prospective randomized trial of fish or EPA supplementation during pregnancy is warranted.

Objective: To determine whether individuals exhibiting the type A behaviour pattern have a different extent of prostacyclin or thromboxane production after relaxation or after a structured interview compared to individuals exhibiting the type B behaviour pattern., Design: Subjects were randomized to receive prospectively either a relaxation session or a mildly stressful interview first. Each then received the alternate treatment second., Setting: Students attending an introductory psychology course at the University of Manitoba received a relaxation session (20 mins lying down listening to a tape) and an interview session (the structured interview of Rosenman)., Outcome Measures: Type A/B behaviour pattern was rated using the structured interview of Rosenman. Production of 6-keto prostaglandin F1-alpha and thromboxane B2, metabolites of prostacyclin and thromboxane A2, respectively, were measured in response to a standardized vascular injury-bleeding time., Main Results: No significant differences were observed in the length of bleeding time, in bleeding time thromboxane production or in prostacyclin production after relaxation between individuals exhibiting type A and B behaviour patterns. Prostacyclin production after the interview was lower in type A individuals (3.29 +/- 0.29 pg/min) than in individuals exhibiting type B behaviour (4.76 +/- 0.63 pg/min) (P = 0.04). No significant post interview differences in bleeding time or in thromboxane production were seen., Conclusion: After relaxation, type A and B subjects are similar in their prostacyclin and thromboxane responses to vascular injury. However, type A individuals show a less favorable prostacyclin response than type B when confronted with the structured interview; type A individuals responded in an aggressive or hostile fashion, while the type B individuals exhibited a more relaxed response.

A questionnaire, designed to assess bleeding/bruising tendencies, was administered to 251 otherwise healthy children undergoing a tonsillectomy and/or adenoidectomy. 23 children with excessive bleeding during or after the operation, with a long bleeding time or who reported taking aspirin recently were excluded, to give a population of 228 non-bleeders. For comparative purposes, 31 patients with bleeding disorders (von Willebrand's disease and/or platelet function defects) were studied. A considerable proportion of "non-bleeding" children reported easy bruising (24%), had bruises at least once a week (36%) and suffered from nosebleeds (39%). The respective frequencies (67%, 68% and 69%) for children with bleeding disorders were significantly higher. Occurrence of bruises usually on more than one part of the body, frequent large bruises or hematomas were rare in "non-bleeders" (4.9%, 3.5% and 2.7% respectively), but more common in "bleeders" (38.5%, 29.6% and 21.7% respectively).

In previous reports, we have provided evidence indicating that newly formed histamine is an intracellular messenger in human platelets. The involvement of protein kinase C (PKC) and intracellular calcium (Ca2+i) in the synthesis of histamine was investigated. Human platelets were stimulated by phorbol 12-myristate 13-acetate (PMA), collagen and the Ca2+ ionophore A23187, with or without the PKC inhibitor staurosporine. Aggregation, histamine synthesis and phosphorylation of pleckstrin (47 kDa; P47) and myosin light chain (20 kDa; P20) proteins were monitored. Staurosporine inhibited PMA- and collagen-induced aggregation, histamine synthesis and phosphorylation of 47 kDa and 20 kDa proteins in a dose-dependent manner. For PMA, median inhibitory concentrations (IC50 values) for staurosporine inhibition of aggregation, histamine synthesis and phosphorylation were similar, suggesting that histamine synthesis induced by this agonist may be a consequence of PKC activation. Conversely, collagen-stimulated histamine synthesis was inhibited by staurosporine at concentrations significantly higher than those required to inhibit aggregation (P less than 0.005) or pleckstrin phosphorylation (P less than 0.01), indicating the possible involvement of non-PKC mechanism(s) in the synthesis of histamine induced by this agonist. A23187 failed to induce the synthesis of intracellular histamine in platelets, whereas staurosporine blocked A23187-induced aggregation and phosphorylation of the 20 kDa protein at significantly higher concentrations than those needed to inhibit PKC. When platelets were stimulated with a combination of A23187 and PMA, the increase in platelet histamine was less than that with PMA alone. The results provide evidence that the synthesis of intracellular histamine in platelets occurs as a consequence of PKC activation and may be down-regulated under conditions where there is a substantial rise in [Ca2+]i.

SUMMARY. The role of intracellular histamine in the activation of human platelets was explored using the novel histamine antagonist N,N-diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine (DPPE). DPPE inhibited aggregation, [(32)P]-phosphatidic acid production (an index of phospholipase C activity) and the increase in cytosolic calcium ([Ca(2+)]i) in response to collagen. In contrast, while at higher concentrations DPPE inhibited aggregation in response to ADP and the thromboxane mimetic EP171, it failed to inhibit EP171-induced [(32)P]-phosphatidic acid production or increases in [Ca(2+)]i elicted by either ADP or EP171. Collagen but neither ADP nor EP171 caused the significant formation of intracellular histamine. The biochemical changes induced by collagen have been shown previously to be, at least partly, secondary to thromboxane generation. Collagen-induced arachidonic acid release was therefore assessed. DPPE inhibited the release of arachidonic acid in response to collagen but had no effect on its subsequent conversion to thromboxane. The findings imply that intracellular histamine acts at an early stage in collagen-induced platelet activation, most likely prior to arachidonic acid release, and further that DPPE inhibits ADP and EP171-induced activation by a non-histamine related effect.

