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Biomedical subjects

E F Lüscher

Publications and source records attributed to E F Lüscher.

At least 19 recordsLinked to original sources
PubMed1996

Thrombospondin measured in whole blood--an indicator of platelet activation.

Abnormal platelet activation may be involved in prethrombotic states and lead to thromboembolism. When platelets become activated, they release thrombospondin (TSP) from their alpha-granules which binds mainly to the surface of activated platelets, platelet-derived microparticles and other blood cells. To determine bound as well as free TSP in a single assay, we developed an indirect ELISA to measure TSP in fixed whole blood. The intra-assay variance was less than 5% and 97% of purified standard TSP, added to whole blood samples, was recovered with the ELISA. Blood collected with a 20G needle into a syringe resulted in lower "whole blood TSP' values than blood collected with the Vacutainer system. Whole blood TSP levels were measured in 66 healthy blood donors (20F, 46M) aged 25-75 years. The mean whole blood TSP concentration was 33 +/- 19 ng/ml. No significant difference in whole blood TSP was found between healthy females and males (35 +/- 23 ng/ml vs. 33 +/- 17 ng/ml).

Adult↗
PubMed1993

The formation of the haemostatic plug--a special case of platelet aggregation. An experiment and a survey of the literature.

The formation of the haemostatic plug is an extremely fast process. This excludes, at least in its first phase, the involvement of soluble activating agents released from or produced by the platelets. An experiment with ADP-activated, formaldehyde-fixed platelets shows that platelets with activated fibrinogen receptors will bind inactive platelets in the presence of fibrinogen and Ca(2+)-ions. A survey of the literature shows that platelet activation is accompanied by the clustering of the fibrinogen receptors. The surface of an activated platelet, which makes part of the growing haemostatic plug therefore is covered with patches of tightly packed fibrinogen. This allows the multisite combination with the statistically distributed low affinity receptors of the newly arriving platelets. Tightly packed fibrinogen, as present on clusters of the activated GP IIb/IIIa receptors as well as when absorbed to artificial surfaces acts as an activator of platelets. Thus, the propagation of the activation process is possible without a requirement for other, external activators. Such agents, which are released from platelets and, finally, thrombin formation, are nonetheless of vital importance, not for the formation but for the consolidation of the haemostatic plug.

Adenosine Diphosphate↗
PubMed1992

Induction of vascular haemostasis by Nd:YAG laser light in melanin-rich and melanin-free tissue.

Haemostasis was effected in vessels of melanin-rich (MR: choroid) and melanin-free (MF: mesentery) rabbit tissue irradiated with a cw-Nd:YAG laser. The following parameters were employed: - pulse duration: 200 ms (MR) and 100 ms (MF); focal spot diameter: 200 microns (MR) and 80 microns (MF); pulse energies: 100-250 mJ (MR) and 0.5-1 J (MF); irradiances: 1.6-4.0 kW cm-2 (MR) and 1-2 x 10(2) kW cm-2 (MF). In melanin-rich tissue, laser energy is absorbed principally by melanin granules contained within the stromal melanocytes. The heat generated in these structures radiates into the surrounding tissue where it is dissipated. The damage thus incurred by the endothelium of blood vessels encompassed within this field triggers the haemostatic mechanism whereby blood flow is arrested. This effect is realized by the formation of an occluding plug of platelets, which is stabilized by the deposition of fibrin, particularly in capillaries, and to a lesser degree in larger vessels of the vascular lamina. In melanin-free tissue, haemoglobin serves as the primary site of energy absorption, which is thus shifted from the stroma to the vessel lumen. Irradiation of vessels in such tissue leads to thermocoagulation of plasma proteins and consequent stasis of blood flow.

Animals↗
PubMed1986

Morphology of the interaction of collagen fibrils with normal human platelets and thrombasthenic platelets.

The ultrastructure of the platelet contacts with collagen fibrils (CF) as well as the course taken by CF on the platelet surface were studied on ultrathin sections of platelets and CF. Platelets from normal donors and from a patient with thrombasthenia were incubated in citrated plasma with collagen. For electron microscopy a protein-stabilizing fixation procedure was applied. Platelet-collagen contacts (PCC) are characterized by a distance of 7 +/- 3 nm between the platelet membrane and the CF; the gap contains electron-dense bridges. The PCC of normal and thrombasthenic platelets are morphologically identical. Hence, it is unlikely that the glycoproteins IIb/IIIa-complex, which is absent in thrombasthenic platelets, plays a significant role in the platelet collagen interaction. The CF induce random movements of the platelets and their pseudopods, whereby the fibrils, which often show multisite attachment, coil up around the platelet surface; they become bent and often display drastic directional changes. CF are found inside invaginations of the platelet membrane as well as in depressions of the platelet surface. These processes require an involvement of the platelet's contractile system, which appears to interact reversibly with the platelet plasma membrane.

