Esophageal disease in AIDS is associated with pathologic processes rather than mucosal human immunodeficiency virus type 1.

Twenty-five patients with AIDS and esophageal symptoms were evaluated for the presence of esophageal disease and human immunodeficiency virus type 1 (HIV-1) in the esophageal mucosa. A single infectious process caused by Candida albicans, cytomegalovirus, herpes simplex virus, varicella-zoster virus, or Mycobacterium avium-intracellulare complex or a single noninfectious process caused by Kaposi's sarcoma or reflux esophagitis was identified in 20 patients. Two processes were identified in 5 patients. HIV-1 mRNA was detected by in situ hybridization in mononuclear cells in esophageal lamina propria in 36% of patients. The presence of HIV-1 in the esophageal mucosa was not associated with a specific esophageal symptom, mucosal inflammation or ulceration, Kaposi's sarcoma, specific opportunistic infection, or response of the infection(s) to therapy. Esophageal disease in patients with AIDS appears to be associated with specific pathologic processes rather than the presence of HIV-1 in esophageal mucosa.

Further characterization of the loop structure of platelet glycoprotein IIIa: partial mapping of functionally significant glycoprotein IIIa epitopes.

Glycoprotein (GP) IIb-IIIa serves as the platelet fibrinogen receptor. Studies of the tertiary structure of GPIIIa have shown that the protein has a large loop structure of at least 325 amino acids in length. To further characterize this loop structure, intact platelets were digested with alpha-chymotrypsin. Digestion products were examined using the anti-GPIIIa monoclonal antibodies (MoAbs) AP3, D3GP3, and C5GP3, as well as the human alloantibody, anti-PLA1. AP3 recognized GPIIIa digestion products of 109, 95, and 68 Kd. D3GP3 and C5GP3 recognized an additional band of 51 Kd. Time course digestions demonstrated that the 51-Kd fragment was generated by proteolysis of the 68-Kd peptide. Sequence analysis of the reduced 51-Kd peptide showed that this fragment began at amino acid 422. The nonreduced 51-Kd peptide was reactive with antibodies directed against the first 13 amino acids of GPIIIa, demonstrating the presence of a covalently attached N-terminal peptide. These data suggest that: (1) the minimum length of the loop structure is at least 384 amino acids; (2) the AP3 epitope is formed at least in part by a determinant contained within residues 348 to 421; and (3) the D3GP3 and C5GP3 epitopes are contained within amino acids 422 to 692 of GPIIIa, a region that may be flexible and involved in conformational changes that occur after ligand binding.

The effect of glycoprotein IIb-IIIa receptor occupancy on the cytoskeleton of resting and activated platelets.

The platelet integrin, glycoprotein IIb-IIIa (GPIIb-IIIa), serves as the receptor for fibrinogen. This study examined what effect GPIIb-IIIa receptor occupancy had on the cytoskeleton of resting and activated platelets. Triton X-100-insoluble residues (cytoskeletons) were isolated from resting washed platelets incubated with either 500 microM RGDS or 500 microM RGES and examined for protein content. RGDS did not increase the amount of GPIIb-IIIa associated with the cytoskeletal residues which sedimented at either 15,800 x g or 100,000 x g. To determine the effect of receptor occupancy on the formation of the activated platelet cytoskeleton, stirred and nonstirred RGDS-treated platelets in plasma were activated with ADP. Triton X-100-insoluble residues were isolated and examined for both protein content and retention of GPIIb-IIIa. Further, morphological studies were performed on the RGDS-ADP-stimulated platelets. The results of this study suggest that 1) RGDS peptide receptor occupancy does not lead to GPIIb-IIIa linkage to the cytoskeleton, 2) ADP-stimulated platelet shape change, polymerization of actin, and association of myosin with the cytoskeleton are unaffected by RGDS peptide receptor occupancy. 3) RGDS inhibits an aggregation-dependent incorporation of ABP, alpha-actinin, talin, and GPIIb-IIIa into the Triton-insoluble residue.

cDNA cloning and expression of platelet p24/CD9. Evidence for a new family of multiple membrane-spanning proteins.

