Identification of a nucleotide-binding site on glycoprotein IIb. Relationship to ADP-induced platelet activation

Formalin-fixed platelets have been used to study the binding of adenine nucleotides in order to avoid the complications of nucleotide metabolism and to achieve steady-state binding. Sp-adenosine-5'-(1-thiotriphosphate) (Sp-ATP-alpha-S) binds to platelets at two sites (Kd1 3 nM; 31,000 sites/platelet; Kd2 200 nM; 300,000 sites/platelet) as compared with values for ADP under these conditions (Kd1 30 nM; 25,000 sites/platelet and Kd2 3 microM; 400,000 sites/platelet) (bound/total approximately 0.1). Competition binding experiments showed that both of the ATP-alpha-S sites were accessible to ADP and vice versa. [35S]ATP-alpha-S was photoaffinity cross-linked to unfixed platelets by direct irradiation with ultraviolet light. A single radiolabeled component (120 kDa) was identified and shown to be identical with the alpha subunit of GPIIb based on two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western blotting with anti-GPIIb monoclonal antibodies, by isoelectric focusing (pI 4.5-5.5), by immunoaffinity adsorption using monoclonal anti-GPIIb/IIIa antibodies coupled to Sepharose, and by crossed immunoelectrophoresis. Amino-terminal sequencing of a tryptic fragment labeled with [35S]ATP-alpha-S identified an 18-kDa domain beginning at Tyr-198 in the primary sequence of GPIIb alpha. These studies demonstrate the presence of an adenine nucleotide-binding site on GPIIb alpha.     

ADP receptor-induced activation of guanine-nucleotide-binding proteins in human platelet membranes.

ADP receptor-regulated binding of the labeled GTP analog, guanosine 5'-O-(3-[35S]thiotriphosphate) ([35S]GTP[gamma S]), to guanine-nucleotide-binding proteins (G proteins) was studied in human platelet membranes. The potent ADP receptor agonist, 2-methyl-thio-adenosine 5'-diphosphate (2MeSADP), a non-hydrolyzable analog of ADP, increased the binding of [35S]GTP[gamma S] without apparent lag phase. Under optimal conditions, i.e. in the presence of GDP (1-10 microM), 2MeSADP increased the binding up to about threefold, with half-maximal and maximal increase observed at 10 nM and 1 microM 2MeSADP, respectively. ADP itself increased the binding of [35S]GTP[gamma S] by maximally about twofold, with half-maximal increase occurring at 0.1 microM ADP. The agonist-induced stimulation was competitively antagonized by the ADP receptor(s) antagonist, (1S)-adenosine 5'-O-(1-thiotriphosphate) [(Sp)-ATP[alpha S]]. Other platelet receptor agonists known to act through receptors coupled to G proteins also increased binding of [35S]GTP[gamma S] in human platelet membranes, but without being inhibited by (Sp)-ATP[alpha S]. The data presented indicate that the platelet ADP receptor(s) can interact with and efficiently activate G proteins, the nature of which remains to be identified.     

Alpha2-adrenergic agonist modulation of [35S]GTPgammaS binding to guanine-nucleotide-binding-proteins in human platelet membranes.

Alpha2-adrenoceptor (alpha2-AR)-regulated binding of the labelled GTP analog, guanosine 5'-O-(3-[35S]thiotriphosphate) ([35S]GTPgammaS), to guanine-nucleotide-binding proteins (G proteins) was studied in human platelet membranes. Under optimal conditions, the potent alpha2-AR agonist, 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14304), increased the binding of [35S]GTPgammaS up to approximately 1.8 fold, with half-maximal increase at 60 nM and was competitively inhibited by the alpha2-AR antagonist Rauwolscine. The actions of both UK 14304 and Rauwolscine were modulated by monovalent and divalent cation levels, as well as by the concentrations of GDP. [35S]GTPgammaS binding induced by UK 14304 had a Kd value of 4.5 +/- 0.3 nM and a Bmax value of 4.15 +/- 0.40 pmol/mg protein. The rank order of potencies of adrenergic ligands tested in stimulating [3S]GTPgammaS binding and inhibiting forskolin-stimulated c-AMP accumulation was UK 14304> Guanabenz acetate> Oxymetazoline hydrochloride> B-HT 920 dihydrochloride> p-Aminoclonidine hydrochloride> Clonidine hydrochloride. The data presented indicate that enhancement of [35S]GTPgammaS binding by alpha2-AR in human platelet membranes provides a simple functional measure for receptor activation and can be used for determination of potencies and efficacies of ligands at the alpha2-AR.     

