Inhibition of binding of anti-PLA1 antibodies to platelets with monoclonal antibody LK-4. Evidence for multiple PLA1 receptor sites on platelet GPIIIa

The PLA1 epitope on platelet GPIIIa has a sulfhydryl-dependent conformation and is dependent on a leucine 33/proline33 polymorphism. Monoclonal antibody LK-4 differentiates PLA1/PLA1 from PLA2/PLA2 platelet lysates on solid phase enzyme-linked immunosorbent assay (ELISA), as well as immunoblot. To determine whether LK-4 reacts at or near the binding site(s) for human anti-PLA1, nine such antibodies (Abs) (six neonatal; three posttransfusion) were examined in the presence and absence of LK-4 for binding to platelets, as well as rGPIIIa 1-66, a recombinant glutathione S-transferase fusion peptide. All nine human Abs bound to rGPIIIa 1-66, as well as platelets, in a saturation-dependent manner, employing both solid phase ELISA, as well as flow cytometry. Binding of all nine Abs to rGPIIIa 1-66 or platelets was inhibited by LK-4. IC50's for inhibition of binding of anti-PLA1 to rGPIIIa 1-66 varied from 8 to 160 micrograms/mL (5 x 10(-8)- 1 x 10(-6) mol/L). However, IC50's for LK-4 inhibition of binding to platelets was strikingly different. Six of the nine Abs had IC50's of 1 to 10 micrograms/mL (8-fold to 16-fold greater inhibition than with rGPIIIa 1-66), whereas three neonatal Abs had IC50's of 380 to 1,013 micrograms/mL (6-fold to 48-fold less inhibition than with rGPIIIa 1-66). Similar results were noted with intact GPIIIa, rGPIIIa 1-66 blocked the binding of anti-PLA1 Abs to platelets and served to segregate the nine patients into two groups: a sensitive group of anti-PLA1 Abs from six patients in which binding to platelets was progressively inhibited by increasing concentrations of rGPIIIa 1-66 with inhibition at 1 micrograms/mL of 18% and inhibition at 256 micrograms/mL of 78%; a second resistant group of three anti-PLA1 Abs from three patients in which inhibition was first noted at 16 micrograms/mL of 4% with 35% inhibition at 256 micrograms/mL. Thus, LK-4 binds to GPIIIa at the 1-66 N-terminal region, inhibits binding of anti-PLA1 Ab to platelets, and segregates, anti-PLA1 Abs into two groups. These data are compatible with two or more receptor sites for anti-PLA1 Ab: one that is present on rGPIIIa 1-66 and sensitive to LK-4 inhibition, another that is present on rGPIIIa 1-66, as well as other site(s) on platelet GPIIIa and insensitive to inhibition.     

Differential agonist inhibition identifies multiple epibatidine binding sites in mouse brain

We have charterized a Mycobacterium smegmatis gene encoding a homolog of the ATP-dependent protease Lon (La). Our identification of a Lon homolog, in conjunction with our previous work, identifies M. smegmatis as the first known example of a eubacterium containing both Lon and a complete 20S proteasome (containing both alpha- and beta-subunits). Despite the significant primary sequence divergence between M. smegmatis Lon (Ms-Lon) and E. coli Lon (Ec-Lon), expression of Ms-Lon was only moderately toxic to E. coli cells. The ability of E. coli cells to tolerate expression of Ms-Lon reveals that Ms-Lon does not recognize and degrade essential E. coli proteins. We conclude that discrimination against nonsubstrate proteins is broadly conserved between Ec-Lon and Ms-Lon. Additional conservation of substrate recognition was demonstrated by the ability of Ms-Lon to degrade efficiently RcsA, a natural substrate of Ec-Lon. Purified Ms-Lon displays chymotrypsin-like specificity in peptidase assays that are stimulated by unfolded protein and supported by nonhydrolyzed nucleotide analogs. Maximal peptidase activity requires ATP or dATP. Replacement of Ms-Lon's catalytic Ser with Ala (S675A), Thr (S675T), or Cys (S675C) reduced to background levels Ms-Lon's in vitro peptidase activity. However, by employing a sensitive in vivo assay, based on the degradation of RcsA, we demonstrated that the S675C variant retained specific protease activity. Finally, variants of Ms-Lon, with substututions at or near S675, reduce the enzyme's basal ATPase activity, suggesting a structural interaction between the peptidase and ATPase active sites of Ms-Lon.     

