bluuebunny/crossref_metadata_2025_split huggingface dataset:
ADP-BINDING SITES IN PLATELETS: CHARACTERIZATION BY PHOTOAFFINITY LABELING AND BINDING STUDIES WITH FIXED PLATELETS
Authors: Jefferson J R , Harmon J T , Jamieson G A 
Published: August 2018

Attempts to photoaffinity label platelet ADP receptors with 2-azidoADP have not been successful possibly due to the absence of a spacer arm between the nucleotide and the photolabile group. We have synthesized a probe having a long spacer arm by coupling 2-(3-aminopropylthio)-ADP to succinimidyl 4-3H-azidobenzoate. Labeling competable by ADP could not demonstrated with intact platelets. With isolated platelet membranes, three bands (Mr 140,000, 110,000 and 46,000) were labeled that were not competed by ADP while three other bands (Mr 188,000, 92,000 and 51,000) were competable by 100 uM ADP.Another problem in characterizing ADP receptors has been complications due to ADP metabolism and secretion from the dense granules. To avoid this problem we have measured the binding of ADP and analogues to formalin-fixed platelets. ADP bound to two sites (Kl, 0.35 ± 0.04 uM; R1, 160,000 ± 20,000 sites/platelet; K2 7.9 ± 2.0 uM; R2, 400,000 ± 40,000 sites/platelet) with low non-specific binding: these values are in agreement with ADP concentrations required for activation. Affinity at the high affinity site was in the sequence ADP(0.35 uM)=ATP(0.4 uM)›2-MeS.ADP(6.8 uM)› GDP(49 uM) › AMP(360 uM); adenosine did not compete. Binding at the high affinity site was blocked by pMBS (EC50 250 uM) and 5-fluoro-sulfonylbenzoyladenosine (EC50 1 mM). This is the first report of photoaffinity labeling of putative ADP receptors. Our experiments with fixed platelets suggest that they may be useful in testing agonists, antagonists and inhibitors in the absence of complications due to secretion and metabolism.
ADP-INDUCED CYTOPLASMIC CALCIUM MOBILIZATION AND SHAPE CHANGE IN PLATELETS ARE MEDIATED BY DIFFERENT BINDING SITES
Authors: Koneti Rao A , Kowalska Maria A 
Published: August 2018

Platelet stimulation with ADP results in a number of responses including increase in cytoplasmic ionized calcium concentration [Ca2+]i, shape change, aggregation, secretion, and inhibition of cAMP accumulation caused by PGI2.5'-Fluorosulphonylbenzoyladenosine (FSBA), which covalently labels ADP binding site on platelets, blocks platelet shape change but not inhibition of cyclic AMP levels by ADP, while p-chloromercuribenzenesulfonate (pCMBS), a non-penetrating thiol reagent, blocks ADP-induced inhibition of adenylate cyclase but not shape change. We examined the effect of FSBA and pCMBS on ADP-induced increase in [Ca2+]i to determine whether it is linked to the binding site mediating shape change or that for inhibition of adenylate cyclase. In platelets loaded with Ca2+ indicators, quin 2 or fura 2, and in presence of adenosine deaminase (AD), FSBA (50-200 μM) induced a dose-dependent, rapid rise in [Ca2+]i. from basal levels of 70-90 nM to peak levels of 300-500 nM in the presence of 1 mM external Ca2+ providing direct evidence that FSBA is a platelet agonist. The [Ca2+ ]i. returned to near basal levels over 30 min. The effect of FSBA on [Ca2+]i. was inhibited by ZK 36,374 (40 nM), a stable PGI2 analog. AdP concentrations eliciting similar responses were about 10-fold less than those for FSBA. Platelet incubation with FSBA (50-100 μM) in the presence of AD for 30 min (to ensure optimal covalent labelling of the ADP binding sites) abolished shape change but jjid not inhibit ADP (5, 25 μM)-induced increase in [Ca2+]i. or block the inhibitory effect of ADP on cAMP accumulation in1platelets exposed to ZK 36,374 (50 nM) in.presence of theophylline (7 mM). Incubation with pCMBS (5-100 pM, 2 min) abolished the effect of ADP on [Ca2+]. and on the inhibition of cAMP levels; shape change was not 1 inhabited even at 1 mM. pCMBS (0.5-1 mM) inhibited the rise in [Ca2+ ]. by FSBA alone. These observations suggest that ADP-induced Ca mobilization is mediated by platelet binding sites which are distinct from those mediating shape change but probably the same as those modulating adenylate cyclase.
COMPARISON OF THE PLATELET MEMBRANE SIGNAL TRANSMISSION INDUCED BY THROMBIN OR THE mAb ALB6 (CD9)
Authors: Rendu F , Boucheix C , Lebret M , Soria C , Levy-Toledano S 
Published: August 2018