Blood Platelet Disorders↗
PubMed1986

Identification of the immunoglobulin G receptor of human platelets.

The binding site of IgG on human platelets was studied by the use of the cleavable heterobifunctional cross-linking agent N-succinimidyl (4-azidophenyldithio)propionate. Binding characteristics of the derivatized IgG were similar to normal IgG. Periodate-borohydride treatment of platelets also did not significantly alter their ability to bind IgG. N-Succinimidyl (4-azidophenyldithio)propionate was bound to IgG via a succinimidyl ester and then photolyzed in the presence of intact platelets. Their membrane glycoproteins were first tritiated by the periodate-borohydride method. The cross-linked product was analyzed by two dimensional sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis. The non-reduced first-dimension gels were subjected to 5% 2-mercaptoethanol prior to separation in the second dimension. Such gels were then evaluated by fluorography, silver staining, and counting the radioactivity of sequential gel strips in the area of cross-linking. The protein complexes at the interface between stacking and running gel were further resolved in isoelectric focusing gels. One IgG-containing band could be identified. After reduction, the constituent proteins of the cross-linked complex were analyzed by sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis and subsequent immunoblotting with an antiserum against platelet membrane glycoproteins. All of these studies gave evidence of glycoprotein IIIa as the receptor of IgG. Based on the results of the different experimental approaches, we conclude that glycoprotein IIIa is the IgG receptor in human platelets.

Azides↗
PubMed1986

Stochastic response of human blood platelets to stimulation of shape changes and secretion.

Stopped-flow turbidimetric data indicate that platelets stimulated with low levels of thrombin undergo a shape transformation from disc to "sphere" to smaller spiny sphere that is indistinguishable from the shape change induced by ADP through different membrane receptor sites and a dissimilar receptor trigger mechanism. Under conditions where neither secretion nor aggregation occur, the extinction coefficients for total scattering by each of the three platelet forms are independent of the stimulus applied, and both reaction mechanisms can be described as stochastic (Poisson) processes in which the rate constant for the formation of the transient species is equal to the rate constant for its disappearance. This observation is independent of the shape assignment, and as the concentration of thrombin is increased and various storage organelles secrete increasing amounts of their contents into the external medium, the stochastic pattern persists. Progressively larger decreases in the extinction coefficients of the intermediate and final platelet forms, over and above those that reflect shape alterations alone, accompany or parallel the reaction induced by the higher thrombin concentrations. The excess turbidity decrease observed when full secretion occurs can be wholly accounted for by a decrease in platelet volume equal in magnitude to the fraction of the total platelet volume occupied by alpha granules. Platelet activation, as reported by the whole body light scattering of either shape changes alone or shape changes plus parallel (but not necessarily also stochastic) alpha granule secretion, thus manifests itself as a random series of transient events conceivably with its origins in the superposition of a set of more elementary stochastic processes that could include microtubule depolymerization, actin polymerization, and possibly diffusion. Although the real nature of the control mechanism remains obscure, certain properties of pooled stochastic processes suggest that a reciprocal connection between microtubule fragmentation and the assembly of actin-containing pseudopodal structures and contractile elements--processes that may exhibit reciprocal requirements for calcium--might provide a hypothetical basis for a rate-limiting step.

Adenosine Diphosphate↗
PubMed1985

Evidence that the platelet plasma membrane does not contain a (Ca2+ + Mg2+)-dependent ATPase.

The present study was designed to determine the subcellular distribution of the platelet (Ca2+ + Mg2+)-ATPase. Human platelets were surface labeled by the periodate-boro[3H]hydride method. Plasma membrane vesicles were then isolated to a purity of approx. 90% by a procedure utilizing wheat germ agglutinin affinity chromatography. These membranes were found to be 2.6-fold enriched in surface glycoproteins compared to an unfractionated vesicle fraction and almost 7-fold enriched compared to intact platelets. In contrast, the isolated plasma membranes showed a decreased specific activity of the (Ca2+ + Mg2+)-ATPase compared to the unfractionated vesicle fraction. This decrease in specific activity was found to be similar to that of an endoplasmic reticulum marker, glucose-6-phosphatase, and to that of a platelet inner membrane marker, phospholipase A2. We conclude, therefore, that the (Ca2+ + Mg2+)-ATPase is not located in the platelet plasma membrane but is restricted to membranes of intracellular origin.

