Human Platelet Cytosol Research Articles

Constitutive GDP/GTP Exchange and Secretion-dependent GTP Hydrolysis Activity for Rab27 in Platelets

We have previously demonstrated that Rab27 regulates dense granule secretion in platelets. Here, we analyzed the activation status of Rab27 using the thin layer chromatography method analyzing nucleotides bound to immunoprecipitated Rab27 and the pull-down method quantifying Rab27 bound to the GTP-Rab27-binding domain (synaptotagmin-like protein (Slp)-homology domain) of its specific effector, Slac2-b. We found that Rab27 was predominantly present in the GTP-bound form in unstimulated platelets due to constitutive GDP/GTP exchange activity. The GTP-bound Rab27 level drastically decreased due to enhanced GTP hydrolysis activity upon granule secretion. In permeabilized platelets, increase of Ca(2+) concentration induced dense granule secretion with concomitant decrease of GTP-Rab27, whereas in non-hydrolyzable GTP analogue GppNHp (beta-gamma-imidoguanosine 5'-triphosphate)-loaded permeabilized platelets, the GTP (GppNHp)-Rab27 level did not decrease upon the Ca(2+)-induced secretion. These data suggested that GTP hydrolysis of Rab27 was not necessary for inducing the secretion. Taken together, Rab27 is maintained in the active status in unstimulated platelets, which could function to keep dense granules in a preparative status for secretion.

Analysis of nitric oxide-sensitive guanylyl cyclase in human platelets before and after aggregation

Nitric oxide (NO) inhibits platelet adhesion to vascular endothelium and platelet aggregation through activation of soluble guanylyl cyclase (sGC) and a consequent increase in cGMP. The aim of the present study was to analyze NO-sensitive sGC in human platelets before and after aggregation. NO-sensitive sGC activity was tested in the cytosol and membrane fractions of native human platelets and ADP-induced platelet aggregates in the presence of 3 mM Mn2+ as cofactor. After ADP-induced platelet aggregation there was a significant increase of sGC activity in membranes. Western blot analysis showed a partial translocation of the enzyme to the plasma membrane. These findings support recent data that sGC is associated with cellular membranes in various tissues and cell types and that this membrane association is influenced by the activation state in human platelets (Nat Cell Biol 2002; 4: 307–11). Using 3 mM Mg2+ instead of Mn2+ as cofactor, a sharp decrease of sGC activity was apparent in the cytosol of aggregated platelets. Kinetic analysis of the cytosolic enzyme and concentration–response curves for free Mg2+ showed that platelet aggregation changes binding of free Mg2+ but not binding of the substrate complex Mg·GTP. This effect was specific for free Mg2+ and was not seen for free Mn2+. In addition, changes in free Mg2+ concentration in a physiological range markedly influenced NO-stimulated sGC activity. This provides a possible explanation for the increased platelet aggregability in patients with low intraplatelet Mg2+ levels.

Activation of soluble guanylate cyclase by NO donors— S-nitrosothiols, and dinitrosyl-iron complexes with thiol-containing ligands

We studied the capability of dimeric forms of dinitrosyl-iron complexes and S-nitrosothiols to activate soluble guanylate cyclase (sGC) from human platelet cytosol. The dinitrosyl-iron complexes had the ligands glutathione (DNIC-GS) or N-acetylcysteine (DNIC-NAC). The S-nitrosothiols were S-nitrosoglutathione (GS-NO) or S-nitrosoacetylcysteine (SNAC). For both glutathione and N-acetylcysteine, the DNIC and S-nitrosothiol forms are equally effective activators of sGC. The activation mechanism is strongly affected by the presence of intrinsic metal ions. Pretreatment with the potent iron chelator, disodium salt of bathophenanthroline disulfonic acid (BPDS), suppressed sGC activation by GS-NO: the concentration of GS-NO producing maximal sGC activation was increased by two orders of magnitude. In contrast, activation by DNIC-GS is strongly enhanced by BPDS. When BPDS was added 10 min after supplementation of DNIC-GS or GS-NO at 4 °C, it exerted a similar effect on sGC activation by either NO donor: BPDS only enhanced the sGC stimulation at low concentrations of the NO donors. Our experiments demonstrated that both Fe 2+ and Cu 2+ ions contribute to the decomposition of GS-NO in the presence of ascorbate. The decomposition of GS-NO induced by Fe 2+ ions was accompanied by formation of DNIC. BPDS protected GS-NO against the destructive action of Fe 2+ but not Cu 2+ ions. Additionally, BPDS is a sufficiently strong chelator to remove the iron from DNIC-GS complexes. Based on our data, we propose that S-nitrosothiols activate sGC via a two-step iron-mediated process: In the first step, intrinsic Fe 2+ ions catalyze the formation of DNICs from S-nitrosothiols. In the secondary step, these newly formed DNICs act as the real NO donors responsible for sGC activation.