A CD9 monoclonal antibody described to aggregate human platelets was studied on different platelet functions in order to determine its mechani^i of action. The mAb ALB6 induces a transient decrease in P-phosphatidylinositol-4,5 bisphos-phate (PIP2) after a lag phase of 35 sec. This results in a synthesis of P-phosphatidate (PA), calcium mobilization as measured by quin 2 and phosphorylation of myosin light chain (P20) and of 43 kDa protein (P43). All these biochemical steps allow the release of dense granule, alpha-granule and lysosome constituents. Final biological and metabolic effects of thrombin and ALB6 thus appear similar but three main differences bring additional information.(1) The kinetic of thrombin-induced effects is faster. (2) The thrombin-induced release of dense granules compared to alpha-granules and lyso-somes occurs extremely rapidly (50% of the maximum C 5-HT releasable at 5 sec) and is considerably slowed down by the presence of EDTA (50% release of 14C 5-HT at 20 sec.), whereas the release of the three types of granules occurs concomitantly in the case of ALB6 -induced aggregation whether EDTA is present or not. (3) Myosin light chain is phosphorylated in both cases but external calcium is necessary for maximum phosphorylation by thrombin since it is inhibited by EDTA, which is not the case with ALB6.The ALB6-induced lag phase would result from the fact that the signal transmission has to occur from the external non integral membrane CD9 antigen molecule and the internal phospholipase C. The different requirements in external calcium for the responses induced by the two types of inducers suggest that the early myosin light chain phosphorylation and dense granule release induced by thrombin coulcL be at least partly dependent on the entry of external Ca2+ through a putative Ca2+ channel.
COLLAGEN-DERIVED OCTAPEPTIDE (OP) AND INITIAL EVENTS OF PLATELET ACTIVATION
Authors: Karniguian A , Rendu F , Grelac F , Legrand Y J 
Published: August 2018

A collagen-derived OP was shown to specifically inhibit collagen induced platelet aggregation, fibrinogen binding and prothrombinase activity, suggesting that it affects on the iniating mechanism. In view of this, early biochemical steps were investigating in platelet response to collagen and OP. In the absence of external calcium, a transient decrease in 32Pphosphatidy14,5,bisphosphate (PIP2) occuring in parallel with the shape change, but partially preceding it, was followed by a rapid resynthesis up to 25%. A slightly delayed hydrolysis of PIP also occured within seconds of platelet aggregation. Phosphatidate (PA) was produced only at a small extent , even with high doses of collagen. The phosphorylation of myosin light chain (P20) was minimal, whereas, the phosphorylation of the 43K protein (P43) occured and increased inparallel with the onset of secretion and aggregation, and was collagen dosedependent. Pretreatement by OP reduced the platelet responsiveness and parallely the P43 phosphorylation up to 80%.At concentrations allowing shape change but not aggregation, the breakdown of PIP2 persisted, and only the resynthesis was affected; high doses of OP which prevented any shape change, no PIP2 cleavage was observed.PA formation and P20 phosphorylation were slightly inhibited by 30-50% only at these high doses which completely abolished the PIP2 breakdown.Thus OP could inhibit P43 phosphorylation and platelet aggregation at a concentration which did not affect the ' PIP2-specific phospholipase C. This suggest that collagen can activate proteine kinase C, independently of the initial PIP2 breakdown.
EVIDENCE FOR TWO DIFFERENT Ca2+TRANSPORT SYSTEMS ASSOCIATED WITH PLASMA AND INTRACELLULAR HUMAN PLATELET MEMBRANES
Authors: Enouf J , Breadux R , Bourdeau N , Levy-Toledano S 
Published: August 2018