Mechanism of platelet aggregation induced by a monoclonal antibody requiring Fc portion

A monoclonal antibody designated Apt4, which is IgG1, was produced by fusion of mouse myeloma cells to spleen cells from a BALB/c mouse immunized with normal human platelets. Apt4 whole IgG caused the aggregation of both platelet rich plasma (PRP) and washed platelets from normal subjects and a patient with Bernard Soulier syndrome but not those from two patients with the Type 1 Glanzmann's thrombasthenia. No aggregation was observed when Apt4 F(ab')2 fragments were used. Immunofluorescence study showed that both whole IgG and F(ab')2 fragments of Apt4 bound to fresh or formalin fixed platelets from normal subjects and a patient with Bernard Soulier syndrome but not to those from two patients with Glanzmann's thrombasthenia. Aggregation induced by Apt4 IgG was inhibited by EDTA (10 mM), PGE1 (1 mM), 2-deoxy-D-glucose/antimycin (1.4 uM), and apyrase (20 units/ml). Preincubation of normal PRP with monoclonal anti-GPIIb/IIIa or anti-GPIb antibodies completely or partially inhibited the Apt4-induced aggregation, whereas anti-GPIIIa antibodies have no effects on this activation. Monoclonal ant-Fc gamma RII antibody (IV.3) inhibited Apt4 induced aggregation. Immunoprecipitation of 125I-labeled platelet membrane lysate by Apt4 IgG showed two protein bands with a molecular weight of 145,000 and 95,000 daltons respectively under non-reducing condition, which are corresponding to GPIIb and GPIIIa. In conclusion, Apt4 antibody binds to GPIIb/IIIa complex and induces aggregation, requiring energy metabolism, calcium, ADP release and Fc portion of IgG to interact with Fc receptor, but independent of thromboxane A2 formation.     

Anti-idiotypic monoclonal antibodies to GM1 identify ganglioside binding proteins

Ganglioside stimulated neurite outgrowth may be due to ganglioside binding to membrane proteins or to intercalation into the membrane. To test that ganglioside binding proteins could be found on neuronal surfaces, anti-idiotypic ganglioside monoclonal antibodies (AIG mAbs) were generated to mimic the biological properties of the GM1 ganglioside. The AIG mAbs were identified by their ability to bind to a known GM1 binding protein, the beta-subunit of cholera toxin. For the two AIG mAbs studied, AIG5 and AIG20, binding to beta-CT was blocked most strongly by GM1. This data also suggests that AIG5 and AIG20 mimic different but overlapping epitopes of the ganglioside GM1. Western blotting and immunoprecipitation of mammalian tissues reveals four potential ganglioside binding proteins of molecular weight 93, 66, 57, and 45 kDa. Immunocytochemistry demonstrates neuronal surface label with the AIG mAbs, which suggests that gangliosides, enriched on the neuronal surface membrane, are co-localized with putative ganglioside binding proteins. In bioassays, the AIG mAbs promote neuronal sprouting. This shows that these antibodies can be used to study the biological effects of ganglioside binding to neuronal surface proteins, and the role of gangliosides in the activation of neurite outgrowth.     

Biphasic initial phase of platelet aggregation inhibition after oral aspirin

For one hour after the ingestion of 1 g aspirin the pharmacodynamics of acetylsalicylic acid with regard to the inhibition of platelet aggregation were studied in nine healthy male volunteers. Plasma salicylic acid (SA) and acetylsalicylic acid (ASA) levels were measured, and platelet aggregation was controlled by the collagen-induced aggregation. It took 12 - 24 minutes till the maximum of platelet aggregation inhibition was reached; maximal inhibition was only observed with ASA levels above 4.5 /microgram/ml and total ASA levels above 10 /microgram/ml. At that time already more than 50% of the total ASA were hydrolysed to minimally active SA. In spite of further increasing ASA levels inhibition of platelet aggregation decreased again. The different sensitivity of platelet- and vessel wall cyclooxygenase to aspirin does not explain our findings.     

