bluuebunny/crossref_metadata_2025_split huggingface dataset:
R59022, A DIACYLGLYCEROL (DG) KINASE INHIBITOR POTENTIATES THROMBIN-INDUCED PLATELET AGGREGATION AND GRANULE RELEASE
Authors: Joseph S K , Krishnamurthi S , Kakkar V V 
Published: August 2018

R59022 is a recently described inhibitor of the enzyme DG kinase [1], which converts DG to phosphatidic acid. While R59002 inhibits DG conversion in platelets resulting in enhanced protein kinase C (PrkC) activation [1], little is known on its effect on other platelet responses. In this study, we have examined the effect of R59022 on agonist-induced platelet aggregation and [14C]-5-hydroxytryptamine (5HT) release using washed human platelets. With a sub-maximal concentration of thrombin (T, 0.05U/ml) R59022 (10-30μM) significantly potentiated T-induced platelet aggregation and [14C]-5HT release eg % [14C]-5HT release:- 0.05U/ml T-52±5,30μM R59022+T-76±8. Removal of external Ca2+ (ImM) using EGTA (5mM) reduced T-induced 5HT release but not the potentiation of it by R59022 eg EGTA+ 0.05U/ml T-36±6%, EGTA+R59022+T- 72±5%. These results show that in the presence of EGTA and R59022 the increased DG levels can compensate for the diminished rise in T-induced Ca/2+ mobilisation thus re-emphasizing the importance of DG in promoting granule secretion. In addition to inhibiting DG phosphorylation, R59022 also inhibits the phosphorylation of the DG analogue 1-oleoyl 2-acetylglycerol (OAG) [1]. OAG (63μM) with pre-incubation times of 10-60 sec, significantly potentiated threshold T (0.03U/ml)-induced [l4C]-5HT release, though with longer incubation times, this potentiatory effect was gradually lost eg 0.03U/ml T-l±0.3%, OAG+T (10 sec)- 33±4%, OAG+T (1 min)-11±3%, 0AG+veh.-0%. However, in the presence of R59022 (30μM), OAG retained its potentiatory effect for longer periods eg R59022+0AG+T (1 min)-45+10%, R59022+T-2±l%. With incubation times > 5 min the potentiatory effects of OAG were lost even in the presence of R59022. This is possibly due to the metabolism of OAG by DG lipase. Our results demonstrate that R59022, which has been reported to inhibit DG kinase leading to enhanced PrkC activation, also enhances agonist-induced platelet aggregation and 5HT release. It may therefore be a useful compound in elucidating further the role of DG in terms of both stimulatory and inhibitory effects on platelet activation.[1]. de Chaffoy de Coucelles, D. et al (1985) J Biol Chem 260, 15762.
PROTEIN KINASE C CONTROLS CA2+ MOBILIZATION IN HUMAN PLATELETS
Authors: Siffert W , Scheid P , Akkerman JW N 
Published: August 2018

Platelet stimulation has been shown to result in a rise of cytosolic pH (pHi) as a result of an activation of a Na+/H+ antiport. We have investigated the role of pH in Ca2+ mobilization in human platelets. pHi and free Ca2+, {Ca2+)i, were measured in platelets loaded with the fluorescent indicators BCECF and quin2, respectively. Stimulation of platelets by either thrombin or OAG, an activator of protein kinase C (Pk-C), increased pHi. Pretreatment of platelets with inhibitors of Pk-C, trifluoperazine (TFP) or sphingosine (SPH), blocked the stimulus-induced rise in pHi, suggesting a role of Pk-C in the activation of Na+/H+ exchange. Blocking Na+/H+ exchange by an amiloride analogue or by TFP similarly suppressed the thrombin-induced increase in {Ca2*}i. This effect could be prevented by increasing pHi with the Na+/H+ ionophore monensin or with NH4Cl. The thrombin-induced (0.05 U/ml) rise in {Ca2+}i was more than 3-fold enhanced when the pH was raised from 6.8 to 7.4.Our results demonstrate that pHi controls Ca2+ mobilization in human platelets and suggest that Pk-C contributes to this control by activating the Na+/H+ exchanger.Supported by the Deutsche Forschungsgemeinschaft. No Sche 46/5-2.
Phosphoinositide metabolism in resting and thrombin-stimulated human platelets: Evidence for metabolic homogeneity
Authors: Tysnes Q-B , Verhoeven A J M , Aarbakke G M , Holmsen H 
Published: August 2018