Activation of several receptor types is followed by their binding to a G-protein. Prior to transmission of the agonist signal, the G-protein which had affinity for guanosine 5-diphosphate (GDP) binds guanosine 5-triphosphate (GTP) instead. Because evidence exists that several agonist groups activate their receptors by reduction, we evaluated whether the nucleotide associated with G-proteins could enhance electron flow. Using a model system of ferrous iron and ferric cytochrome c, it was determined that substitution of GTP for GDP led to an enhanced reduction of ferric cytochrome c. These results support the concept that cellular activation by certain receptors may involve reductive activation with the participation of GTP and G-proteins. We speculate that GTP, when bound to G-protein, can facilitate electron transfer perhaps from the receptor or the G-protein to the catalytic subunit of the adenylate cyclase enzyme.

Sixty-six male university students were classified as Type A or B on the basis of the Structured Interview of Rosenman and as hostile or non-hostile on the basis of the Cook-Medley scale. Vascular production of prostacyclin and platelet thromboxane in response to a standard vessel injury was evaluated. Basal thromboxane production, measured as the primary metabolite, thromboxane B2, in blood oozing from the bleeding-time site, was highest among hostile Type A subjects with significantly lower thromboxane production in hostile Type Bs and all non-hostile groups combined. Following an exercise treadmill test hostile subjects produced more thromboxane than non-hostile ones, and hostile Type As had significantly shorter bleeding times than hostile Type Bs. No significant differences on any measure were observed following a stressful color naming task. The observed interaction of hostility and Type A behavior on bleeding time thromboxane formation links behavior to an adverse aspect of a thrombosis-related parameter thought to be involved in the genesis of cardiovascular disease.

Monoclonal antibodies were raised after injecting mice with isolated human dense granules. Several of these monoclonals were found to recognize a 40-Kd dense granule membrane protein. Western blot and immunofluorescent analysis confirmed the dense-granule specificity. After thrombin activation, the protein was found in patches on the external platelet membrane. By Western blot and slot blot analysis, the protein was found to be markedly deficient in a patient with the Hermansky-Pudlak syndrome. Studies of neutrophils and endothelial cells show the presence of immunologically related granule-membrane protein(s). Western blots using four anti-synaptophysin antibodies and three antibodies to the platelet 40-Kd protein suggest that the protein may share some homology with, but is not identical to, the synaptosomal membrane protein synaptophysin.

It has been reported that lymphocytes from peripheral blood (PBL) cultured with interleukin-2 (IL-2) produce predominantly CD16+ lymphokine-activated killer (LAK) cells. We developed a two-step method to generate LAK cells from human PBL in long-term cultures (10-12 days) with recombinant human IL-2 (rhIL-2) and characterized the evolving LAK cell population by testing its phenotype and cytotoxic activity as a function of time. The starting PBL displayed some natural killer (NK) cytotoxicity but no LAK activity. At day 6, the cells were a mixed population of about 80% CD3+ and 6% CD16+ cells. Little proliferation was evident but strong LAK activity was detected. After 10-12 days, major cell expansion had occurred and they were essentially a pure (greater than 90%) CD3+ CD16- CD56- cell population large granular lymphocyte (LGL) by morphology that displayed strong non-MHC-restricted killing activity (greater than 200 lytic units). Over the same period of time, the CD16+ cells had almost completely regressed in these cultures. This preferential induction of CD+ LAK cells was not an effect of IL-2 concentration as 10 U/ml was as effective as 500 U/ml. Further characterization revealed a major population of CD4+ (60%) and CD8+ (30%) with a smaller fraction (less than 9%) of gamma delta + cells. These results indicate that a virtually pure CD3+ LAK cells population was produced with long-term cultures of lymphocytes from peripheral blood in rhIL-2, in which active proliferation of the CD3+ but not CD16+ cells occurred.

The production of thomboxane B2, the primary metabolite of thromboxane A2, and 6-keto prostaglandin F1 alpha, the primary metabolite of prostacyclin, were measured in response to a standardized vascular injury, the bleeding time, in patients with von Willebrand's disease and in patients with platelet function defects. Compared to controls, thromboxane B2 levels in bleeding time blood were significantly lower in subjects with von Willebrand's disease. In patients with platelet function defects associated with a deficient response to thromboxane A2, thromboxane B2 production in bleeding time blood was similar to controls. In subjects with other platelet function defects, thromboxane production was significantly lower than normal. 6-keto PGF1 alpha production in bleeding time blood was not significantly different in patients compared to controls. The results suggest that bleeding time thromboxane production is influenced by the extent of platelet-vessel interaction.

Studies were performed to determine whether endothelium-derived relaxing factor (EDRF) can inhibit platelet aggregation within the vascular lumen, and if so, whether the inhibition persists in the presence of red blood cells (RBCs). Canine femoral arteries mounted in an organ bath were perfused with physiologic saline solution to which acetylsalicylic acid was added to block prostacyclin formation. During contraction with phenylephrine, addition of acetylcholine to the perfusing solution to evoke EDRF release relaxed the vessel wall. Washed human platelets labeled with 14C-5-hydroxytryptamine were added to the perfusing solution, and activated by thrombin infused via a branch vessel. The perfusate was collected downstream and centrifuged; the fraction of 14C-5-hydroxytryptamine appearing in the supernatant reflected the degree of platelet activation. Stimulation of EDRF release with acetylcholine inhibited 14C-5-hydroxytryptamine release. Hemoglobin (Hb) (10(-5) mol/L) blocked vascular relaxation and platelet-inhibition. RBCs at a hematocrit of 10% (treated with echothiophate to block erythrocyte cholinesterase) did not prevent relaxation but reversed the platelet inhibition. Lower hematocrits did not completely block the inhibition. Thus, erythrocyte Hb may modulate the inhibition of intraluminal platelet aggregation by EDRF.