This study was designed to clone, sequence, and express the full-length cDNA for the human platelet p24/CD9 antigen. A 1.3-kilobase cDNA clone was identified that has an open reading frame encoding a mature protein of 228 amino acids (approximately 25,400 Da) containing 10 cysteine residues and four putative transmembrane domains. The identity of the clone was confirmed by: (i) its predicted size, (ii) identity to four peptide sequences from the isolated protein including the NH2 terminus, and (iii) expression of the isolated clone in Xenopus oocytes and Chinese hamster ovary cells. p24/CD9 has sequence identity (24-34%) to four other cell-surface proteins: ME491, a melanoma antigen; CO-029, a carcinoma antigen; CD37, a leukocyte antigen; and SM23, an antigen of the parasitic helminth Schistosoma mansoni. The five proteins have a similar number of amino acids and are characterized by the presence of four putative transmembrane domains. These data indicate the presence of a new family of surface antigens that may function in cellular activation and differentiation.

Hemostasis in Glanzmann's thrombasthenia (GT): GT platelets interfere with the aggregation of normal platelets.

The transfusion requirements for a 6-year-old Glanzmann's thrombasthenia (GT) patient undergoing tonsillectomy and adenoidectomy were studied. Transfusion of pheresed platelets from a single normal donor increased the platelet count by 63 x 10(9)/L but did not correct the bleeding time. Since the ratio of normal:GT platelets in vivo was approximately 1:5, it was possible that GT platelets interfered with the function of normal platelets. To test this hypothesis, mixtures of platelet-rich plasma (PRP) from a normal donor and the patient were studied to determine a ratio of normal:GT platelets that would yield acceptable in vitro aggregation. Normal:GT ratios of 1:4 and 3:2 resulted in 25% and 59% aggregation, respectively. Mixtures of normal and nonfunctional ethylene glycol tetra-acetic acid-treated platelets gave similar results. Aggregates from mixtures of normal and patient platelets were also examined morphologically by light microscopy and were proportional in size to the normal:GT platelet ratio. Transfusion of platelets from the pheresis of four donors increased the patient's platelet count by greater than 300 x 10(9)/L (normal:GT ratio 1:1), produced 53% aggregation, and resulted in satisfactory postoperative hemostasis. The platelet transfusion requirement for this GT patient was much greater than would have been expected in the absence of aggregation-defective platelets.

A conformation-dependent epitope of human platelet glycoprotein IIIa.

This study explores conformational states of human platelet glycoprotein IIIa (GP IIIa) and possible mechanisms of fibrinogen receptor exposure. D3GP3 is an IgG1, kappa monoclonal antibody generated against purified GP IIIa and found to be specific for GP IIIa by immunoprecipitation and Western blot analysis. The binding of D3GP3 to resting platelets caused fibrinogen binding (approximately 5,000 molecules/platelet) and platelet aggregation but not secretion. Platelets express 40,000-50,000 GP IIb-IIIa molecules in their surface membranes. However, resting platelets only bound approximately 5,000 D3GP3 molecules/platelet. D3GP3 binding to platelets could be increased 2-3-fold by dissociation of the GP IIb-IIIa complex with 5 mM EDTA or by occupying the fibrinogen receptor with either RGDS peptides or fibrinogen. Platelet stimulation with ADP in the absence of fibrinogen did not cause increased D3GP3 binding above control levels. These data suggest that 1) GP IIb-IIIa can exist in multiple conformations in the platelet membrane, 2) D3GP3 binding to GP IIIa can expose the fibrinogen receptor, 3) the binding of either RGDS peptides or fibrinogen causes exposure of the D3GP3 epitope, and 4) platelet activation in the absence of ligand does not induce the same conformational changes in GP IIb-IIIa as does receptor occupancy by RGDS peptides or fibrinogen.

The activation of human platelets mediated by anti-human platelet p24/CD9 monoclonal antibodies.