The regulation of Ca2+ concentration in different cells involves two Ca2+ pumps. The presence of such mechanisms in human platelets is still controverted. We then investigated this question by using plasma and intracellular membranes obtained after simultaneous isolation by centrifugation ca 40% sucrose from a mixed 100,000 g membrane fraction.The Ca2+ uptake by the different membrane vesicles has been studied. Both membrane fractions took up Ca2+ and the Ca2+ transport systems exhibited a high affinity towards Ca 2+.However, the two associated Ca2+ transport systems showed a different time course and exhibited different oxalate sensitivity. The plasma membranes are not permeable to potassium oxalate, while the Ca2+ uptake was stimulated by potassium oxalate in intracellular membranes.Two Ca2+ activated ATPase activities are associated with the isolated membrane fractions and appeared different for the following parameters : 1) a different time course of the two enzyme activities; 2)a similar apparent affinity towards Ca2+ (10−7 M), though inhibition of the Ca2+ ATPase activity was only observed in intracellular membranes at 10−6 M Ca2+ ; 3)a different pH dependence with a maximum at pH 7 for the enzyme of intracellular membranes and pH 8 for the enzyme of plasma membranes; 4)a 10 fold difference in the ATP requirement of the enzymes, thus the maximal response was obtained with 20 uM for the intracellular membrane enzyme and with 200 uM for the plasma membrane enzyme ; 5) a different affinity for various nucleotides as energy donors with a higher specificity of the plasma membrane enzyme towards ATP ; 6) a different vanadate inhibition-dose reponse which did not exceed 60% for the plasma enzyme while it reached 100% for the intracellular enzyme.Taken together, these studies agree with the possible role of both a plasma membrane and a dense tubular system Ca2+ -ATPases in the regulation of Ca2+ concentration in human platelets.
MONOCLONAL ANTIBODIES RECOGNISING PROTEINS OF THE OUTER AND INNER SURFACE OF THE PLATELET PLASMA MEMBRANE
Authors: Wilkinson J M , Hack N , Thorsen L I , Thomas J A 
Published: August 2018

Platelet membrane preparations can be fractionated into two major subpopulations by free flow electrophoresis and these have been shown to correspond to the plasma membrane and the endoplasmic reticulum of the platelet. The plasma membrane fraction can be shown, by two-dimensional electrophoresis, to contain the major surface glycoproteins together with considerable amounts of actin and actin-associated proteins such as the 250 kDa actin-binding protein (filamin), P235 (talin), myosin, α-actinin and tropomyosin (Hack, N. … Crawford, N., Biochem. J. 222, 235 (1984). These cytoskeletal proteins are associated with the cytoplasmic face of the plasma membrane and probably interact with transmembrane glycoproteins. We have raised monoclonal antibodies to the purified plasma membrane preparation in order to investigate the nature of these glycoprotein-cytoskeletal interactions. In two fusion experiments, out of 804 tested, 104 hybrids secreted antibody to the membrane preparation and of these 24 were selected for further study. Initial assays were by ELISA using either the membrane preparation or whole fixed platelets as the target antigen. The specificity of the antibodies was investigated further by immunoblotting of SDS gels of total platelet proteins prepared under reducing and nonreducing conditions, by immunofluorescence, by immunohisto-chemistry and by crossed immunoelectrophoresis. The majority of the antibodies recognise major surface glycoproteins; of these, four bind to glycoprotein Ib under all conditions examined while another seven recognise the glycoprotein IIb/IIIa complex as detected by crossed immunoelectrophoresis. Three antibodies recognise the actin binding protein and these cross-react with the smooth muscle protein filamin in a number of different species. Further characterisation of these antibodies in both structural and functional terms will be presented.We are grateful to the Smith and Nephew Foundation for financial support for these studies
PLATELET INDUCED VASOSPASM
Authors: Drouet L , Pignaud G , Baladier L , Paris C 
Published: August 2018