Identification of Protein Kinase Cα as an Essential, but Not Sufficient, Cytosolic Factor for Ca2+-induced α- and Dense-core Granule Secretion in Platelets

Upon activation, platelets release many active substances. Here, we have analyzed the mechanism governing Ca(2+)-induced secretion of von Willebrand factor stored in alpha-granules and 5-hydroxytryptamine in dense-core granules in permeabilized human platelets. Both secretions were dependent on ATP and cytosol. An essential factor for both granule secretions was purified from rat brain cytosol and identified to be protein kinase Calpha (PKCalpha) by partial amino acid sequencing. Purified PKCalpha efficiently stimulated both secretions in the presence of cytosol, whereas PKCalpha alone did not support the secretion of either type of granules, suggesting that PKCalpha is not a sufficient factor. Finally, in human platelet cytosol fractionated by a gel filtration column, the stimulatory activity for dense-core granule secretion paralleled with the concentration of PKC, suggesting that PKC could also be such a stimulatory factor in platelet cytosol. Thus, we identified PKCalpha as an essential, but not sufficient, cytosolic factor for the Ca(2+)-induced secretions of both alpha- and dense-core granules in platelets.

The inositol polyphosphate 4-phosphatase forms a complex with phosphatidylinositol 3-kinase in human platelet cytosol.

Inositol polyphosphate 4-phosphatase (4-phosphatase) is an enzyme that catalyses the hydrolysis of the 4-position phosphate from phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P2]. In human platelets the formation of this phosphatidylinositol, by the actions of phosphatidylinositol 3-kinase (PI 3-kinase), correlates with irreversible platelet aggregation. We have shown previously that a phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase forms a complex with the p85 subunit of PI 3-kinase. In this study we investigated whether PI 3-kinase also forms a complex with the 4-phosphatase in human platelets. Immunoprecipitates of the p85 subunit of PI 3-kinase from human platelet cytosol contained 4-phosphatase enzyme activity and a 104-kDa polypeptide recognized by specific 4-phosphatase antibodies. Similarly, immunoprecipitates made using 4-phosphatase-specific antibodies contained PI 3-kinase enzyme activity and an 85-kDa polypeptide recognized by antibodies to the p85 adapter subunit of PI 3-kinase. After thrombin activation, the 4-phosphatase translocated to the actin cytoskeleton along with PI 3-kinase in an integrin- and aggregation-dependent manner. The majority of the PI 3-kinase/4-phosphatase complex (75%) remained in the cytosolic fraction. We propose that the complex formed between the two enzymes serves to localize the 4-phosphatase to sites of PtdIns(3,4)P2 production.

Stimulation by G Protein βγ Subunits of Phospholipase C β Isoforms in Human Platelets

SummaryDifferent phospholipase C (PLC) isoforms were located in human platelet cytosol and membranes. PLCγ2 and PLCβ3b were mainly located in the cytosol and PLCβ2 and PLCβ3a were in both cytosol and membranes by using specific antibodies against PLC isozymes (Banno Y, Nakashima S, Ohzawa M, Nozawa Y. J Biol Chem 1996; 271: 14989-94). Three PLC fractions activated by G protein βγ subunits were purified from human platelet cytosol and membrane fractions. Two PLC fractions from membranes were identified as PLCβ2 and PLCβ3a, and one from cytosol was PLCβ3b. These PLCβ isoforms were activated by the purified βγ subunits of brain G proteins in the order PLCβ3b > PLCβ3a > PLCβ2. Western blot analysis of γ subunits of the purified platelet G proteins with antibodies against various standard γ subunits revealed that the major component of the γ subunit of Gi2 and Gq was γ5, and that γ7 was a minor component. Studies using various subtypes of βγ subunits, βγ2, βγ3, and βγ7 purified from bovine brain, βγ5 from bovine lung, or βγ12 from bovine spleen, failed to show differences in their ability to stimulate the isolated platelet PLCβ isoforms. These results suggest that the βγ subunits of Gi2 and Gq have similar efficacy in regulation of effectors in human platelets.