Binding of the snake venom-derived proteins applaggin and echistatin to the arginine-glycine-aspartic acid recognition site(s) on platelet glycoprotein IIb.IIIa complex inhibits receptor function

In the present report we describe the platelet-binding characteristics of applaggin and echistatin, potent inhibitors of fibrinogen-dependent platelet aggregation derived from Agkistrodon piscivorus piscivorus and Echis carinatus snake venoms, respectively. Both molecules bound to unstimulated platelets in a specific and saturable manner. At saturation there were 37,100 +/- 3,150 (mean, +/- S.D.) molecules of applaggin and 27,200 +/- 2,816 molecules of echistatin bound/platelet, with dissociation constants (Kd) of 1.4 +/- 0.6 x 10(-7) M and 4.9 +/- 1.2 x 10(-7) M, respectively. Stimulation of platelets with ADP (10 microM) + epinephrine (2 microM) resulted in an increase in the number of molecules bound at saturation to 42,300 +/- 2,105 for applaggin and 32,185 +/- 3,180 for echistatin, with a Kd of 5.6 +/- 0.3 x 10(-8) M and 1.8 +/- 0.6 x 10(-7) M, respectively. The synthetic peptide (Arg)8-Gly-Asp-Val was a competitive antagonist of applaggin and echistatin binding to unstimulated platelets (Ki = 25 and 36 microM, respectively). Applaggin and echistatin inhibited the binding of fibrinogen to stimulated platelets in a dose-dependent manner, with an IC50 of 9 and 25 nM, respectively. In concert with inhibition of platelet aggregation, applaggin and echistatin inhibited platelet secretion and synthesis of thromboxane A2 induced by ADP, collagen, and human gamma-thrombin. The monclonal antibody, LJ-CP3, which inhibits the binding of Arg-Gly-Asp containing ligands to platelet GPIIb.IIIa, also inhibited applaggin binding to unstimulated platelets in a competitive manner (Ki = 4.5 microM). Thus, applaggin and echistatin bind to the platelet GPIIb.IIIa complex, and the Arg-Gly-Asp sequence plays a central role in mediating this interaction.     

Unique guanine nucleotide binding properties of the human placental GTP-binding protein Gp

Gp is a major GTP-binding protein of human placenta and platelets [Evans, T., Brown, M. L., Fraser, E. D., & Northup, J. K. (1986) J. Biol. Chem. 261, 7052-7059]. High-affinity guanine nucleotide binding is associated with a polypeptide migrating identically with H-ras on SDS-PAGE. We have characterized the interactions of preparations of purified human placental Gp with guanine nucleotides in detergent solution. Equilibrium binding studies with [35S]GTP gamma S, [3H]Gpp(NH)p, and [3H]GTP identified a single class of sites with a dissociation constant of 10 +/- 1, 153 +/- 61, and 125 +/- 77 nM for the ligands, respectively. These three ligands were mutually competitive with Ki values consistent with the Kd values from direct binding experiments. Competition for the binding of [3H]Gpp(NH)p was used to determine the specificity of the site. Ki values determined from this assay were 14 nM for GTP gamma S, 143 nM for Gpp(NH)p, 3.3 microM for GDP beta S, 69 nM for GTP, and 64 nM for GDP. ATP, ADP, cAMP, cGMP, and NAD+ had no detectable affinity for this site. While the equilibrium binding data fit well to a single class of sites, association kinetics of these ligands were better fit to two rate constants. Dissociation kinetics, however, were not clearly resolved into two rates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on the hepatic alpha 1-adrenergic receptor. Modulation of guanine nucleotide effects by calcium, temperature, and age