Homotypic aggregation of ovine leucocyte induced by anti-leucosialin (CD43) monoclonal antibodies

Four monoclonal antibodies directed against ovine/bovine leucocyte antigens (Co.44B8, Co.11F10, Co.33B3 and Co.26F4) were produced and all recognized an antigen with an apparent molecular weight of 105 kDa under reducing conditions. This molecule was considered to be the ovine/bovine analogue of human, rat and mouse CD43 or leucosialin. This conclusion was based on (a) the cellular distribution of the antigen which was similar to that described in other species, (b) a decrease of apparent M(r) (150 kDa) after treatment with neuraminidase, and (c) production of cell aggregation by the four monoclonal antibodies and the influence of temperature and metabolic inhibitors on this phenomenon. Co.44B8, Co.11F10, Co.33B3 and Co.26F4 all recognized and immunoprecipitated the molecule after neuraminidase treatment of the cells. The data indicate an important function of CD43 as a mediator of cell aggregation for ruminant cells.     

Synergistic inhibition of thrombin-induced platelet aggregation by the novel nitric oxide-donor GEA 3175 and adenosine

1. The influence of the novel nitric oxide-donor GEA 3175 on thrombin- and ionomycin-stimulated human platelets was investigated. The effect of GEA 3175 was compared with that of adenosine, an activator of platelet adenylyl cyclase. 2. GEA 3175 inhibited thrombin-induced secretion of ATP but did not affect aggregation; similar results were obtained with adenosine. 3. Thrombin-stimulated rises in the cytosolic free Ca2+ concentration, [Ca2+]i, were dose-dependently inhibited by GEA 3175 and adenosine. GEA 3175 and adenosine maximally reduced the initial rise in [Ca2+]i by 41% and 35%, respectively. 4. Simultaneous exposure to GEA 3175 and adenosine nearly abolished both the functional responses (i.e. aggregation and degranulation) and the rises in [Ca2+]i in thrombin-stimulated platelets. 5. Aggregation and increases in [Ca2+]i triggered in platelets by the Ca(2+)-ionophore ionomycin were only marginally affected by a combination of GEA 3175 and adenosine. 6. GEA 3175 potently increased the guanosine 3':5'-cyclic monophosphate (cyclic GMP) content in platelets but did not affect adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels. Adenosine did not increase either the cyclic AMP or the cyclic GMP levels in platelets. However, adenosine and GEA 3175 combined significantly elevated the platelet cyclic AMP content. 7. The results show that simultaneous exposure to GEA 3175 and adenosine promotes potent anti-aggregatory properties in platelets in vitro. The findings suggest that blockage of the cytosolic Ca(2+)-signal, which is probably mediated by an amplified cyclic nucleotide response, is an important event during the synergistic inhibition of thrombin-induced aggregation.     

Characterization and regional binding of human sperm monoclonal antibodies

Therapeutic effect of superoxide dismutase (SOD) and three derivatives: a conjugate with polyethylene glycol (SOD-PEG2), a cationized derivative (cSOD), and a mannosylated derivative (Man-SOD), on acute renal failure induced by ischemia/reperfusion was studied in rats. SOD and derivatives were administered intravenously to the rat after nephrectomy of the right kidney and before and after 60 min occlusion of the left renal artery. At 48 hr after reperfusion, the renal function was evaluated by determining the urinary excretion rate of 14C-inulin injected intravenously. No therapeutic effect on the impaired renal function was shown in the case of low dose SOD (2600 unit/kg) treatment. In contrast, administration of cSOD which was shown to be taken up by the isolated perfused kidney from its capillary side and SOD-PEG2 which maintained high plasma concentration exhibited significant therapeutic effect, as did SOD at ten-fold higher dose (26,000 unit/kg). On the other hand, renal damage was promoted by Man-SOD. Thus, the present study demonstrated that chemical modification may improve the therapeutic effect of SOD on the ischemic acute renal failure and increased SOD concentration in the renal vascular space is an important factor for the improved effect.     