Antibodies specific for proteolyzed forms of protein kinase C α

The activation of protein kinase C (PKC) is irreversibly regulated by limited proteolysis catalyzed by a calcium-activated neutral cysteine protease, calpain. Calpain cleaves PKC α at specific sites in the hinge region between the catalytic and the regulatory domains of this kinase. Here we show a novel method for production of antibodies that bind specifically to the catalytic fragment of PKC α but not to the unproteolyzed protein. To detect proteolyzed PKC α ‘cleavage site-directed antibodies,’ which recognize amino-terminal regions in the nascent catalytic fragments and do not cross-react with the unproteolyzed enzymes, were raised using synthetic peptides corresponding to the amino-terminal sequences. The synthetic peptides used in this study were the sequences of human PKC α at the cleavage sites by m- and μ-types of calpains (LGPAGNKV and VISPSEDRKQPSNNLDRVKLT, respectively) and they are designated as CFα2, CFα4, in this order. Each synthetic peptide was injected into rabbit after conjugation with a carrier protein. The antibodies thus obtained (anti-CFα2 or -CFα4) specifically reacted with either the 46- or 45-kDa catalytic fragment of PKCα, respectively, whereas they did not cross-react with other fragments. Furthermore, the antibodies did not bind to the unproteolyzed enzyme nor fragments of PKCα obtained by treatment with other proteinases unless the fragment carried the same amino-terminal sequence. When human platelets were treated with calcium ionophore, the catalytic fragments of PKCα (45- and 46-kDa) were detected in the cytosol by immunoblotting with the antibodies. However, these antibodies did not bind unproteolyzed 80-kDa PKCα, although this form was dominant in the cytosol of the calcium ionophore-treated human platelets. In addition, the 45-kDa catalytic fragment of PKCα was detected in an apoptotic human fibroblast TIG-3 cells cultured in serum-free medium. Our method is applicable for analysis of proteolysis in various cellular states.

Photoaffinity labelling of cyclic GMP-inhibited phosphodiesterase (PDE III) in human and rat platelets and rat tissues: effects of phosphodiesterase inhibitors

Ultraviolet irradation of human platelet cytosol in the presence of 32P-labelled cyclic GMP (cGMP) can specifically label 110, 80, 55, 49 and 38 kDa proteins; the 110 kDa species is the subunit of cGMP-inhibited phosphodiesterase (PDE III) and the 80 kDa species that of cGMP-dependent protein kinase (Tang et al., 1993, Biochem. J. 294, 329). We have now shown that although photolabelling of platelet PDE III was inhibited by unlabelled cGMP, 8-bromo-cGMP and cyclic AMP (cAMP), it was not affected by phosphorothioate analogues of these cyclic nucleotides. Specific concentration-dependent inhibitions of the photolabelling of PDE III were observed with the following PDE inhibitors: trequinsin (IC 50 = 13 ± 2 nM), lixazone (IC 50 = 22 ± 4 nM), milrinone (IC 50 = 56 ± 12 nM), cilostamide (IC 50 = 70 ± 9 nM), siguazodan (IC 50 = 117 ± 29 nM) and 3-isobutyl 1-methylxanthine (IBMX) (IC 50 = 3950 ± 22 nM). Thus, measurements of the inhibitory effects of compounds on the photolabelling of platelet PDE III provide a simple quantitative means of investigating their actions at a molecular level that avoids the need to purify the enzyme. Photolabelling of rat platelet lysate or rat heart homogenate by [ 32P]cGMP showed that the 110 kDa PDE III present in human material was replaced by a 115 kDa protein, labelling of which was also blocked by PDE III inhibitors. Heart and other rat tissues contained much less of this putative 115 kDa PDE III than rat platelets. In contrast, the 80 kDa protein was labelled much less in platelets than in many other rat tissue homogenates (e.g., heart, aorta, uterus and lung). Thus, comparison of the relative amounts of specific photolabelled proteins in different cells may provide an indication of different patterns of cyclic nucleotide action. We compared the abilities of phosphodiesterase inhibitors to block the photolabelling of PDE III in human platelet cytosol and to increase the iloprost-stimulated accumulation of cAMP in intact platelets. Whereas trequinsin (EC 50 = 19 ± 3 nM), lixazinone (EC 50 = 122 ± 8 nM), milrinone (EC 50 = 5320 ± 970 nM) and siguazodan (EC 50 = 18880 ± 3110 nM) all increased platelet cAMP to the same maximum extent, cilostamide and IBMX increased cAMP further, indicating that they inhibited a PDE isozyme in addition to PDE III. Differences in membrane permeability of these compounds may account for discrepancies between their abilities to inhibit photolabelling of PDE III and to increase platelet cAMP.