A new model of experimental thrombosis on 100 to 300 micron diameter rat mesenteric arteries has been designed.The focal specific deendothelialisation is induced by a tunable dye pulsed laser. All changes in the vascular lumen and of the vascular wall are observed under a stereozoom magnifier. This model allows in-time observation and quantification of induced phenomena after digitalisation and image processing of the video signal of the observation system. Besides thrombus formation and possibilities of its pharmacological manipulation, we focussed this study on the downstream vasospasm induced by platelet activation during thromboformation. The vasospasm is platelet dependent because it can be observed only when a platelet thrombus takes place . The vascular reaction is mediated through the endothelium, as the endothelium has been localy injured only on a 50 micron diameter spot at the place where the thrombus developpes and is intact at the level where the spasm is observed (more than 500 micro downstream). The role of platelet factors putatively involved in the vascular reaction has been evaluated by the use of specific inhibitors or antagonists (namely of ADP, serotonin, catecholamins, thromboxane A2 and PAF acether). Proofs are given that at least both platelet serotonin and thromboxane A2 are actively involved. These two intermediaries would sequentially participate : serotonin in the early phase and thromboxane A2 in the secondary phase. Vascular synthesis of prostacyclin does not seem to be a major intervenant in the regulation of the vascular tone.
SYNERGISM OF Gi-DISSOCIATION AND PROTEIN KINASE C STIMULATION IN PLATELET ACTIVATION
Authors: Siess W , Lapetina E G 
Published: August 2018

Epinephrine or UK 14304 (a specific (α2-adrenoceptor agonist) synergizes with phorbol esters (phorbol 12,13-dibutyrate, PdBu) or bioactive diacylglycerols (sn-1,2-dioctanoylglycerol, DiC8) to induce aggregation and ATP-secretion of platelets. The effect on aggregation is more pronounced than on secretion, and it is observed in aspirinized platelet-rich plasma or suspensions of washed platelets containing ADP-scavengers. No prior shape change is found. In the presence of epinephrine, DiCg induces reversible aggregation and PdBu evokes irreversible aggregation that correlates with the effects, on protein phosphorylation. Epinephrine and UK 14304 neither induce nor enhance the phosphorylation of myosin light chain (20kDa), the substrate of protein kinase C (47kDa), or a 38kDa protein evoked by DiCg) or PdBu. Epinephrine does not cause, stimulation of phospholipase C as reflected by the production of inositol mono-, bis- and tris-phosphate or phosphatidic acid. Even under conditions of maximal aggregation induced by epinephrine plus PdBu, formation of 32p-phOSphatidic acid is not observed. The synergistic action of epinephrine and PdBu does not depend on extracellular Ca2+. Primary aggregation induced by epinephrine, but not platelet aggregation induced by PdBu plus epinephrine, is inhibited by high intracellular concentrations of the calcium chelator quin2. Prostacyclin prevents platelet aggregation but does not affect protein phosphorylation induced by PdBu plus epinephrine.The experiments indicate that α2-adrenoceptor agonists may induce primary aggregation by a mechanism involving release of membrane-bound Ca2+. The synergism with protein kinase C is, however, caused by a mechanism that occurs distally to protein phosphorylation and is not related to Phospholipase C activation and Ca2+-fluxes across the Dlasma membrane or in the cvtosol. Evidence is presented suoportina the view that this mechanism miqht be related to the dissociation of Gi caused by α2-adrenoceptor activation.
POSSIBLE RELATIONSHIP BETWEEN THE 23-kDa PHOSPHOPROTEIN AND THE IP3 -INDUCED Ca2+RELEASE IN HUMAN PLATELETS
Authors: Enouf J , Bredoux R , Giraud A , Bourdeau N , Levy-Toledano S 
Published: August 2018