The effects of guanine nucleotides on the hepatic alpha 1-adrenergic receptor were studied using norepinephrine (NE) displacement of [3H]prazosin binding to rat liver plasma membranes. Nonhydrolyzable GTP analogues caused large rightward shifts of norepinephrine displacement curves of [3H]prazosin binding in EGTA-treated membranes, but only small shifts in membranes prepared with Ca2+. The effect of a brief Ca2+ exposure on NE displacement curves was not reversed by adding excess EGTA prior to binding experiments. Analysis of the curves showed that the EGTA membranes had an increased number of high affinity agonist sites (Kd, 42 nM) and that guanyl-5'-yl imidodiphosphate (GppNHp) converted these to low affinity sites (Kd, 1039 nM). When binding was carried out at 2 degrees C, the norepinephrine displacement curves were shifted to the left, and GppNHp was without effect. Neither EGTA, Ca2+, nor 2 degrees C treatment altered [3H]prazosin binding per se. Attempts were made to differentiate the potency order of GTP analogues which alter glucagon receptor binding (presumably mediated by the stimulatory GTP-binding protein, Na, of the adenylate cyclase system) from the potency order of GTP analogues which alter alpha 1-receptor agonist binding (presumably mediated by a yet uncharacterized GTP-binding protein which some have speculated may be distinct from Ns). However, the potency series of GTP analogues to alter norepinephrine binding was GTP gamma S greater than GppNHp greater than or equal to GTP greater than or equal to GDP greater than or equal to GppCHp greater than GMP (where GTP gamma S represents guanosine 5'-O-(thiotriphosphate) and GppCHp represents guanyl-5'-yl (beta, gamma-methylene)diphosphonate) and was identical to that for inhibition of [125I]iodoglucagon binding. The ability of GppNHp to alter norepinephrine displacement of [3H]prazosin binding increased with the age of the rat from which membranes were prepared. This was due to the fact that juvenile rats (50-75 g) had few alpha 1-receptors in the high affinity state, whereas in old rats (430-490 g) more of the receptors were in this form. Age has previously been shown to increase alpha 1-adrenergic stimulation of cAMP in isolated hepatocytes (Morgan, N.G., Blackmore, P. F., and Exton, J. H. (1983) J. Biol. Chem. 258, 5103-5109) but did not affect the dose-response curves for norepinephrine-induced Ca2+ mobilization and phosphorylase activation in these cells. These data suggest that alpha 1-adrenergic receptors can become coupled to a guanine nucleotide-responsive moiety in hepatic plasma membranes and that this may be similar to Ns.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of the Fc gamma receptor on human platelets

IgG-containing immune complexes may play a role in the immune destruction of human platelets by interacting with an Fc gamma receptor on the platelet surface. We studied the platelet Fc gamma receptor and characterized its interaction with IgG ligand and anti-Fc gamma receptor monoclonal antibodies. Oligomers of IgG, but not monomeric IgG, bound to platelets and the number of binding sites was significantly increased at low ionic strength. Ligand-binding studies indicated that normal human platelets express a single Fc gamma receptor (Fc gamma RII) with 8559 +/- 852 sites per cell, Kd = 12.5 +/- 1.7 X 10(-8) M using trimeric IgG. Results of studies with bivalent and Fab monoclonal anti-Fc gamma RII were consistent with each Fc gamma receptor expressing two epitopes recognized by the antibody. The number of Fc gamma binding sites and affinity of binding were unchanged by the presence of 2.0 mM Mg2+ or 10 micrograms/ml cytochalasin B. Platelet stimulation with thrombin or ADP in the presence of fibrinogen also did not alter the number of Fc gamma binding sites or the affinity of binding. However, platelets preincubated with 5 microM dexamethasone expressed a decreased number of Fc gamma binding sites as well as decreased IgG-dependent platelet aggregation. Platelets from patients with Glanzmann's thrombasthenia and from patients with the Bernard Soulier syndrome expressed a normal number and affinity of Fc gamma binding sites. The data suggest that platelet Fc gamma RII binding of trimeric IgG occurs independent of actin filament interaction, Mg2+, ADP, or thrombin and does not require GPIIb/IIIa or GPIIb/IIIa-fibrinogen interaction. Furthermore, this receptor appears to be normally expressed on GPIb-deficient platelets and susceptible to modulation by glucocorticoids. Finally, the Fc gamma-binding protein was isolated from whole platelets as a 220-kDa protein which upon reduction dissociates into 50,000 Mr subunits.     