Structure and binding properties of monoclonal antibodies to core histones from autoimmune mice

Histones are frequent targets of self-reactive antibodies during autoimmune syndromes. We report the specificities and V region genes of three IgG anti-histone MAbs obtained from autoimmune mice. Each of the MAbs, named LG2-1, LG2-2 and BWA3, is directed against a different determinant located in the basic amino-terminal domain of core histones. LG2-1 reacts with a peptide from histone H3 (residues 30-45), LG2-2 recognizes the amino-terminus of H2B (residues 1-13) and BWA3 binds an epitope corresponding to a region of high sequence similarity between H2A and H4 (residues 1-20 and 1-29, respectively). The analysis of their V region sequences indicates that the H chain CDRs of these MAbs are remarkable for the presence of negatively charged amino acid residues that may play a role in the binding to cationic histones. The H chain importance in conferring reactivity to histones is corroborated by the observation that each of the VH gene segments of these MAbs is very similar to VH genes of previously described murine anti-histone antibodies.     

A novel surface-active peptide derivative exhibits in vitro inhibition of platelet aggregation

A tetrapeptide corresponding to a region of the N-terminal portion of lactotransferrin with hydrophobic alkyl groups at the terminal ends was synthesized and its physicochemical properties as well as its effect on thrombin-stimulated platelet aggregation were examined. The tetrapeptide derivative, in the aggregated state, produced inhibitory effect on platelet aggregation. The concentration dependent activity of the peptide was analyzed in the light of micelle formation, with the micellar aggregate comprising four tetrapeptide units. The unique action of this peptide derivative on the inhibition of platelet aggregation might be useful in the development of potent antithrombotic drugs.     

Mediation of endothelin-1-induced inhibition of platelet aggregation via the ETB receptor

1. The effects of FR139317 (ETA antagonist) or PD145065 (non-selective ETA/ETB antagonist) on endothelin-1 (ET-1)-induced changes in blood pressure and inhibition of ex vivo platelet aggregation were investigated in the anaesthetized rabbit. 2. ET-1 (1 nmol kg-1, i.a. bolus) caused a sustained increase in mean arterial pressure (MAP) (peak increase 47 +/- 5 mmHg, n = 8). Intravenous infusion of FR139317 at 0.2 (n = 4) or 0.6 mg kg-1 min-1 (n = 4) inhibited the ET-1 pressor response by 83 or 89%, respectively. Infusion of PD145065 at 0.2 (n = 4) or 0.6 mg kg-1 min-1 (n = 4) inhibited the ET-1-induced increase in MAP by 79 or 75%, respectively. 3. The transient depressor response (-16 +/- 3 mmHg) which preceded the rise in blood pressure induced by ET-1 (1 nmol kg-1, i.a., n = 8) was enhanced by an intravenous infusion of FR139317 (0.6 mg kg-1 min-1) to -35 +/- 5 mmHg (P < 0.05, n = 4). This enhancement was abolished by indomethacin (5 mg kg-1, i.v.) pretreatment (-17 +/- 1 mmHg, n = 4). PD145065 (0.2 mg kg-1 min-1, i.v.) attenuated the ET-1-induced fall in blood pressure to -9 +/- 1 mmHg (n = 4), while a higher dose of this antagonist (0.6 mg kg-1 min-1, i.v.) completely abolished the ET-1-mediated depressor response. 4. ET-1 (1 nmol kg-1, n = 8) inhibited ex vivo platelet aggregation by 96% at 5 min after injection of the peptide. FR139317 (0.2 or 0.6 mg kg-1 min-1, i.v.) or PD145065 (0.2mg kg-1 min-1, i.v.) did not affect the inhibition of ex vivo platelet aggregation in response to ET-1. In contrast, intravenous infusion of PD145065 (0.6 mg kg-1 min-1) abolished the anti-aggregatory effects of ET-1.5. Thus, FR139317 inhibits the pressor, but not the depressor actions of ET-1 and has no effect on the ET-l-induced inhibition of ex vivo platelet aggregation. In contrast, PD145065 antagonizes the pressor and depressor responses to ET-1 and abolishes the anti-aggregatory effects of the peptide.6. These results strongly suggest that ET-1-induced vasoconstriction in the anaesthetized rabbit is primarily mediated via the ETA receptor while the depressor and antiaggregatory actions of ET-1 are due to activation of the ETB receptor.     