Blood Platelets↗
PubMed1984

Distribution of platelet glycoproteins and phosphoproteins in hydrophobic and hydrophilic phases in Triton X-114 phase partition.

Platelets, either unlabelled, surface-labelled by the periodate NaB3H4 method or metabolically labelled with 32P were solubilized in Triton X-114 and partitioned into aqueous and detergent phases. The phases were analysed by two-dimensional polyacrylamide gel electrophoresis followed by silver-staining, fluorography or indirect autoradiography. Each of the phases contains a distinct set of proteins. The surface-labelled glycoproteins partition into the hydrophobic phase with the notable exceptions of glycoproteins Ib and GP17(5.8-6.5) and minor amounts of a few others. The phosphoproteins which undergo increased phosphorylation on platelet activation in general separate in the hydrophobic phase, while higher molecular weight phosphoproteins were principally in the hydrophilic phase. This method might be used as a first step in purifying many platelet components.

Blood Platelets↗
PubMed1984

Human blood platelet secretion: optical multichannel analyzer measurements using acriflavine as a release indicator.

Blood platelets preloaded with the fluorescent amine acriflavine release the trapped fluorophore after stimulation with thrombin or the divalent cation ionophore A23187. Release was detected by an increase in acriflavine fluorescence, which is otherwise strongly quenched in the platelet, by using an optical multichannel analyzer to monitor the spectral and temporal reaction parameters. The secretion of [14C]serotonin and acriflavine is well correlated, suggesting that acriflavine, like serotonin and the closely related fluorescent drugs mepacrine and acridine orange, is accumulated in and released from platelet dense bodies. Acriflavine secretion at 37 degrees C in the absence of external calcium is characterized by a short delay, followed by a rapid biphasic increase in fluorescence that implies at least a three-stage secretory process. For saturating levels of thrombin the delay was 1.5 s and release was 90% complete within 6-7 s. The delay could not be shortened by prestimulation under conditions that induce shape changes but not release, i.e., with ADP, arachidonic acid, or low levels of thrombin or A23187. Acriflavine secretion induced by A23187 was similar but less effective; the reaction was slower, the yield was smaller, and, in contrast to thrombin, the longer lag period could be significantly shortened by prestimulation.

Acriflavine↗
PubMed1984

Platelet activation studied by fluorescence polarization.

Platelet activation was elevated by changes in the fluorescence anisotropy of the sulfhydryl-reactive fluorescent probe, (5-[2-(iodoacetyl) aminacetyl]aminonaphthalene-1-sulfonic acid. The membrane-permeable fluorophore was shown to bind to a multitude of cytoplasmic and membrane proteins. Platelets were stimulated by addition of thrombin, arachidonic acid or ADP under conditions that did not induce aggregation. A sudden increase in the fluorescence anisotropy, r of moderate degree (25-33%) occurred during the first 60 s after exposure of platelets to the aggregating agents and was sustained during the entire period of observation (15-18 min). Phenylmethylsulfonyl thrombin was unable to produce these changes in fluorescence anisotropy. Preincubation of platelets with colchicine reduced r within 30-60 s after platelets were exposed to thrombin. These findings are interpreted as an indication of a general decrease in the 'motional freedom' of the fluorophores and indirectly their ligand molecules.

Adenosine Diphosphate↗
PubMed1984

Optical shape change analysis in stirred and unstirred human platelet suspensions. A comparison of aggregometric and stopped-flow turbidimetric measurements.

To monitor the discoidity of platelets in an aggregometer, a relative, stirring-dependent change in extinction (delta ES) is defined. delta ES is large in highly discoid and small in ADP-activated platelets. The platelet shape change, as monitored by the changes in delta ES, not only precedes ADP-induced aggregation but also outlasts disaggregation. In unstirred samples, ADP induces insignificant changes in extinction in the aggregometer but biphasic changes in extinction in a "zero degree" stopped-flow turbidimeter. This discrepancy apparently arises from differences in the amount of scattered light collected by the optical systems and from wavelength-dependent differences in sensitivity. Similar progress curves were observed in both instruments for the biphasic changes in extinction which accompany the release reaction induced by thrombin or concanavalin A in pre-sphered platelets. The aggregometer is advantageous for monitoring rheooptical effects of the asymmetric platelets while the stopped-flow laser turbidimeter is superior in quantifying the changes in extinction according to the light scattering theory.