A G-protein beta gamma-subunit-responsive phosphoinositide 3-kinase activity in human platelet cytosol

Thrombin activates phosphoinositide 3-kinase (PI 3-kinase) in platelets via a mechanism involving G-proteins, possibly of both the heterotrimeric and the low molecular weight families. We have investigated the regulation of PI 3-kinase present in platelet cytosol, and we show that this activity can be stimulated by a mixed preparation of G-protein beta gamma-subunits. This stimulation is reversed by preincubation of the beta gamma-subunits with GDP-liganded alpha-subunits. The beta gamma-stimulated activity is inhibited by wortmannin, a recently identified inhibitor of PI 3-kinase in other systems. In addition, the activity associates with PDGF receptor phosphotyrosyl peptide and monoclonal antibody designed to interact with the p85 subunit of PI 3-kinase. We suggest that this beta gamma-sensitive activity is related to previously identified forms of PI 3-kinase.

Glyceraldehyde-3-phosphate dehydrogenase is required for the transport of nitric oxide in platelets.

Nitric oxide (NO) or NO-generating compounds like sodium nitroprusside (SNP) increase cellular levels of cGMP and produce S-nitrosylation of glyceraldehyde-3-phosphate dehydrogenase [GAPDH; D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12]. In search of a reagent that could discriminate between these two effects, we used the sesquiterpene antibiotic koningic acid, which binds to GAPDH at the Cys-149 of the active site. Koningic acid inhibited basal and sodium nitroprusside-stimulated NAD-dependent covalent modification of purified rabbit muscle GAPDH in a dose-dependent manner. Furthermore, we tested the effect of koningic acid on human platelets. Approximately 90% of GAPDH is present in the cytosol of human platelets, and the exposure of platelet cytosol to koningic acid inhibited GAPDH activity, while the soluble guanylyl cyclase (basal and sodium nitroprusside-stimulated) activity remained unaltered. Pretreatment of intact platelets with koningic acid slowed the rate of aggregation induced by a submaximal concentration of thrombin. In addition, the antibiotic also inhibited the cGMP increases triggered by SNP, S-nitroso-N-acetylpenicillamine (SNAP), and 3-morpholinosyndomidine (SIN-1) but failed to prevent an increase in cGMP caused by nitrosylated albumin. Under the same conditions, koningic acid also inhibited basal and SNP- SNAP-, and SIN-1-stimulated NAD-dependent modification of GAPDH and its enzymatic activity. These results suggest that the mechanism of delivery of NO from SNP, SNAP, and SIN-1 to platelets may require the active form of GAPDH. When NO is delivered by nitrosylated albumin, active GAPDH was not necessary.