On the basis of differences in specific radioactivity (SA), separate pools of phosphoinositides have recently been proposed in platelets. Human platelets were labelled for 60 min with [32p]p- and subsequently transferred to a phosphate- and Ca2+free Tyrode’s solution by gel-filtration. Thereafter, the platelets were either incubated at 37°C for 120 min, a condition which induces increase in specific labelling of the diester phosphate of phosphatitylinositol (PI), or stimulated with 0.5 U/ml of thrombin. The changes in SA of both diester and monoester phosphates of the phosphoinositides were detrmined. Immediately after the gel filtration, the SA of the diester phosphate of phosphatidylinositol-4-phosphate (PIP) and phosphatidylinositol-4,5-bisphosphate (PIP2) were both similar to that of PI and amounted to 4% or the SA of the monoester groups of PIP and PIP2- Whereas the SA of the monoester phosphate essentially remained constant and the same for PIP and PIP2 during the entire incubation, the SA of their diester phosphates increased gradually in parallel to that of PI, and reached 20% of the monoster groups after 120 min. The effect of thrombin was studied at 15, 60 and 180 sec after the addition. The absolute radioactivity of both diester and monoester phosphates of all phosphoinositides increased conciderably after an initial decrease. However, for the monoester groups, the changes in radioactivity were parallelled by the changes in mass for both PIP and PIP2. Thrombin therefore induced no changes in SA of the monoester phosphates. In contrast, the SA of the diester phosphates increased 5-fold and remained similar for all three phosphoinositides during the 180 sec of stimulation.In conclusion, our results demonstrate close metabolic equilibrium between all three phosphoinositides.Thrombin-induced changes in SA of PIP and PIP2 are purely secondary to changes in specific labelling of the diester phosphate.
ADRENALINE POTENTIATES Ca2+ INFLUX AND INTRACELLULAR MOBILIZATION INDUCED BY PAF AND THROMBIN IN QUIN 2 LOADED PLATELETS
Authors: Powling M J , Hardisty R M 
Published: August 2018

In a medium containing 1 mM extracellular calcium (Ca2+o), sub-threshold concentrations of thrombin (0.01-0.02 u/ml) and PAF (0.1-0.2 nM) induced shape change but not aggregation and raised [Ca2+i] about 200 nM above the basal level of 80 nM. Addition of 0.5 uM adrenaline (which produced no effect alone) 15-30 seconds before those concentrations of second agonist induced rapid aggregation and substantial ATP secretion. In parallel, the rate and magnitude of the Ca2+i flux were both increased: each approximately doubled in response to thrombin while in response to PAF the rate increased about 2.5 fold and the maximum response by about 50%. In the presence of Fab fragments of Ml48, a monoclonal antibody against GPIIb/IIIa, adrenaline produced the same effect on the Ca2+i response although aggregation and ATP secretion were completely abolished.In the presence of 2 mM EGTA ([Ca2+o] = <100 nM) the threshold concentrations of PAF and thrombin raised the [Ca2+i] from 60 nM only to about 110 nM. Under these conditions pre-incubation with 0.5 uM adrenaline did not induce aggregation or ATP secretion, nor was there a significant increase in the maximal extent of Ca2+i flux.The rate of Ca2+i mobilization, however, was approximately doubled in response to thrombin and increased by about 50% in response to PAF.Blockade of cyclo-oxygenase with aspirin did not inhibit the adrenaline-induced potentiation of Ca2+i mobilization whether Ca2+o was 1 nM or <100 nM.It appears that adrenaline may exert its synergistic effects on low doses of other agonists by accelerating Ca2+ influx and intracellular mobilization.
RELATIONSHIP BETWEEN ELEVATION OF CYCLIC-3∲,5∲-GMP (cGMP) AND AGGREGATE FORMATION IN HUMAN PLATELETS
Authors: Edgecombe M , Scrutton M C , Kerry R 
Published: August 2018