The regulation of Ca2+ concentration in human platelets involves intracellular membranes i.e. dense tubular system (DTS). Agonist-induced platelet activation generates inositol 1,4,5 trisphosphate (IP3) which is responsible for Ca2+ mobilization from DTS. However, its mechanism of action is still unknown. cAMP has been shown to regulate Ca2+ transport by isolated membrane vesicles. This effect was correlated with the phosphorylation of a 23 kDa protein. We investigated whether this phosphorylation could play a role in the mechanism of IP3-induced Ca release.We isolated a membrane fraction enriched in intracellular membranes, which actively sequesters Ca2++. The Ca2+ uptake was mediated by a characterized (Ca2+ + Mg2+)-ATPase of a molecular weight 120 kDa. As well, the characterization of the 23-kDa protein phosphorylation by the catalytic subunit of the cAMP dependent protein kinase (C. Sub.) has been achieved.
IP^-induced Ca release was tested on our membrane preparations. The transient effect was maximal at one minute and a dose-response curve was obtained.The cAMP dependent phosphorylation of the 23-kDa protein increased the Ca2+ liberation induced by IP by two fold whatever the IP3 concentration. The addition on the protein kinase inhibitor inhibited the IP3 -induced Ca2+ release.The effect of IP3 on the cAMP-mediated phosphorylation of the 23-kDa protein has been examinated.A dose dependent stimu-ulation of the 23-kDa protein phosphorylation in the presence of C. Sub. was initiated by IP3. The maximal effect was observed after 1-2 min and obtained with an IP3 concentration similar to that producing the maximal calcium release. The stimulation of the phosphorylation by IP3 was detected in the absence of Ca2+ and in the presence of phosphatase inhibitors.Therefore, we suggest a possible correlation between cAMP dependent phosphorylation of the 23-kDa protein and the IP3-induced Ca2+ release in human platelet membrane vesicles.
THROMBIN, PAF AND VASOPRESSIN-INDUCED RISE OF PLATELET INTRACELLULAR CALCIUM, AGGREGATION AND ATP SECRETION ARE REDUCED BY ANTIOXIDANTS
Authors: Doni M G , Deana D , Alexandre A 
Published: August 2018

Physiological agonists induce a rapid increase of cytosolic free Ca2+ concentration ([Ca2+]i) in platelets, as well as shape change, aggregation and1 secretion. They activate a phosphodiesterase which specifically splits polyphosphoinositides, generating the second messengers diacylglycerol, a protein kinase C ativator, and inositol 1,4,5 triphosphate, which releases Ca2+ from the intracellular stores - [Ca2+]i rise and protein kinase C activation are responsible for aggregation and exocytosis.We have now studied the effects of the synthetic antioxidants: butyl-hydroxytoluene (BHT), butyl- hydroxyanisole (BHA), nordihydroguaiaretic acid (NDGA) and the one electron donor 1-1' dimethylferrocene plus ascorbate (FC) on the increase of [Ca2+]i, aggregation and ATP secretion. Human platelets were loaded1 with 20μM quin-2-acetoxymethylester and incubated with aspirin (100μM). Changes in the [Ca2+]. were measured with a spectrofluorimeter. Platelet aggregation and ATP secretion (luciferin/luciferase system) were evaluated in parallel. In the presence of 1mM external Ca2+,0.15 u/ml thrombin induced a [Ca2+]. increase to about 1μM, that was inhibited by 50% and abolished by 25μM and 100μM BHT respectively; [Ca2+]. increase induced by 75 nM PAF, 1μM vasopressin, 10 μM ADP was also inhibited by BHA, NDGA and FC in a range of 30-150 μM. Shape change, aggregation and ATP secretion were also inhibited. In the absence of external Ca2+ (1mM ECTA), 80μM BHT inhibits the [Ca2+]i increase originating only from the intracellular stores. Tumor-promoting phorbol esters induce aggregation and secretion without raising [Ca2+]i; 80 μM BHT induced 80-90% inhibition of aggregation and ATP secretion by 0.12nM TPA. Our results suggest that some free radical dependent reactions are involved both in the processes of platelet activation leading to the increase of [Ca2+]i and in those leading to aggregation and exocytosis.
SUB-SECOND CALCIUM DYNAMICS IN ADP AND THROMBIN-STIMULATED PLATELETS; ASSESSED BY A C0NTINU0US-EL0W APPROACH
Authors: Gear A R L , Jones G D 
Published: August 2018