Purification and characterization of a low Mr GTP-binding protein, c25KG, from human platelet membranes

A low M r GTP-binding protein with a M r of 26 000 has been purified from a sodium cholate extract of human platelet membranes by using an antibody raised against a synthetic peptide of c25KG, which was previously purified from human platelet cytosol (Nagata, N., et. al. (1989) J. Biol. Chem. 264, 17000–17005). The M r of membranous c25KG (m-c25KG) was slightly higher than that from cytosolic c25KG ( M r 25 000) and calculated to be 26 000. It was suggested that m-c25KG contains an equimolar amount of GDP. The purified protein could bind approx. 1 mol of [ 35S]guanosine 5′- O-(thiotriphosphate)(GTPγS)/mol of protein, with a K d value of 50 nM. [ 35S]GTPγ S-binding to this protein was inhiibited by GTP and GDP, but not by ATP and ADP, showing that the binding is specific for guanine. In the presence of 10 mM Mg 2+, the dissociation of [8,5′- 3H]GDP from the m-c25KG occurred with a rate of 0.01 min −1. The rate of release of P i from [γ- 32P]GTP-bound m-c25KG was calculated to be 0.03 min −1. These results indicate that c25KG is also present in membrane fraction of human platelet which has very similar biochemical properties in those of the cytosolic type.

Nitric oxide causes ADP-ribosylation and inhibition of glyceraldehyde-3-phosphate dehydrogenase.

Nitric oxide and nitric oxide-generating agents like 3-morpholinosydnonimine (SIN-1) stimulate the mono-ADP-ribosylation of a cytosolic, 39-kDa protein in various tissues. This protein was purified from human platelet cytosol by conventional and fast protein liquid chromatography techniques. N-terminal sequence analysis identified the isolated protein as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Nitric oxide stimulates the auto-ADP-ribosylation of GAPDH in a time and concentration-dependent manner with maximal effects after about 60 min. Associated with ADP-ribosylation is a loss of enzymatic activity. NAD(+)-free enzyme is not inhibited by SIN-1, indicating the absolute requirement of NAD+ as the substrate of the ADP-ribosylation reaction. Inhibition of the glycolytic enzyme GAPDH may be relevant as a cytotoxic effect of NO complementary to its inhibitory actions on iron-sulfur enzymes like aconitase and electron transport proteins of the respiratory chain.

Effects of gelsolin on human platelet cytosolic phosphoinositide-phospholipase C isozymes.

The effective resolution of human platelet cytosolic phosphoinositide-phospholipase C (PLC) revealed five distinct activity peaks by Q-Sepharose and heparin-Sepharose column chromatographies when assayed using phosphatidylinositol (PI) and phosphatidylinositol 4,5-bisphosphate (PIP2). The results of Western blotting analysis with various antibodies against PLC isozymes showed that peak-Ia (PLC-delta type), peak-Ib (PLC-gamma 1 type), and peak-IIc (PLC-beta type) and two unidentified activity peaks (PLC-IIa and PLC-IIb) were present in human platelet cytosol. A protein with guanosine 5'-3-O-(thio)triphosphate-binding activity was coeluted with the PLC-IIa and was purified to homogeneity. It exhibited 86- and 42-kDa polypeptide bands upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis which were identified as gelsolin and actin by immunostaining, respectively. Large amounts of gelsolin/actin (1:1) complex "gelsolin complex" were detected in the PLC-delta and PLC-gamma 1 fractions. The PLC-gamma 1 and the gelsolin complex were co-immunoprecipitated by the antibody raised against PLC-gamma 1. Furthermore, the partially purified bovine brain PLC-gamma 1 fraction also was found to be associated with the gelsolin complex and the association was released by the addition of 1% sodium cholate. This finding has prompted us to examine effects of the gelsolin complex and the free gelsolin on activities of the above PLC isoforms from platelet cytosol. The gelsolin complex did not affect the PIP2 hydrolyzing activities of all PLC isoforms. In contrast, the purified gelsolin inhibited distinctly PIP2 hydrolyses by PLC-Ia (delta), PLC-Ib (gamma 1), and PLC-IIa (unidentified), whereas the inhibitory effects for PLC-IIb (unidentified) and PLC-IIc (beta) were moderate. The inhibitory effect of gelsolin on PIP2-hydrolysis by PLC-gamma 1 was diminished by a large amount of PIP2 substrate. These results suggested that the inhibition of PLC by gelsolin is due to sequestration of substrate PIP2 by its competitive binding.