Novel monoclonal antibodies to putative selectin carbohydrate ligands that inhibit selectin binding to myeloid cells

Four newly developed monoclonal antibodies (MAbs) are characterized using flowcytometry, enzyme-linked immunoadsorbent assay (ELISA), immunoprecipitation and Western blots, carbohydrate epitope mapping, glycosidase cleavage, and competition binding assays. Their effects on selectin binding to myeloid cells was tested. These MAbs react only with myeloid cells. MAbs CI-1, BU60, and HIM95 recognize epitopes expressed by CD11/CD18 (beta2) integrins, while HI247 and CSLEX1 do not. The epitopes require Lewis x [Galbeta1-4 (Fucalpha1-3)GlcNAc] based on reactivity with oligosaccharide-polyacrylamide-biotin or oligosaccharide-BSA conjugates. MAb HI247 recognizes a related structure, sialyl-Lewis x, NeuAcalpha2-3GaLbeta1-4(Fucalpha1-3)GlcNAc. The three MAbs against Lewis x show some minor differences in their reactivity such as recognizing their antigens on CD11/CD18 integrins after endo-beta-galactosidase treatment and recognizing free Lewis x. The hydroxyl group on C-3 of the terminal galactose is important for recognition by MAb CI-1, BU60, and HIM95 as its substitution with sulfo group of sialic acid abolishes the binding of these MAbs. The C-3 sialic acid is crucial for the binding of MAb HI247. Its replacement by sulphate or its cleavage by sialidase eliminates recognition by this MAb. MAbs HI247 and CSLEX-1 did not react in ELISA with immobilized CD11/CD18, suggesting that the majority of sialyl Lewis x on CD11/CD18 molecules may have sialic acid 6-linked rather than 3-linked to galactose. Unexpectedly, MAb BU60 inhibited binding of P-selectin mu chain chimera to HL-60 or U937 cells, while CI-1, HIM95 and three other defined anti-Lewis x MAbs (6C7, M6-1 and LeuM1) did not. MAb HI247 inhibited binding of both E- and P-selectin chimeras to these cell lines more effectively than several characterized MAbs (CSLEX-1, FH6, HECA-452) to sialyl Lewis x and related oligosaccharides. Certain combinations of these anticarbohydrate MAbs had additive inhibitory effects on selectin binding, suggesting a potential application of these new MAbs in cell adhesion/migration and tumor metastasis studies.     

Detection of platelet adhesion/aggregation to immobilized ligands on microbeads by an aggregometer

Platelet aggregation is believed to follow platelet adhesion to vascular injury sites. We have developed a turbidimetric assay for platelet aggregation following platelet adhesion to immobilized ligands using an aggregometer. The addition of polystyrene beads coated with von Willebrand factor (vWF) or fibrinogen (Fg) to platelet suspensions caused prompt aggregation of beads and platelets, which was detected as an increase in light transmission. Electron microscopic analysis revealed that platelets adhered to the bead surfaces and that additional platelets adhered to already adhering platelets, leading to the formation of platelet aggregates. vWF-coated beads induced larger aggregates than Fg-coated beads. The interaction of vWF-coated beads with platelets was abolished by both GPIb and GPIIb-IIIa blockers, while that of Fg-coated beads was abolished by GPIIb-IIIa blockers. vWF-coated beads induced modest secretion of granules from platelets but no thromboxane B2 synthesis. Fg-coated beads induced neither reaction. However, pleckstrin phosphorylation and protein tyrosine phosphorylation was induced by both types of bead. Platelet aggregation following platelet adhesion to both types of bead was inhibited by ADP scavengers, a protein kinase C inhibitor and a tyrosine kinase inhibitor, but not by aspirin. These findings suggest that vWF- and Fg-coated beads can induce platelet aggregation following platelet adhesion through specific ligand-receptor interactions and intracellular signaling. Our simple assay using these beads may represent a useful test for immobilized ligand-induced platelet adhesion and aggregation.     

