Arg-Gly-Asp-dependent occupancy of GPIIb/IIIa by applaggin: evidence for internalization and cycling of a platelet integrin

Using indirect immunofluorescence microscopy we examined the distribution and cycling of GPIIb/IIIa after binding to applaggin, a high-affinity Arg-Gly-Asp (RGD)--containing ligand. Resting, unfixed platelets were incubated with applaggin for 30 minutes at 37 degrees C, and bound applaggin was detected by an affinity-purified rabbit anti- applaggin antibody. Examination of intact cells showed a rim pattern for applaggin, consistent with its binding to the platelet surface. Staining of Triton X-100--permeabilized cells showed an intracellular pool of applaggin. Competition of applaggin binding by either AP-2, an anti-GPIIb/IIIa monoclonal antibody (MoAb) that blocks fibrinogen binding, or the synthetic peptide RGDW eliminated both surface and intracellular staining, indicating that applaggin is binding to GPIIb/IIIa in an RGD-dependent manner. Inhibition of platelet activation by PGE1 and theophylline had no effect on the observed staining patterns, indicating that cellular activation is not required for surface binding and subsequent internalization. To evaluate whether occupancy of functional binding sites on GPIIb/IIIa is required for internalization, we used mAb15, an anti-GPIIIa antibody that neither blocks fibrinogen binding nor induces the expression of ligand-induced binding sites on GPIIb/IIIa. In these studies mAb15 was internalized in a manner analogous to both AP-2 and applaggin, showing that occupancy of the RGD binding site is not required to initiate receptor internalization. To estimate the size of the newly internalized pool of applaggin, 125I-applaggin--binding studies were performed. Displacement of bound 125I-applaggin by excess unlabeled applaggin or EDTA showed that at least 17% of bound applaggin was nondisplaceable when binding was performed under conditions permitting membrane flow and internalization. These data indicate that GPIIb/IIIa is internalized in unstimulated platelets independent of cellular activation or occupancy of the functional binding site(s) of GPIIb/IIIa by RGD-containing ligands. Thus, internalization of GPIIb/IIIa may represent a mechanism by which the surface expression of this adhesion receptor is regulated.     

Transfected leukocyte integrin CD11b/CD18 (Mac-1) mediates phorbol ester-activated, homotypic cell:cell adherence in the K562 cell line

The CD11b/CD18 leukocyte integrin molecule mediates diverse neutrophil adherence-related functions, including cell:cell and cell:extracellular matrix attachments. To study the individual role of this leukocyte integrin in cell adherence in hematopoietic cells, we expressed the CD11b/CD18 complex on the surface of K562 cells, a cell line derived from an individual with chronic myelogenous leukemia in blast crisis. We used an amphotrophic retroviral vector designated LCD18SN, harboring the complete coding sequence for the CD18 subunit, to transfer the CD18 cDNA into K562 cells and select stable cell lines. The CD11b subunit in the expression plasmid pREP4 was transfected into these K562/CD18 cells by electroporation and stable cell clones were selected. These K562 cells possessed RNA and intracellular protein for each subunit, and they expressed the CD11b/CD18 heterodimer on the cell surface. When CD11b/CD18 expressing K562 cells were stimulated with phorbol myristate acetate (50 ng/mL) for 24 to 48 hours, these K562 cells formed dense cell:cell aggregates. This homotypic aggregation required both activation of the CD11b/CD18 complex and the induction of the counter- receptor for CD11b/CD18 on the conjugate cell. This cell line will (1) enable the structure-function relationships between cell activation and homotypic adherence to be assessed, (2) provide the opportunity to identify accessory molecules required for activation of the CD11b/CD18 complex, and (3) facilitate the identification of novel ligands for the CD11b/CD18 complex.     