Isolation of a Polyphosphoinositide-phospholipase C (Type β) from Cytosolic and Membrane Fractions of Human Platelets

Human platelet cytosol contains several phosphoinositide-specific phospholipases C (PLCs) separable by stepwize ion-exchange chromatographies [Banno, Yu, Nakashima, Homma, Takenawa and Nozawa Biochem Biophys Res Commun 1990 167; 396-401]. In the present study, one type of PLC (cPIP(2)-PLC) for which phosphatidylinositol 4,5-bisphosphate (PIP(2)) was the preferred substrate, was isolated from human platelet cytosol as a truncated form (100 kDa). A mPIP(2)-PLC was purified to near homogeneity from the cholate extract of human platelet membranes. The purified 150-kDa mPIP(2)-PLC was truncated during purification, and ran as 100-kDa and 45-kDa polypeptides on SDS-polyacrylamide gel electrophoresis. The 100-kDa component of cytosolic PLC (cPIP(2)-PLC) and the 150-kDa, 100-kDa and 45-kDa polypeptides of mPIP(2)-PLC were all recognized by the antibody raised against PLC-β. The 150-kDa enzyme was immunoprecipitated by anti-PLC-β antibody from freshly prepared human platelet cytosol. The catalytic properties of the platelet 100-kDa form were observed to be very similar to those of 100-kDa form derived from bovine brain PLC-β. These results indicate that β-type PLC exists in both cytosol and membranes of human platelets.

Catabolism of platelet‐activating factor and its acyl analog

Recent investigations have shown the presence of 1-acyl-2-acetyl-sn-glycero-3-phosphocholine, i.e. the acyl analog of platelet-activating factor (PAF), in unstimulated tissues as well as its formation along with platelet-activating factor upon stimulation of a variety of cells. We demonstrate here that this acyl analog of PAF can be catabolized by purified lysophospholipases I and II from bovine liver with near stoichiometric formation of 2-acetyl-sn-glycero-3-phosphocholine. Lysophospholipase II also deacetylated PAF to lysoPAF and evidence is presented to show that this is an intrinsic activity of this enzyme. This suggested that some lysophospholipases may contribute to intracellular inactivation of PAF by deacetylation. Anion-exchange chromatography of rat liver cytosol confirmed this possibility. However, similar experiments with rat kidney cytosol and rat and human platelet cytosol clearly separated lysophospholipase activities without PAF acetylhydrolase activity from specific PAF acetylhydrolases not having lysophospholipase activity. Thus, lysophospholipases are clearly involved in the metabolism of the acyl analog of PAF and in some tissues, such as liver, may even contribute to abolishing the biological activity of PAF through deacetylation.

Catalytic properties of human platelet 12-lipoxygenase as compared with the enzymes of other origins

Arachiidonate 12-lipoxygenase of porcine and bovine leukocytes were different in substrate specificity and immunogenicity from the enzyme of bovine platelets (Arch. Biochem. Biophys. (1988) 266, 613). In order to extend the comparative studies on the two types of 12-lipoxygenase, we purified the enzyme from the cytosol of human platelets by immunoaffinity chromatography to a specific activity of about 0.3 μmol/min per mg protein at 37°C. The purified enzyme was active with eicosapolyenoic acids and docosahexaenoic acid. Linoleic and linolenic acids were poor substrates in contrast to the high reactivity of the leukocyte enzymes with these octadecapolyenoic acids. The finding that the human platelets enzyme catalyzed 15-oxygenation of 5S-hydroxy-6,8,11,14-eicosatetraenoic acid, raised a question if lipoxins were produced by incubation of the enzyme with leukotriene A 4. Howeverm the leukogtriene A 4 was scarcely transformed to lipoxin isomers by 12-lipoxygenases of human and bvvine platelets. In sharp contrast, the porcine and bovine leukocyte enzymes converted leukotriene A 4 to various lipoxin isomers by the reaction rates of 3% and 2% of the arachidonate 12-oxygenation. Thus, 12-lipoxygenase of human and bovine platelets were catalytically distinct from the porcine and bovine leukocyte enzymes in terms of their reactivities not only with linoleic and linolenic acids, but also with leukotriene A 4 as lipoxin precursor.