Guanine nucleotide-sensitive interaction of a radiolabeled agonist with a phospholipase C-linked P2y-purinergic receptor

Analogs of ATP and ADP produce a guanine nucleotide-dependent activation of phospholipase C in turkey erythrocyte membranes with pharmacological properties consistent with those of a P2y-purinergic receptor (Boyer, J. L., Downes, C. P., and Harden, T.K. (1989) J. Biol. Chem. 264, 884-890). This study describes the interaction of adenosine-5'-O-2-thio[35S] diphosphate ([35S]ADP beta S) with this putative P2y-purinergic receptor on purified plasma membranes prepared from turkey erythrocytes. In binding assays performed at 30 degrees C, the association rate constant of [35S] was 1.1 x 10(7) M-1 min-1 and the dissociation rate constant was 3.8 x 10(-2) min-1. [35S]ADP beta S bound with high affinity (Kd = 6-10 nM) to an apparently homogeneous population of sites (Bmax = 2-4 pmol/mg protein). ATP and ADP analogs (2-methylthio ATP, ADP beta S, ATP, ADP, 5'-adenylyl imidodiphosphate, alpha, beta-methylene adenosine-5'-triphosphate, and beta, gamma-methylene adenosine 5'-triphosphate) inhibited the binding of [35S]ADP beta S with properties consistent with ligand interaction by simple law of mass action kinetics at a single site. The rank order of potency for inhibition of [35S]ADP beta S binding was identical to the potency order observed for these same agonists for stimulation of phospholipase C in turkey erythrocyte ghosts. Guanine nucleotides inhibited [35S]ADP beta S binding in a noncompetitive manner with the following potency order: guanosine 5'-O-(3-thiotriphosphate) greater than 5'-guanylyl imidodiphosphate greater than GTP = GDP greater than guanosine 5'-O-2-(thiodiphosphate). The data are consistent with the idea that [35S]ADP beta S may be used to radiolabel the P2y-purinergic receptor linked to activation of phospholipase C in turkey erythrocyte membranes. In addition, interaction of radiolabeled agonist with the receptor is modified by guanine nucleotides, providing evidence that an agonist-induced receptor/guanine nucleotide regulatory protein complex may be involved in P2y-receptor action.     

Comparative platelet binding and kinetic studies with normal and variant factor IXa molecules

We have recently shown that thrombin-stimulated human platelets have specific, saturable receptors for factor IXa, occupancy of which promotes factor X activation (Ahmad, S. S., Rawala-Sheikh, R., and Walsh, P. N. (1989) J. Biol. Chem. 264: 3244-3251, 20012-20016; Rawala-Sheikh, R., Ahmad, S. S., and Walsh, P. N. (1990) Biochemistry 29, 2606-2611). To study the structural requirements for factor IXa binding to platelets, equilibrium binding studies and kinetic studies of factor X activation were carried out with normal factor IXa and with two variant proteins: factor IXaAlabama (FIXaAL; Asp47----Gly substitution) and factor IXaChapel Hill (FIXaCH; Arg145----His substitution). In the absence of factors VIIIa and X, there were 331 binding sites/platelet for FIXaCH (Kdapp = 2.8 nM), and 540 sites/platelet for FIXaAL (Kdapp = 3.2 nM), compared with 540 sites/platelet (Kdapp = 2.3 nM) for normal factor IXa. The addition of factors VIIIa and X, both at saturating concentrations, had no effect on the number of binding sites for either normal or variant factor IXa, resulted in a decrease in the Kd for normal factor IXa to 0.67 nM, resulted in a suboptimal decrease in Kd for FIXaAL (1.4 nM), and had no effect on the Kd for FIXaCH. Kinetic studies of factor X activation at variable factor IXa concentration confirmed these values of Kd in the presence of factors VIIIa and X. Determination of rates of factor X activation at variable substrate concentrations yielded normal values of catalytic efficiency (kcat/Km) for the variant proteins, thereby indicating that the abnormally low rates of factor X activation obtained were a consequence of the low affinity binding of FIXaAL and FIXaCH to thrombin-activated platelets in the presence of factors VIIIa and X. These studies suggest that the presence of Asp47 and the cleavage of factor IX at Arg145-Ala146 are important structural features required for specific, high affinity factor IXa binding to platelets in the presence of factors VIIIa and X.     