Anti-human platelet p24/CD9 (p24/monoclonal antibody 7) causes the activation of platelets and in the presence of calcium induces platelet aggregation. Our studies suggest that platelet response to this antibody is mediated at least in part by the pertussis toxin-sensitive guanine nucleotide-binding proteins (G proteins) that stimulate phosphoinositide hydrolysis and inhibit adenylate cyclase. Prior exposure of saponin-treated platelets to anti-p24/CD9 inhibited the [32P] ADP-ribosylation of the alpha 41 protein by pertussis toxin. Platelet aggregation induced by this antibody is preceded by and/or accompanied by accelerated phosphatidylinositol turnover, the generation of inositol phosphates and diacylglycerol (DAG), calcium mobilization, and protein phosphorylation. The production of inositol phosphate(s) was measurable within 15 s of either anti-p24/CD9 or thrombin addition. Within 10 s of antibody addition (10 micrograms/ml), the level of DAG was 200% over that of the control and similar to that observed with 2 units/ml thrombin (201% over that of the control). Therefore, as it appears to be true for thrombin, platelet response upon binding of anti-p24/CD9 is primarily mediated by the activation of phospholipase C. When platelets pretreated with aspirin (200 microM) and apyrase (1 mg/ml) were subsequently exposed to anti-p24/CD9, aggregation still occurred. This indicates that neither secreted ADP nor thromboxane generation is required for this aggregation response. Using indo-1 and ratio cytofluorometry, we observed that an increase in platelet cytosolic calcium is a relatively early event and occurs in either the presence or absence of calcium in the external media. Phosphorylation studies of platelet proteins showed that anti-p24/CD9 binding to platelets caused increased phosphorylation of four proteins with apparent molecular masses of 50,000, 47,000, 36,000, and 20,000 daltons. These studies suggest that platelet activation mediated by the surface protein p24/CD9 is mainly through the stimulation of a phospholipase C, the activation of which is responsible for the generation of second messengers inositol trisphosphate and DAG.

Calcium mobilization in human platelets using indo-1 and flow cytometry.

Regulation of cytoplasmic free calcium concentration is believed to be important in the response of platelets to external stimuli. A relatively new fluorescent calcium indicator, indo-1, has properties by which alterations of cytoplasmic calcium can be evaluated in single platelets by flow cytometry. Activation of platelets at a temperature lower than 37 degrees C allows examination of the heterogeneity of intracellular free calcium levels and can distinguish variations among platelets in the initiation, duration, and magnitude of calcium fluxes. The clear advantage of flow cytometric analysis of platelet cytosolic calcium is that stimulus-response coupling can now be studied on a single cell basis. Platelets were activated by addition of human alpha-thrombin or ADP at 37 degrees C or at room temperature (22 degrees C). Activation at 37 degrees C approaches more closely an in vivo response and, as expected, increases in cytosolic calcium occurred within seconds of agonist addition. Transient increases in cytoplasmic calcium levels occurred when platelets were challenged with a low concentration of agonist. Heterogeneity in cytoplasmic calcium levels was also observed at 10(-5) mol/L ADP and 0.1 U/mL alpha-thrombin. Some of this heterogeneity was no longer observed at higher concentrations of agonist (10(-4) mol/L ADP and 0.5 U/mL thrombin), suggesting that a sufficient magnitude of signal is required to induce changes in platelet cytosolic calcium. Light-scatter properties of the activated platelets were also monitored simultaneously and showed changes in response to both agonists. The ability to measure changes in cytoplasmic free calcium by ratio flow cytofluorimetry provides a new approach to study of the role of alterations in intracellular calcium in response to agonists acting through different membrane receptors as well as providing a sensitive technique to detect functional subpopulations of platelets.

Decreased progesterone binding and attenuated progesterone action in cultured human breast carcinoma cells treated with epidermal growth factor.