Maximal 3- to 5-fold increases in platelet cGMP levels as measured by specific radioimmunoassay are observed on stimulation of aspirin-treated platelet-rich plasma with saturating doses of ADP, adrenaline, 5HT, PAF, thrombin, 9,11-epoxymethano-PGH2 (U44069), collagen, ristocetin and the Ca2+-ionophore ionomycin. The dose/response curves for the elevation in cGMP induced by these agents are either superimposable on, or lie to the right of, those describing the rate or extent of the aggregatory response as measured by an increase in light transmittance. The increase in cGMP induced by ADP is totally inhibited by addition of PGI2 or forskolin with dose/response relationships superimposable on those observed for inhibition of the aggregatory response. No increase in cGMP is observed if platelets are stimulated by PAF or ionomycin in an unstirred system or when aggregation induced by ADP is prevented by addition of a monoclonal antibody which recognises the glycoprotein IIb/IIIa complex.Addition of the fibrinogen γ-chain C-terminal decapeptide (γ402-411) or α-Chain tetrapeptide ARG-GLY-ASP-SER prevents aggregation and the increase in cGMP induced by PAF. The γ-chain decapeptide also completely prevents the increase in cGMP induced by ristocetin, but the a-chain tetrapeptide is ineffective in this respect. Both peptides inhibit to some extent aggregate formation induced by ristocetin.The data demonstrates a strong correlation between aggregate formaticfn and the increase in the platelet cGMP levels and support the previous postulate that platelet-platelet contact causes, activation of guanylate cyclase. No relationship is apparent between the effects of the various agents tested on cGMP levels and their known ability to increase cytosolic Ca2+ concentration. (Supported by SERC and Ciba-Geigy.)
AUSTRALIAN SNAKE VENOMS AND THEIR EFFECT UPON HUMAN PLATELETS
Authors: Marshall L R , Herrmann R P 
Published: August 2018

The in vitro effect of Australian snake venoms on human citrated plasma has been documented and the majority induce coagulation, in keeping with the common clinical presentation of D.I.C. following envenomation. The effect of these venoms upon platelets in vitro has hitherto not been studied extensively and clinical evidence is conflicting, some cases with thrombocyto-paenia have been reported. Twenty Australian venoms were tested, 19 elapids and one hydrophiid (Enhydrina schistosa). Four crotalid snake venoms from the Americas and S.E. Asia were also tested. All of the venoms (1 mg/ml) were investigated for t^eir ability to aggregate both fresh washed platelets (200 × 109/l) resuspended in modified Ardlie’s buffer pH 7.35 and formaldehyde fixed platelets (200 × 109/l) in phosphate buffered saline pH 7.35 using a dual channel Chronolog aggregometer. Samples were taken for electron microscopy (EM).All elapid venoms induced aggregation in fresh platelets, some only minimally and often after a long lag phase. EM studies revealed only clumping without degranulation of the platelets. This was in marked contrast to the crotalid venoms where rapid aggregation and gross degranulation occurred. The hydrophiid venom failed to induce aggregation of the fresh platelets, however upon addition of normal plasma gross aggregation and degranulation was demonstrated. Aggregation of fixed platelets was negligible in the presence of the majority of elapid and the hydrophiid venoms. The crotalid venoms however did induce aggregation, although to a lesser extent than with the fresh platelets.The elapid venoms, along with the others studied, required metabolically active platelets to exert their maximal effect. Crotalid and hydrophiid venoms were more active against platelets than the elapid venoms. The hydrophiid venom’s action on platelets was unique in that a plasma co-factor appeared to be required and this is the subject of further investigations.
INHIBITION OF HUMAN PLATELET AGGREGATION BY THE THROMBOXANE MIMETIC, U46619
Authors: Stratton L , Hornby E 
Published: August 2018

Recent publications suggest that high concentrations (10-100μM) of the stable analogues of PGH2, U46619 and U44069 stimulate adenylate cyclase (Avdonin et al.. 1985) and cause an elevation in cAMP in intact platelets (Best et al., 1979). The effect of high concentrations of U46619 on responses to ADP [1.0-30μM] and vasopressin [1-10nM] was investigated in human platelet rich plasma preincubated with the thromboxane antagonist, GR32191, [10μM] (Lumley et al., this meeting) and aspirin [0.1mM]. Concentration-effect curves to ADP were not affected by preincubation with O.lmM U46619 in the presence of GR32191 [10μM]. Under the same conditions, aggregation to vasopressin was inhibited. U46619 at 30μM had no effect on the vasopressin concentration-effect curve, but at 0.1mM, it resulted in an eight-fold rightwards shift and also reduced the maximum response by 30%. This was equivalent to the inhibition of vasopressin-induced aggregation achieved with prostacyclin at a concentration of 1nM in the same experiments. At 0.3mM, U46619 caused total inhibition of aggregation to vasopressin, equivalent to that seen with prostacyclin at 3nM. The failure of U44619 to inhibit ADP-induced aggregation may reflect inhibition of adenylate cyclase by ADP (Cusack et al. 1982). Aggregation induced by vasopressin is independent of inhibition of adenylate cyclase (Thomas et al.. 1983) and under these conditions, U46619 may induce increases in cAMP which exert an inhibitory action on platelet function. These results indicate that experiments using U44619 as an aggregating agent should be interpreted with caution. The high concentrations of U46619 required to induce aggregation in the presence of thromboxane antagonists may also cause stimulation of adenylate cyclase, thereby making an additional contribution to the inhibition observed. Avdonin P.V. et al.f Thromb. Res., 40, 101-112, 1985.Best L.C. et al.. Biochim, Biophys. Acta, 583. 344-351, 1979.Thomas M.E. et al.. Thromb. Res., 32, 557-566, 1983.Cusack et al.. Br. J. Pharmac., 76, 221-227, 1982.
ABNORMALITIES OF PLATELET AGGREGATION AND ENHANCED FACTOR X ACTIVATOR ACTIVITY OF WASHED PLATELETS IN SICKLE CELL DISEASE
Authors: Chamone D A F , Hoshikawa-Fujimura A Y , Massumoto C , Bellotti G , Arashiro F , Jamra M 
Published: August 2018