Adenosine Diphosphate↗
PubMed1984

Fluorescence anisotropy changes in platelet membranes during activation.

Dynamic changes in platelet membrane components were evaluated by two fluorescent probes, the anion channel blocker 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) and the membrane-impermeant stachyose derivative of pyrenebutyryl hydrazide (SPBH). Fluorescence anisotropy, r, was measured in intact platelets treated with either fluorophore. Activation of platelets by thrombin, arachidonic acid, and ADP under nonaggregating conditions increased the anisotropy values of DIDS within 60-120 s. A slow return to base-line values occurred after 8-10 min. Thrombin produced an initial transient reduction of r during the first 60 s. Its effect was specific as inactivated enzyme did not induce any changes. The latter could also be prevented by omitting Ca2+ from the platelet suspension. Treatment of platelets with SPBH, a fluorophore inserted into the lipid leaflet of membranes, revealed an activation-induced increase of its fluorescence anisotropy during the first 120 s. It was followed by a 6-8 min lasting decline of r when thrombin and ADP were the stimulants. Preexposure of platelets to colchicine did not change significantly the fluorescence anisotropy pattern of either fluorophore, but cytochalasin B inhibited such changes almost completely. The findings are interpreted as demonstrating greater motional freedom in the lipid bilayer but a decrease in this parameter in membrane proteins upon stimulation of platelets.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1984

Sequential determination of circulating immune complexes during progressive and regressive phases of mouse mastocytoma.

Circulating immune complexes were determined by 125I-C1q-binding and Raji cell-binding sequentially during distinct phases of progression and regression of a weakly immunogenic murine tumour. No increase in levels of circulating immune complexes was found at any time during tumour development, although reference complexes formed between tumour cell membrane antigens and a murine histocompatibility antigen-directed alloantibody were easily detected by both tests. These findings parallel the absence of humoral antibody during tumour development but are in some contradiction to the pronounced B cell proliferation which was observed in this tumour model. The results add evidence for a more critical evaluation of the role which levels of circulating immune complexes might play in tumour diagnosis and prognosis.

Animals↗
PubMed1983

Improvement of the periodate-borohydride surface-labeling method for human blood platelets.

The periodate/sodium boro[3H]hydride ([3H]-NaBH4) method is extensively used for the specific labeling of cell surface glycoproteins. Reduction with tritiated borohydride is also used in other surface-labeling techniques, the neuraminidase/galactose oxidase/[3H]-NaBH4 method (specific for terminal galactose and N-acetyl-galactosamine residues) and the pyridoxal phosphate/[3H]-NaBH4 method (specific for protein). By modification of the reaction conditions during the periodate-oxidation and borohydride-reduction, the ratio of the incorporated to the total added radioactivity could be increased by a factor of 50, while the specific activity of the labeled material was twice as high as in the original method. Alternatively, by another modification, the specific activity of the labeled material could be increased about 10-fold. The influence of the most important parameters was investigated in detail. Sodium dodecyl sulfate gel electrophoresis and fluorography demonstrate that the labeling pattern of the membrane glycoproteins is the same as with the conventional method.

Blood Platelets↗
PubMed1983

Stimulation of different pathways of T-cell functions by syngeneic tumor cells and soluble membrane proteins.

Cells from the draining lymph nodes of DBA/2 mice bearing syngeneic intradermal P-815 tumors represent an excellent responding cell population for secondary stimulation in vitro, despite the virtual absence of response by spleen cells of the same animals. Cytotoxicity is a result of stimulation with intact mitomycin-treated tumor cells. Isolated tumor cell membranes, in the form of small vesicles, stimulated cytotoxicity to a very limited extent and inhibited the development of cytotoxic T lymphocytes over a wide range of concentrations. Membrane proteins solubilized with deoxycholate and papain had only a suppressive effect. Soluble proteins exerted their effect during the induction of cytotoxic T lymphocytes and were ineffective when present during the effector phase of cytotoxic T lymphocytes. The suppressive capacity was shown to reside in a cell population which was sensitive to treatment with monoclonal Lyt-2.1 antibody and complement but not with Lyt-1.1 antibody. This phenotype was compatible with a specific rather than a promiscuous type of suppressor effector cell.

Animals↗