Specific progesterone binding by cultured human breast carcinoma T47D, MCF-7, and ZR75-1 cells was decreased 25-40% by epidermal growth factor (EGF), with a 50% effective dose of 0.1 nM EGF. Studies with the soluble and particulate fractions prepared after homogenization of T47D cells grown in glass roller bottles revealed equivalent EGF-induced decreases in progesterone binding to receptors in both fractions. Equilibrium progesterone binding studies with these soluble and particulate fractions revealed that EGF decreased the receptor number, but had no effect on affinity. With cells grown adherent to plastic dishes, EGF treatment induced a greater decrease in binding to receptors recovered in the particulate fraction, than to receptors recovered in the soluble fraction. The decrease in progesterone binding induced by 20 nM EGF was maximal after 2 min of cellular EGF treatment for receptors recovered in the soluble fraction, but was only half-maximal after 15 min for receptors recovered in the particulate fraction. Decreased progesterone binding persisted for at least 8 days in cells cultured with 1 nM EGF. Either insulin or EGF stimulated T47D cell proliferation by two- to threefold with a 50% effective dose of 100 nM for insulin and 0.1 nM for EGF. The progestin, R5020, decreased T47D cell growth by 30% with a 50% effective dose of 1 nM. Either EGF or insulin antagonized the inhibitory effect of R5020 on cell reproduction, but progestins did not antagonize the growth stimulatory response of cells to EGF. Progestins increased the number of EGF receptors within 12 h of their addition to T47D cells, but this response was lost after 6 days. These data show that EGF or progesterone can regulate the receptor number of the other, but for cell reproduction, the effect of EGF is dominant over that of progestins.

Resolution of high and low affinity progesterone receptors from human breast carcinoma T47D cells.

Each of four independent experimental approaches showed that human breast carcinoma T47D cells contain both high and low affinity progesterone receptors. (i) Equilibrium-specific [3H]progesterone binding to adherent cultured cells revealed dissociation constants (Kd) of 1.5 and 60 nM and 0.33 and 2.4 X 10(6) sites/cell, respectively. Both the high and low affinity receptors were specific for progestins as demonstrated by steroid binding competition studies conducted at 5 and 50 nM [3H]progesterone. (ii) Equilibrium [3H]progesterone binding to the resolved soluble and particulate fractions from a cell homogenate sedimented at 40,000 X g.min revealed Kd = 1.4 nM high affinity binding sites exclusively in the supernatant fraction and Kd = 24 nM low affinity sites exclusively in the particulate fraction. Extraction of the particulate fraction with a high ionic strength buffer solubilized the low affinity receptors stoichiometrically; but once solubilized, they displayed Kd = 2.4 nM high affinity progesterone binding. Characterizations of 3H-ligand bound specifically to progesterone receptors in intact cells or resolved subcellular fractions revealed no [3H]progesterone metabolites that could account for the low affinity binding. (iii) Calculations based on the rate constants of [3H] progesterone association with or dissociation from adherent cells revealed the same dissociation constants for both high and low affinity binding as those determined by equilibrium measurements. (iv) Nonionic detergent extraction of cells incubated with a wide range of [3H]progesterone concentrations revealed high affinity progesterone binding to receptors in the detergent-soluble fraction and low affinity binding associated primarily with the particulate residue, consistent with the data on equilibrium progesterone binding to resolved cell homogenate fractions. The rate of extraction of the high affinity receptor-progesterone complex with nonionic detergent (t1/2 = 1 min at 0 degrees C) equaled the rate of extraction of a representative lysosomal enzyme, beta-acetylglucosaminidase.

Altered glucocorticoid binding and action in response to epidermal growth factor in HBL100 cells.

Incubation of adherent human breast epithelial HBL100 cells with epidermal growth factor (EGF) decreased [3H]dexamethasone binding by 35% with no effect on affinity. Maximal inhibition was obtained at 3 nM EGF and the 50% effective dose was 0.2 nM EGF. Decreased dexamethasone binding induced by 3 nM EGF was maximal by 5 min of treatment and, in the continuous presence of EGF, persisted at a constant level over 4 days. The action of EGF was antagonized by 12-O-tetradecanoylphorbol-13-acetate, which did not inhibit dexamethasone binding significantly, and by concanavalin A. In homogenates of EGF-treated cells, decreased dexamethasone binding was observed only in the cytosolic fraction. Saturation dexamethasone binding inhibited the growth rate of HBL100 cells by approximately 50%, but concurrent treatment with EGF overcame this inhibition. The effect of EGF on dexamethasone-inhibited cell growth also was antagonized by 12-O-tetradecanoylphorbol-13-acetate.