The occurence of microvascular occlusion is one of the most prominent pathologic features of sickle cell anemia. The mechanism of vaso occlusion has generally been attributed to the abnormal shape and reduced deformability of the sickled erithrocy tes. However, the involvement of vascular endothelium, platelets and their interactions with coagulation factors may also be of pathogenic significance in microvascular occlusive crises.We investigated the interaction between vascular endothelium, platelets and blood coagulation factors in 23 patients with Sickle Cell Disease (SCD) and in normal volunteers.Factor X activator activity in washed platelets was performed according to Semeraro and Vermylen (1977), thromboxane B2 (TXB2) and 6-keto-PGF1β were determined using specific radioimmunoassays.. PAF-acether from platelets was determined according to Chignard et al (Nature, 1979, 279:799). Platelet aggregation was performed with a Chrono-Log Aggregometer (Model 440) on platelet rich plasma (PRP) using the Born method. Prostacyclin release from endothelium was performed according to Mon-cada et al (Lancet i:18, 1977).Our results showed that platelets from patients with SCD ha ve enhanced factor X activator activity (p < 0.0001), produce mo re PAF-acether than controls (p < 0.02) and showed hyperaggregability in these patients as compared to normal volunteers (p < 0.00001).We concluded that platelets from homozygous sicklers have enhanced factor X activator activity as well as increased capacity for PAF-acether production. These abnormalities may contribute to the incidence of vaso occlusive crises in these patients.
TRANSITORY INFLUENCE OF FIBRINOGEN/FIBRIN DEGRADATION PRODUCTS (FDPs) ON PLATELET AGGREGATION
Authors: Nelson S D , Moriarty A J , Hughes R , Balnave K 
Published: August 2018

This paper describes a pilot study to investigate the influence of FDPs on platelet aggregation in a small cohort of patients (N = 12) undergoing systemic thrombolytic therapy with streptokinase (600,000 I.U. or 1,500,000 I.U. delivered over 30 minutes) for acute myocardial infarction.Serial pre- and post-therapy blood samples were anticoagulated with sodium citrate, and whole blood aggregation studies carried out over 24 hours using a Crono-log 540 aggregometer and the standard adenosine diphosphate (ADP), adrenalin (A), collagen (C) and ristocetin (R) aggregating agents.Results, in the form of mean percentage voltage change from baseline voltage change, measured at 8 minutes after addition of aggregant, are presented for the cohort at times in Figure 1. Serial aggregometry tracings for one representative patient are shown in Figure 2.Clearly comparison of the 1 hour and 18 hour results for each aggregating agent shows a variable but consistent return towards baseline (at FDP < 8 μg/ml) as the FDP concentration drops. This implies that, provided the same platelet population is involved, there is no generalised permanent platelet defect consequent on systemic STK therapy. Coulter counter measurements do not indicate the increase in platelet number that would suggest a large influx of new platelets.
PREPARATION OF A REPRESENTATIVE PLATELET POPULATION BY A SINGLE STEP SLOW CENTRIFUGATION
Authors: Denfors I , Wadenvik H , Kutti J 
Published: August 2018