Characterization of the binding of human tissue-type plasminogen activator to platelets

Cell surface binding sites for the constituent proteins of the fibrinolytic system may play a role in the localization and regulation of fibrinolysis. In the present study, specific binding of recombinant human tissue-type plasminogen activator (rt-PA) to human blood platelets was identified and characterized. 125I-labeled rt-PA was found to bind specifically, saturably, and reversibly to the surface of gel-filtered platelets, reaching equilibrium within 5 min at 22 degrees C. Scatchard analysis revealed a single class of binding sites. Unstimulated platelets bound 120,000 +/- 24,000 (mean +/- S.D.) molecules/platelet with an apparent Kd of 340 +/- 25 nM, whereas thrombin-stimulated platelets bound 290,000 +/- 32,000 molecules/platelet with an apparent Kd of 800 +/- 60 nM. Binding of 0.1 microM 125I-rt-PA was greater than 90% reversible by a 50-fold excess of unlabeled rt-PA. Binding was not inhibited by fibrinogen or single chain urokinase-type plasminogen activator, but plasminogen partially competed for binding of 125I-rt-PA to platelets (up to 40% displacement). These findings indicate that the platelet surface possesses a large number of specific, low affinity binding sites for t-PA and provide further evidence for the role of platelets in localization and regulation of fibrinolysis.     

Binding and hydrolysis of guanine nucleotides by Sec4p, a yeast protein involved in the regulation of vesicular traffic

The 23.5-kDa Sec4 protein is required for vesicular transport between the Golgi apparatus and the plasma membrane in Saccharomyces cerevisiae. In order to analyze its biochemical properties, we have purified the soluble pool of the wild-type protein from an overproducing yeast strain. At 30 degrees C, Sec4p bound [35S] guanosine 5'-O-(thiotriphosphate) (GTP gamma S) with a rate of 0.18 min-1 in a reaction requiring micromolar concentration of free magnesium ions. The protein had high affinity for guanine nucleotides with Kd values for GTP gamma S and GTP of 3.7 nM and 3.5 nM, respectively, and that for GDP of 77 nM. The dissociation of [3H] GDP from Sec4p occurred with a rate of 0.21 min-1 suggesting that the association of GTP gamma S was the result of exchange for prebound GDP. The release of GTP from Sec4p was slow and correlated with a low inherent GTPase activity of 0.0012 min-1. By analogy with other classes of GTP binding proteins, both the nucleotide exchange and hydrolysis activities of Sec4p may be modulated in vivo to facilitate its role in the regulation of intercompartmental membrane traffic.     

Down regulation of alpha 2 adrenergic receptor numbers in platelets by insulin.

Incubation of platelets from normal volunteers, who had not taken any medication at least for 2 weeks, with insulin (200 mu units/ml), resulted in the inhibition of the potentiation of ADP-induced platelet aggregation in the presence of (-)-epinephrine by 50-60% when compared with the control. The inhibitory effect of insulin was not related to the increased cyclic AMP level or decreased thromboxane A2 synthesis in these cells. It was found that the treatment of platelets with insulin decreased alpha 2 adrenergic receptors number of these cells from 413 +/- 92/platelet to 206 +/- 84/platelet as determined by Scatchard analysis of [3H]yohimbine binding. The affinity of the receptors (1.05 +/- 0.02 nM) remained essentially unchanged due to the treatment of platelets with the hormone (1.40 +/- 0.60 nM).     

Characterization and site-directed mutagenesis of a low M(r) GTP-binding protein, ram p25, expressed in Escherichia coli.