ヒト血小板由来中球接着能阻害因子

Localization and characterization of human platelet-derived adhernce-inhibiting factors (AIFs) were studied. Subcellular fractionation experiments showed that AIFs were present in both cytosol and granule fractions of human platelets. When cytosol and granule fractions of human platelets were applied to a Superose 6 column, the adherence inhibiting activity of the cytosol fraction was eluted at two different positions (2, 600 and 480 kDa), whereas that of the granule fraction was eluted as a single peak (2, 600 kDa) . Thrombinstimulated platelets released granular AIF extracellularly without releasing a cytosolic marker.When the purified AIF was analyzed on SDSPAGE, the 340 kDa protein band and the other large protein bands were observed. Under reducing conditions, AIF was separated into two components (340 and 190 kDa) . The purified AIF inhibited human neutrophil adherence to glass, plastic and type IV collagen-coated plastic, whereas it did not affect neutrophil adherence to fibronectin- or plasma-coated plastic. AIF inhibited neutrophil spreading on glass. Neither monocyte adherence nor monocyte spreading was affected by AIF.These results indicate that AIF selectively affects neutrophil adherence and spreading among phagocytic cells.

Platelet-derived neutrophil adherence-inhibiting factor in humans

The effect of constituents of human platelets on leukocyte adherence was examined. Adherence-inhibiting factors (AIFs), which strongly inhibited neutrophil adherence to glass, were present in both cytosol and granule fractions of human platelets. On the Superose 6 gel chromatography (Pharmacia LKB Biotechnology, Uppsala, Sweden), the granular AIF was eluted as a single active peak (2,600 Kd), whereas cytosolic AIFs were eluted at two different positions (2,600 and 480 Kd). When platelets were stimulated by thrombin, granular AIF was released extracellularly without releasing a cytosolic marker. Using DE32 anion exchange chromatography and Superose 6 gel filtration, granular AIF was completely purified. Sodium dodecyl sulfate- polyacrylamide gel electrophoresis analysis suggests that granular AIF consists of two subunits with molecular masses of approximately 340 and 190 Kd. Purified granular AIF inhibited human neutrophil adherence to glass, plastic, and type IV collagen-coated plastic, whereas it did not affect monocyte adherence. These results suggest that granular AIF inhibits neutrophil adherence not only via nonspecific adsorption sites, but also via type IV collagen receptors.

Platelet-Derived Neutrophil Adherence-Inhibiting Factor in Humans

The effect of constituents of human platelets on leukocyte adherence was examined. Adherence-inhibiting factors (AIFs), which strongly inhibited neutrophil adherence to glass, were present in both cytosol and granule fractions of human platelets. On the Superose 6 gel chromatography (Pharmacia LKB Biotechnology, Uppsala, Sweden), the granular AIF was eluted as a single active peak (2,600 Kd), whereas cytosolic AIFs were eluted at two different positions (2,600 and 480 Kd). When platelets were stimulated by thrombin, granular AIF was released extracellularly without releasing a cytosolic marker. Using DE32 anion exchange chromatography and Superose 6 gel filtration, granular AIF was completely purified. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis suggests that granular AIF consists of two subunits with molecular masses of approximately 340 and 190 Kd. Purified granular AIF inhibited human neutrophil adherence to glass, plastic, and type IV collagen-coated plastic, whereas it did not affect monocyte adherence. These results suggest that granular AIF inhibits neutrophil adherence not only via nonspecific adsorption sites, but also via type IV collagen receptors.

The ram: A novel low molecular weight GTP-binding protein cDNA from a rat megakaryocyte library

A novel low M r GTP-binding protein cDNA was isolated from a rat megakaryocyte cDNA library with a synthetic oligonucleotide probe corresponding to an 8-amino acid sequence specific for c25KG, a GTP-binding protein previously isolated from human platelet cytosol fraction [(1989) J. Biol. Chem. 264, 17000–17005]. The cDNA has an open reading frame encoding a protein of 221 amino acids with a calculated M r of 25068. The protein is designated as ram ( ras-related gene from megakaryocyte) protein ( ram p25). The amino acid sequence deduced from the ram cDNA contains the consensus sequences for GTP-binding and GTPase domains. ram p25 shares about 23%, 39% and 80% amino acid homology with the H- ras, smg25A and c25KG proteins, respectively. The 3.5-kb ram mRNA was detected abundantly in spleen cells.