There is now evidence that many platelet reactions begin within 1 sec of platelet stimulation. These include "shape change," aggregation and biochemical events such as protein phosphorylation. Our laboratory has devised quenched-flow approaches for following such early events (J Lab Clin Med 100, 866, 1982) and we have extended these to fluorimetric analyses of rapid calcium changes. A micro, flow-through cell, with a sensing volume of 0.1 μ1, is placed on line from the quenched-flow apparatus. Indo-1 loaded, human platelets are pumped through the system and reaction times from 0.25 sec can be followed. Ratioing emission changes at 400 and 480 nm, after excitation at 355 nm, provides an index of free calcium. ADP (10 μM) induced a rapid increase in Ca++ to about 1 μM by 1.5 sec, beginning near 0.3 sec. This was faster and greater than the first increase caused by thrombin (10U/ml). However, thrombin induced a second (> 5s) and larger increase in free platelet calcium. Control experiments where the Indo-1 loaded platelets were simply pumped through the 0.3 mm ID reaction tubing, revealed a slight increase above resting calcium values, indicating some shear-induced activation. The use of the continuous-flow fluorescent cell coupled to the quenched-flow apparatus enables following calcium dynamics under Theological conditions very close to those iui vivo.Correlations with other early events, such as protein phosphorylation, become possible. Supported by NIH HL-27014.
AGGREGATION RESPONSE OF PLATELETS DURING INHIBITION OF PHOSPHOLIPASE A2
Authors: Labow R S , Meek E , Adams G A , Rock G 
Published: August 2018

Arachidonic acid (AA) is liberated from platelet membrane phospholipids during stimulation and promotes cellular aggregation through the synthesis of thromboxane A2. Two pathways; phospholipase A2 (PLA2) or phospholipase C (PLC) followed by the action of acylglycerolipases, are thought to be activated during platelet stimulation and supply the necessary AA. We have reported that mono (2-ethylhexyl)phthalate (MEHP), a physiological metabolite of the plasticizer di(2-ethylhexyl)-phthalate (DEHP), commonly used in a variety of medical devices and storage containers, inhibits PLA2, but not PLC in platelet lysates. The effects of MEHP on intact platelets were studied. PLA2 activity in intact platelets or lysates was assayed by incubating them with 2-14C-arachidonyl-phosphatidylcholine and measuring formation of free 14C-arachidonic acid in 10 min. Platelet lysates hydrolyzed 10% of the substrate while 2.6% was hydrolyzed by intact platelets. The amount of MEHP needed to inhibit 14C-AA liberation was 0.35 mM for platelet lysates and 0.7 mM for intact platelets. Platelet aggregation induced by collagen was inhibited by MEHP (1 mM), although responses to adenosine diphosphate, AA and ionophore were unaffected. Identical effects on platelet aggregation were found when indomethacin (0.1 mM) was added. Higher concentrations of MEHP blocked platelet aggregation induced by adenosine diphosphate or AA but not ionophore or synergistic pairs of these stimuli, indicating a more generalized membrane disruption at higher MEHP concentrations. These results suggest that MEHP acts in a similar manner to indomethacin to block PLA2-mediated liberation of arachidonate during platelet aggregation(supported by MRC and NHRDP, Canada)