The ram gene encodes a GTP-binding protein with a M(r) of 25,068 (Nagata, K., Satoh, T., Itoh, H., Kozasa, T., Okano, Y., Doi, T., Kaziro, Y., and Nozawa, Y. (1990) FEBS Lett. 275, 29-32). It has a putative effector domain very similar to that of yeast SEC4 protein, and shares 40% identity and 60% homology with it, respectively. In order to analyze the biochemical properties, ram cDNA was engineered and inserted into a bacterial expression vector; this allowed the production at a high level of soluble recombinant ram p25 in Escherichia coli. The purified ram p25 contained an equimolar amount of GDP. The purified protein bound approximately 1 mol of [35S]guanosine 5'-O-(thiotriphosphate) GTP gamma S)/mol of protein, with a Kd value of 120 nM. [35S]GTP gamma S binding to this protein was inhibited by GTP and GDP, but not by ATP and ADP. In the presence of 10 mM Mg2+, the dissociation of [8,5'-3H]GDP and [35S]GTP gamma S from ram p25 occurred with rates of 0.015 min-1 and 0.004 min-1, respectively, showing that the ram p25 has a higher affinity for GTP than GDP. The rate of release of Pi from [gamma-32P]GTP-bound ram p25 was calculated to be 0.011 min-1. The contribution of guanine nucleotide-binding and GTP-hydrolysis domains of the protein to its biochemical activities was investigated by site-directed mutagenesis. Substitution of Val for Gly at position 19 resulted in disappearance of [35S]GTP gamma S- and [3H]GDP-binding activity in spite of good expression of the protein. Mutations of Thr41 to Ser, Ala76 to Thr, and Asn133 to His slightly increased the rates of [35S] GTP gamma S binding and [3H]GDP dissociation, but had almost no effects on the manner of [gamma-32P]GTP hydrolysis. Replacement of Gln78 with Leu significantly increased the [3H]GDP dissociation rate (7-fold) and decreased GTP hydrolytic activity considerably.     

Binding of a thromboxane A2/prostaglandin H2 agonist [3H]U46619 to washed human platelets

The binding characteristics of [3H]U46619 to washed human platelets were studied. [3H]U46619 binding to washed human platelets was saturable and displaceable. Kinetic studies yielded a Kd of 11 +/- 4 nM (n = 4). Scatchard analysis of equilibrium binding studies revealed one class of high affinity binding sites with a Kd of 20 +/- 7 nM and a Bmax of 9.1 +/- 2.3 fmole/10(7) platelets (550 +/- 141 binding sites per platelet) (n = 4). A number of compounds that act as either agonists or antagonists of the TXA2/PGH2 receptor were tested for their ability to inhibit the binding of [3H]U46619 to washed human platelets. The Kds of the agonists and antagonists were similar to their potencies to induce or inhibit platelet aggregation. These data provide some evidence that [3H]U46619 binds to the putative human platelet TXA2/PGH2 receptor.     

Platelet activation by thrombin in the absence of the high-affinity thrombin receptor

The receptor status of the moderate-affinity platelet binding site for alpha-thrombin has been established by treating platelets with Serratia marcescens protease under conditions causing cleavage of 95-97% glycoprotein Ib (2.5 micrograms for 30 min). High-affinity binding was lost under these conditions, but the platelets continued to show moderate-affinity binding (Kd1 = 16 +/- 5 nM; 930 +/- 300 sites/platelet) and low-affinity binding (Kd2 = 4.6 +/- 3 microM; 170,000 +/- 90,000 sites/platelet), in good agreement with the values previously obtained for moderate- and low-affinity binding in intact platelets [Harmon, J.T., & Jamieson, G.A. (1986) J. Biol. Chem. 261, 15928-15933]. Platelets treated with Serratia protease under these conditions were about 4-fold less sensitive to activation by alpha-thrombin, as measured by serotonin secretion. Crossover studies with analogues showed that binding of alpha-thrombin was compatible by both D-phenyl-alanyl-L-prolyl-L-arginine chloromethyl ketone treated thrombin and N alpha-p-tosyl-L-lysine chloromethyl ketone treated thrombin, and both analogues were capable of inhibiting activation of Serratia-proteolyzed platelets by alpha-thrombin. These studies establish that the moderate-affinity platelet binding site for alpha-thrombin is a receptor, occupancy of which is required for platelet activation in the absence of the high-affinity receptor.     