Circulating platelets differ with respect to size, density and functional ability. In several experimental settings it is of vital importance that a representative platelet-rich plasma (PRP) is extracted from the anticoagulated blood. We therefore investigated the effect of centrifugation time and gravitational force on platelet yield and platelet volume distribution in PRP obtained by a single step slow centrifugation.Methods. From each of 12 healthy male blood donors, 357 ml of venous blood were mixed with 63 ml citrate phosphate dextrose in a plastic bag. 20 ml aliquots of the anticoagulated blood were transferred into 30 ml screwcap polycarbonate tubes (Nalgene Labware). PRP was prepared by a single step slow centrifugation in a microprocessor-controlled bench centrifuge (Hettich Rotanta/RP). The gravitational force was calculated at the bottom of the tube. Centrifugations were performed at 180, 200, 220, 240, 260 g for 10 min, at 180, 200, 220, 240, 260 g for 15 min and at 120, 140, 160, 180, 200 g for 20 min. Three hours after blood collection, the platelet count, mean platelet volume (MPV) and platelet distribution width were determined in PRP and anticoagulated blood with an impedance cell counter (Coulter Counter Model S-Plus VI). Student's t-test was employed for comparison of mean values.Comments. Very precise centrifugation conditions can be provided by using a microprocessor-controlled centrifuge and a "fully representative" platelet population can be isolated with a single step slow centrifugation method.
MEASURING WHOLE BLOOD PLATELET AGGREGATION AND ATP-RELEASE WITH A CHRONO-LOG WHOLE BLOOD LUMI-AGGREGOMETER: EFFECT OF STORAGE TIME OF BLOOD SAMPLES
Authors: Sieders F C , Houwelingen A C v , Hornstra G 
Published: August 2018

The influence of storing blood for either one or two hours after blood sampling, on whole blood platelet aggregation and ATP-release was measured with a Chrono-log whole blood lumi-aggregometer, in 21 healthy male volunteers. Storage of blood samples, gently revolving at 37 °C in an incubator for one hour, caused a significant increase in aggregation and release as compared with results obtained immediately after sampling. After two hours' storage, the values had returned to their initial levels.Significant positive correlations were seen between values obtained before and after storage of blood, and between various aggregation and release parameters. In this study, bleeding time nor hematocrit values were significantly correlated with the aggregation and release parameters. The considerable influence of storage time on whole blood platelet aggregation and ATP-release underlines the importance of performing these determinations immediately after sampling, or possibly after a standardized storage time. Otherwise, comparison of results -obtained either in clinical situations or in trials - will increase variability as a result of which false conclusions may be obtained. This will be illustrated in a small trial using paracetamol.
INTRACELLULAR CA++ MOBILIZATION IN GRAY PLATELET SYNDROME. ELECTRONMICROSCOPIC STUDIES ON AEQUORIN LOADED PLATELETS
Authors: Mori K , Suzuki S , Sugai K , Akutsu Y , Ishikawa M , Sakai H , Hiwatashi K 
Published: August 2018

Light microscopic examinations on platelets in Gray Platelet Syndrome(GPS) showed peculiar gray colored platelets due to deficiency in ^-granules on the peripheral blood smear by May-Grunwald-Giemsa stain. Besides α-granule deficiency, however, several morphological abnormalities, especially abnormal features of dense tubular system(DTS) etc., were recognized in the transmission electronmicroscopic examinations. In the platelet function tests, release abnormalities rather than storage-pool deficiency were noted. We were strongly interested in the relationships between these morphological and functional abnormalities, because DTS in the platelets have been thought to be main storage sites of intracellular Ca ion.We examined the intracellular Ca++ mobilization using aequo-rin loaded platelets by means of Lumi-aggregometer(Salzman's method) under the stimulation of A-23187 and thrombin, and also morphological changes of platelets during the process of platelet aggregation by light |ipd transmission electronmicroscope.Intracellular Ca concentration increased dose-dependently after addition of A-23187 in both normal and GPS platelets. Namely, besides the first peak of emission which located at the same site as normal control, the slowly appearing second peak were recognized on the trace line by the addition of A-23187 and also abnormal by thrombin in GPS platelets. Transmission electronmi-crographs showed insufficient contraction of platelet-aggregates and malformation or wide appearance of pseudopods by the addition of A-23187 and thrombin. Most of the contractile gels, which were usually seen in the center of the platelets, were slightly enlarged and eccentric in the position. Delayed intracellular Ca mobilization were also noted even in the buffer solution containing EGTA.From above mentioned results, intracellular Ca++ mobilization were abnormal and these low and delayed mobilization were thou -ght to be related with prominent abnormal morphology, especially abnormalities of DTS in the GPS platelets.