口服右旋索他洛尔生存研究(SWORD)

标题　ａ）口服左旋－Ｓｏｔａｔｏｌ治疗心肌梗死后左室功能不全患者生存研究（ＳＷＯＲＤ）：试验依据、设计和方法　　　ｂ）右旋－Ｓｏｔａｔｏｌ对亚急性和陈旧性心肌梗死后左室功能不全患者病死率的影响研究作者　ＷａｌｄｏＡＬ,ＣａｍｍＡＪ,ｄｅｒｕｙｔｅｒＨ,等ａ）ＡｍＪＣａｒｄｉｏｌ,１９９５,７５：１０２３～１０２７ｂ）Ｌａｎｃｅｔ,１９９６,３４８：７～１２　　研究疾病：充血性心力衰竭。目的：右旋－Ｓｏｔａｔｏｌ为一种具有专一性钾通道阻滞作用的抗心律失常药物。本研究将对其降低心肌梗死后左室功能不全…     

Obtaining accurate patient‐specific perioperative anaesthetic costs in orthopaedic anaesthesia

□ The aim of the study was to obtain perioperative patient‐specific costs associated with orthopaedic anaesthesia □ A prospective observational study design was used to determine fixed, semi‐fixed, and variable costs □ The preliminary results of the first 60 patients reports a mean total perioperative anaesthetic cost of £291 (SD: £103.9, median: £278.09, IQR: £219.3–£369.5) □ Fixed and semi‐fixed costs are a major cost component of anaesthesia; variable costs account for only 12 per cent of total cost □ The results of this study will be used in an economic evaluation examining the use of different anaesthetic techniques in orthopaedics     

Chronic daily headache with migrainous features due to papilledema-negative idiopathic intracranial hypertension

Patients with the syndrome of chronic daily headache often report migrainous symptoms and consequently are diagnosed as having a primary headache syndrome. We report two cases of idiopathic intracranial hypertension causing chronic daily headache with migrainous features in the absence of associated papilledema.     

Dual energy X-ray absorptiometry of the hand and wrist–a possible technique to assess skeletal maturation: methodology and data in normal youths

Ninety five normal Caucasian subjects (51F, 44 M) aged from 2 to 25 y were measured at the hand and wrist level with a small DXA system (pDEXA™) in order to obtain the normal values of the bone mineral content (BMC), density (BMD) and projected area (A) of carpal (c) and metacarpal (m) bones. BMDc ranged from 0.065 ± 0.007 g/cm to 0.365 ± 0.035 g/cm in females and 0.425 ± 0.040 g/cm in males. It presented a sharp change of increase rate at 15.5 and 17 y of age in girls and boys, respectively. Ac presented the same kind of evolution as BMDc, but had a larger value dispersion. The second metacarpal bone had the highest BMCm value in 85% of females and 90% of males. The sum of BMCmi or Ami values (i = 1–5) and the projected mean density of the 5 metacarpal bones was well correlated with BMCc, Ac and BMDc, respectively ( r > 0.90). A volumetric mineral density, dmi, calculated for each of these bones, approximated to a cylinder, was correlated with age ( r > 0.85).     

Effect of acute plasma volume expansion on peripheral arteriolar tone in healthy subjects.

1. Using venous occlusion plethysmography, we have investigated the forearm blood flow response in healthy subjects to the acute plasma volume expansion caused by a rapid intravenous infusion of saline. The contribution made to this response by nitric oxide has been investigated using local intra-arterial infusions of the nitric oxide synthase inhibitor NG-monomethyl-L-arginine. 2. The infusion of 1000 ml of saline over 25 min caused plasma volume to increase by about 7%, and resulted in a rise in forearm blood flow, with no change in arterial blood pressure. The onset of the blood flow response occurred within 10 min and blood flow remained elevated above baseline 20 min after the end of the saline infusion. 3. Local intra-arterial infusion of NG-monomethyl-L-arginine alone caused a reduction in forearm blood flow which was maximal at the end of the infusion and gradually recovered to baseline levels over 40 min. 4. When local intra-arterial infusion of NG-monomethyl-L-arginine was followed by plasma volume expansion, the calculated effect of NG-monomethyl-L-arginine was such as to abolish the vasodilator response to saline. 5. The effect of local intra-arterial infusion of NG-monomethyl-L-arginine on forearm blood flow was greater when the drug was given after volume expansion had occurred, than when it was given before the administration of saline. However, in control experiments the vasoconstrictor response to noradrenaline was also enhanced after the administration of the volume load in comparison with the response to noradrenaline given alone.(ABSTRACT TRUNCATED AT 250 WORDS)