Steady-state binding of adenine nucleotides ATP, ADP and AMP to rat liver and adipose plasma membranes

Binding of native adenine nucleotides to rat liver and adipose plasma membranes was studied under steady-state conditions using EDTA/Na for inhibition of ecto-nucleotidase activity. [3H]-labelled ATP, ADP and AMP are able to interact with specific binding sites with respective Kd values of 88 +/- 9, 278 +/- 29 and 495 +/- 40 nmol/l for liver membranes; and of 64 +/- 7, 231 +/- 36 and 2050 +/- 290 nmol/l for adipose membranes. The nucleotide-binding capacity (Bmax) varied from 15 to 18 pmol/mg protein in the case of [3H]ATP and [3H]ADP-binding studies and from 22 to 26 pmol/mg protein for [3H]AMP-binding sites. Both 2-MeSATP and ADP inhibited [3H]ATP-binding to membranes with respective IC50 values of 60 +/- 7 and 285 +/- 30 nM. Other purinergic agents suramin, Reactive blue 2, alpha,beta-MeATP and beta,gamma-MeATP were less potent competitors of [3H]ATP binding, whereas AMP, adenosine, GTP, UTP, and CTP did not cause any displacement effect at concentrations of 10(-6)-10(-5) M. It is suggested that the described ATP/ADP-binding sites are linked to G protein-coupled P2Y receptors, whereas AMP-binding sites may represent a substrate-binding component of the membrane ecto-5'-nucleotidase.     

Identification of GTP-binding proteins in the plasma membrane of higher plants

Antisera raised against a highly conserved amino acid sequence (G alpha-common peptide) of animal Gs alpha, Gi alpha, Go alpha and Gt alpha recognize, in plasma membranes of several higher plants, sets of proteins of Mr = 37 and 31 kDa (Vicia faba), 36 and 31 kDa (Arabidopsis thaliana) and 38 and 34 kDa (Commelina communis). The A. thaliana proteins were solubilized and partially purified. They bind [35S]GTP gamma S with high affinity (apparent Kd approximately 10 nM) and, with lower affinity, GTP but not the other nucleotides tested (ATP, CTP, ITP, UTP).     

Comparative interactions of factor IX and factor IXa with human platelets

Both factor IX and factor IXa were bound to gel filtered platelets in the presence of CaCl2 (2-20 mM) and human alpha-thrombin (0.06-0.2 units/ml) with maximal binding occurring in 10-20 min at 37 degrees C, and rapid reversibility was observed when unlabeled ligands were added in 100-fold molar excess. Competition studies with various coagulation proteins revealed that neither factor XI nor high molecular weight kininogen, at 300-fold molar excess, could compete with 125I-labeled factor IXa for binding sites on thrombin-activated platelets, whereas prothrombin and factor X, in 450-fold molar excess, could displace approximately 15 and 35%, respectively, of bound factor IXa in the absence of added factor VIII. Analysis of saturation binding data in the presence of CaCl2 and thrombin without factors VIII and X indicated the presence of 306 (+/- 57) binding sites per platelet for factor IX (Kd(app) = 2.68 +/- 0.25 nM) and 515 (+/- 39) sites per platelet for factor IXa (Kd = 2.57 +/- 0.14 nM). In the presence of thrombin-activated factor VIII (1-5 units/ml) and factor X (0.15-1.5 microM), the number of sites for factor IX was 316 (+/- 50) with Kd = 2.44 (+/- 0.30) nM and for factor IXa 551 (+/- 48) sites per platelet (Kd = 0.56 +/- 0.05 nM). Studies of competition for bound factor IXa by excess unlabeled factor IX or factor IXa, and direct 125I-labeled factor IXa binding studies in the presence of large molar excesses of factor IX, confirmed the conclusion from these studies that factor IX and factor IXa share approximately 300 low-affinity binding sites per thrombin-activated platelet in the presence of Ca2+ and in the absence of factor VIII and factor X, with an additional 200-250 sites for factor IXa with Kd(app) similar to that for factor IX. The presence of factor VIII and factor X increases by 5-fold the affinity of receptors on thrombin-activated platelets for factor IXa that participate in factor X activation.     