Cellular progesterone receptor phosphorylation in response to ligands activating protein kinases.

Progesterone receptors were immunoprecipitated with monoclonal antibodies KD68 from lysates of human breast carcinoma T47D cells labelled to steady state specific activity with 32Pi. The 120 kDa 32P-labelled progesterone receptor band was resolved by polyacrylamide gel electrophoresis and identified by autoradiography. Phosphoamino acid analysis revealed serine phosphorylation, but no threonine or tyrosine phosphorylation. Treatment of the 32Pi-labelled cells with EGF, TPA or dibutyryl cAMP had no significant quantitative effect on progesterone receptor phosphorylation, though the EGF receptor and the cAMP-dependent protein kinases have been reported to catalyze phosphorylation of purified avian progesterone receptor preparations in cell free systems. Progesterone receptor phosphorylation on serine residues was increased by 2-fold in cells treated with 10 nM progesterone; EGF had no effect on progesterone-mediated progesterone receptor phosphorylation.

Epidermal growth factor stimulates tyrosine phosphorylation of human glucocorticoid receptor in cultured cells.

Human breast epithelial HBL100 cells, which bind both epidermal growth factor (EGF) and glucocorticoids, were labelled to steady state specific activity with 32Pi and the glucocorticoid receptor was immunoprecipitated from cell lysates with polyclonal antiserum GR884. Immunoprecipitated receptor was resolved by NaDodSO4-polyacrylamide gel electrophoresis and identified by autoradiography. Immunoprecipitated receptor also was characterized by western blot analysis and affinity labelling with [3H]dexamethasone-21-mesylate. Phosphoamino acid analysis of 32P-glucocorticoid receptor revealed 89% phosphoserine and 11% phosphotyrosine. Treatment of steady state 32Pi-labelled cells with EGF stimulated total and alkali-stable phosphorylation in the 97 kDa receptor band by about 35%. Prior incubation with dexamethasone inhibited EGF stimulated, alkali-stable phosphorylation of the 97 kDa glucocorticoid receptor band.

Differential phosphorylation of the progesterone receptor by insulin, epidermal growth factor, and platelet-derived growth factor receptor tyrosine protein kinases.

Purified preparations of insulin, epidermal growth factor (EGF), and platelet-derived growth factor (PDGF) receptors were compared for their abilities to phosphorylate purified hen oviduct progesterone receptors. The specific activities of all three peptide hormone-induced receptor kinases were first defined using a synthetic tridecapeptide tyrosine protein kinase substrate. Next, equivalent ligand-activated activities of the three receptor kinases were tested for their abilities to phosphorylate hen oviduct progesterone receptor. Both the insulin and EGF receptors phosphorylated progesterone receptor at high affinity, exclusively at tyrosine residues and with maximal stoichiometries that were near unity. In contrast, the PDGF receptor did not recognize progesterone receptor as a substrate. Insulin decreased the Km of the insulin receptor for progesterone receptor subunits as substrates, but had no significant effect on Vmax values. On the other hand, EGF increased the Vmax of the EGF receptor for progesterone receptor subunits as substrates. Phosphorylation of progesterone receptor by the insulin and EGF receptor kinases differed in two additional ways. 1) EGF-activated receptor phosphorylated the 80- and 105-kDa progesterone receptor subunits to an equal extent, whereas insulin-activated receptor preferentially phosphorylated the 80-kDa subunit. 2) Phosphopeptide fingerprinting analyses revealed that while insulin and EGF receptors phosphorylated one identical major site on both progesterone receptor subunits, they differed in their specificities for other sites.