Heterogenous binding of 3H-remoxipride to membranes of rat liver and brain

To assess the characteristics of sucrose as a pain-reducing substance, crying in 72 newborn humans during and after blood collection via heel prick was determined. In the first study infants drank 2 ml of water or 2 ml of a 0.17-0.34- or 0.51-M sucrose solution 1 min prior to blood collection. In the second experiment, a delay of 30, 60, 90, 120 or 240 s was imposed between sucrose intake and the initiation of blood collection. The dose-response function for concentration was flat. The most effective time delay was 120 s. The effectiveness of the 2-min interval accords with previous findings of endogenous opioid release caused by sucrose taste. The flat dose-response function extends findings in rats and humans that the calming and pain-reducing effects of sucrose are not influenced by either concentration or volume, suggesting that the transduction from gustatory afferent to opioid-mediated efferent is of an on-off nature and not graded.     

Preparation of mouse monoclonal antibodies to okadaic acid and their binding activity in organic solvents

Seven of 20 mouse monoclonal antibodies to OA, OA8-2, OA10-8, OA22-22, OA227-11, OA296-1, OA423-3, and OA958-2, were studied as to their binding to OA in organic solvents. OA423-3 (IgG1-kappa) and OA958-2 (IgG1-kappa) in 90-100% methanol retained their binding activities with both immobilized and free antibodies. Whereas OA8-2 (IgG2a-kappa), OA10-8 (IgG1-kappa), OA22-22 (IgG2a-kappa), OA227-11 (IgG1-kappa), and OA296-1 (IgM-kappa) did not bind to OA in over 50-60% methanol. The results of a non-competitive inhibition assay for OA indicated that in a methanolic or ethanolic solution, the binding ability of immobilized OA423-3 decreased as the concentration of each alcohol increased. The concentration of OA at the midpoint between the upper and lower plateaus of the inhibition curve was 0.18 ng/ml in 0% methanol and 570 ng/ml in 100%, respectively. In 0-50% of each of acetone, diethyl ether, and benzene in methanol, the binding ability of OA423-3 remained at the level in 100% methanol. OA958-2 showed similar binding properties to OA423-3. No relationship between the subclass of the immunoglobulin and the binding activity of the antibody in organic solvents was observed. These results indicate that the OA423-3 and OA958-2 antibodies are useful for the development of a new ELISA method for OA in organic solvents.     

Measurement of platelet aggregation peptide inhibitors by ultrasonic interferometry

In healthy subjects, basal hepatic glucose production is (partly) regulated by paracrine intrahepatic factors. It is unknown if these paracrine factors also influence basal glucose production in infectious diseases with increased glucose production. We compared the effects of 150 mg indomethacin (n = 9), a nonendocrine stimulator of glucose production in healthy adults, and placebo (n = 7) on hepatic glucose production in Vietnamese adults with uncomplicated falciparum malaria. Glucose production was measured by primed, continuous infusion of [6,6-2H2]glucose. After indomethacin, the plasma glucose concentration and glucose production increased in all subjects from 5.3 +/- 0.1 mmol/L to a maximum of 7.1 +/- 0.3 mmol/L (P < .05) and from 17.6 +/- 0.8 micromol x kg(-1) x min(-1) to a maximum of 26.2 +/- 2.5 micromol x kg(-1) x min(-1) (P < .05), respectively. In the control group, the plasma glucose concentration and glucose production declined gradually during 4 hours from 5.4 +/- 0.2 mmol/L to 5.1 +/- 0.1 mmol/L (P < .05) and from 17.1 +/- 0.8 micromol x kg(-1) x min(-1) to 15.1 +/- 1.0 micromol x kg(-1) x min(-1) (P < .05), respectively. There were no differences in plasma concentrations of insulin, counterregulatory hormones, or cytokines between the groups. We conclude that indomethacin administration results in a transient increase in glucose production in patients with uncomplicated falciparum malaria in the absence of changes in plasma concentrations of glucoregulatory hormones or cytokines. Thus, this study indicates that in uncomplicated falciparum malaria, the rate of basal hepatic glucose production is also regulated by paracrine intrahepatic factors.