Binding of ATP to eukaryotic initiation factor 2. Differential modulation of mRNA-binding activity and GTP-dependent binding of methionyl-tRNAMetf

Eukaryotic initiation factor 2 (eIF-2) is shown to bind ATP with high affinity. Binding of ATP to eIF-2 induces loss of the ability to form a ternary complex with Met-tRNAf and GTP, while still allowing, and even stimulating, the binding of mRNA. Ternary complex formation between eIF-2, GTP, and Met-tRNAf is inhibited effectively by ATP, but not by CTP or UTP. Hydrolysis of ATP is not required for inhibition, for adenyl-5'-yl imidodiphosphate (AMP-PNP), a nonhydrolyzable analogue of ATP, is as active an inhibitor; adenosine 5'-O-(thiotriphosphate) (ATP gamma S) inhibits far more weakly. Ternary complex formation is inhibited effectively by ATP, dATP, or ADP, but not by AMP and adenosine. Hence, the gamma-phosphate of ATP and its 3'-OH group are not required for inhibition, but the beta-phosphate is indispensible. Specific complex formation between ATP and eIF-2 is shown 1) by effective retention of Met-tRNAf- and mRNA-binding activities on ATP-agarose and by the ability of free ATP, but not GTP, CTP, or UTP, to effect elution of eIF-2 from this substrate; 2) by eIF-2-dependent retention of [alpha-32P]ATP or dATP on nitrocellulose filters and its inhibition by excess ATP, but not by GTP, CTP, or UTP. Upon elution from ATP-agarose by high salt concentrations, eIF-2 recovers its ability to form a ternary complex with Met-tRNAf and GTP. ATP-induced inhibition of ternary complex formation is relieved by excess Met-tRNAf, but not by excess GTP or guanyl-5'-yl imidodiphosphate (GMP-PNP). Thus, ATP does not act by inhibiting binding of GTP to eIF-2. Instead, ATP causes Met-tRNAf in ternary complex to dissociate from eIF-2. Conversely, affinity of eIF-2 for ATP is high in the absence of GTP and Met-tRNAf (Kd less than or equal to 10(-12) M), but decreases greatly in conditions of ternary complex formation. These results support the concept that eIF-2 assumes distinct conformations for ternary complex formation and for binding of mRNA, and that these are affected differently by ATP. Interaction of ATP with an eIF-2 molecule in ternary complex with Met-tRNAf and GTP promotes displacement of Met-tRNAf from eIF-2, inducing a state favorable for binding of mRNA. ATP may thus regulate the dual binding activities of eIF-2 during initiation of translation.     

Interaction of thrombospondin with resting and stimulated human platelets

The interaction of isolated and radioiodinated thrombospondin with washed human platelets has been characterized. The ligand bound to nonstimulated and thrombin-stimulated platelets in a time-dependent manner, and apparent steady state was reached within 25 min. Binding was not due to iodination of the ligand and was inhibited by nonlabeled thrombospondin but not by unrelated proteins, and bound ligand was identical with thrombospondin in terms of subunit structure. Nonlinear curve-fitting analyses of binding to resting platelets suggested the presence of a single class of sites which bound 3,100 +/- 1,000 molecules/platelet with an apparent Kd of 50 +/- 20 nM. This interaction was not attributable to contaminating cells or inadvertant platelet activation. Binding to thrombin-stimulated platelets had a lower apparent affinity (Kd = 250 +/- 100 nM) and higher apparent capacity (35,600 +/- 9,600 molecules/platelet). Thrombin-enhanced binding was dependent upon agonist dose and platelet stimulation. Fibrinogen, a monoclonal antibody to GPIIb-IIIa, temperature, and divalent ions had differential effects upon thrombospondin binding to resting and stimulated platelets, suggesting the presence of two distinct mechanisms of thrombospondin binding to platelets. While thrombospondin binding to thrombin-stimulated platelets occurs with characteristics similar to those observed for fibrinogen, fibronectin, and von Willebrand Factor, its high affinity interaction with resting platelets is unique to this adhesive glycoprotein.