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Biomedical subjects

J N George

Publications and source records attributed to J N George.

At least 19 recordsLinked to original sources
PubMed1992

Changes in platelet membrane glycoproteins during blood bank storage.

The literature on membrane glycoprotein changes occurring during platelet storage is reviewed. Technical problems which may have biased reports are clarified, and the consensus of current studies is established. The membrane alterations that occur during blood bank storage are reminiscent of the surface changes which result from platelet secretion: an increased surface concentration of GP IIb-IIIa and GMP-140, and the appearance of platelet membrane microparticles in the supernatant plasma. The clinical importance of these changes is unknown. Future directions for research on the structure and function of stored platelets are discussed.

Blood Platelets↗
PubMed1991

Platelet IgG: measurement, interpretation, and clinical significance.

Platelet IgG has been studied primarily to develop an aid for clinical diagnosis--a search that has proven elusive (48). However, studies of the interaction of IgG and other plasma proteins with platelets have provided new insight into the intracellular pathways for the acquisition of proteins into regulated secretory granules. From a clinical perspective, it is important not to make the assumption that all platelet IgG is antiplatelet antibody. Data on platelet IgG must be distinguished between measurements of alpha-granule IgG, which comprises almost all of the total platelet IgG content, and platelet surface IgG, representing less than 1 percent of total platelet IgG. alpha-granule IgG does not appear to be antiplatelet antibody; platelet surface IgG is likely to be at least partly antiplatelet antibody. The total platelet (alpha-granule) content of IgG mirrors the concentration and composition of plasma IgG. It is increased in patients with hypergammaglobulinemia of any cause and absent in agammaglobulinemia. The platelet alpha-granule content of IgG, IgA, and albumin is proportional to their concentrations in plasma, and the platelet content of IgG, IgA and albumin also reflects their plasma concentration over a wide range of plasma abnormalities. These observations suggest that platelets acquire IgG, IgA, and albumin by fluid phase endocytosis. Total platelet IgG is consistently increased in patients with ITP and is also commonly increased in patients with thrombocytopenia due to increased platelet destruction of apparently nonimmune mechanisms. The total platelet IgG content does not appear to be increased in patients who have thrombocytopenia due to marrow failure. Therefore, the measurement of total platelet IgG may be analogous to the reticulocyte count, which is increased in patients who have accelerated erythropoiesis caused by either immune or nonimmune hemolytic anemia. The increased concentration of total platelet IgG in patients with ITP is parallel to the appearance of larger platelets produced during the stress of increased thrombopoietic stimulation. Therefore, determinations of mean platelet volume may yield clinical information equivalent to the measurement of total platelet IgG. The concentration of platelet surface IgG is greatly increased in patients with ITP, and concentrations are highest in patients with the most acute ITP. Platelet surface IgG may represent antiplatelet antibody in these patients. However, for unknown reasons, modest elevations of platelet surface IgG are found in many patients with nonimmune thrombocytopenia.(ABSTRACT TRUNCATED AT 400 WORDS)

Autoantibodies↗
PubMed1990

Solubilization and partial purification of the rabbit parotid Na/K/Cl-dependent bumetanide binding site.

We demonstrate that the high affinity bumetanide binding site of the rabbit parotid acinar cell can be extracted from a basolateral membrane fraction using relatively low concentrations (0.07%, wt/vol; 1 mg membrane protein/ml) of the nonionic detergent Triton X-100. This extracted site cannot be sedimented by ultracentrifugation at 100,000 x g x 1 hr. Bumetanide binding to this site retains the ionic characteristics of bumetanide binding to native membranes but shows a fivefold increase in binding affinity (Kd = 0.57 +/- 0.15 microM vs. Kd = 3.3 +/- 0.7 microM for native membranes). Inactivation of the extracted bumetanide binding site observed at detergent/protein ratios greater than 1 can be prevented or (partially) reversed by the addition of exogenous lipid (0.2% soybean phosphatidylcholine). When the 0.07% Triton extract is fractionated by sucrose density gradient centrifugation in 0.24% Triton X-100, 0.2% exogenous lipid and 200 mM salt, the high affinity bumetanide binding site sediments as a single band with S20,w = 8.8 +/- 0.8 S. This corresponds to a molecular weight approximately 200 kDa for the bumetanide binding protein-detergent-lipid complex and represents a sevenfold purification of this site relative to the starting membrane fraction. In contrast to previous attempts to purify Na/K/Cl cotransport proteins and their associated bumetanide binding sites, the present method avoids harsh detergent treatment as well as direct covalent modification (inactivation) of the transporter itself. As a consequence, one can follow the still active protein through a series of extraction and purification steps by directly monitoring its bumetanide binding properties.

Animals↗
PubMed1989

Human platelets contain mRNA transcripts for platelet factor 4 and actin.

RNAs from a number of cells, including platelets, were analyzed by Northern blotting for the presence of transcripts to four platelet proteins-actin, thrombospondin, fibronectin, and platelet factor 4. RNA from platelets contains considerable amounts of mRNA for platelet factor 4, easily detectable mRNA for actin, and traces of mRNA for thrombospondin. mRNA for platelet factor 4 was not detected in human lymphocytes or in any of 5 human cell lines.

Actins↗
PubMed1989

Inactivation of the rabbit parotid Na/K/Cl cotransporter by N-ethylmaleimide.

The inactivation of the rabbit parotid Na/K/Cl cotransporter by the irreversible sulfhydryl reagent N-ethylmaleimide (NEM) is studied by monitoring its effect on high affinity bumetanide binding to the carrier. NEM reduces the number of bumetanide binding sites with no significant change in the affinity of those remaining. NEM also reduces KCl-dependent 22Na flux via the cotransporter by the same factor as the reduction in bumetanide binding sites. Both bumetanide and its analogue furosemide can protect against the effect of NEM. The concentration range over which this protection occurs is in good agreement with affinities of these two compounds for the high affinity bumetanide binding site (2.6 and 8.5 microM, respectively), indicating an association of this site with the site of action of NEM. Also consistent with this hypothesis are the observations that (i) sodium and potassium, both of which are required for high affinity bumetanide binding, increase the rate of inactivation of binding by NEM and (ii) chloride, at concentrations previously shown to competitively inhibit bumetanide binding, protects the cotransporter against NEM. The effects of NEM on bumetanide binding are mimicked by another highly specific sulfhydryl reagent, methyl methanethiolsulfonate. The apparent rate constant for inactivation of high affinity bumetanide binding by NEM is a hyperbolic function of NEM concentration consistent with a model in which the inactivation reaction is first order in [NEM] and proceeds through an intermediate adsorptive complex. The data indicate that the presence of a reduced sulfhydryl group at or closely related to the bumetanide binding site is essential for the operation of the parotid Na/K/Cl cotransporter.

Animals↗
PubMed1987

Gray platelet syndrome. Demonstration of alpha granule membranes that can fuse with the cell surface.

Platelets from patients with the gray platelet syndrome have decreased recognizable alpha granules and are markedly deficient in some alpha-granule secretory proteins. Using immunocytochemical techniques with antibodies to an alpha-granule membrane protein, GMP-140, we identified the membranes of intracellular vesicles in gray platelets as alpha-granule membranes. Gray platelets contained normal amounts of GMP-140 as measured by electroimmunoassay. The activation of gray platelets with thrombin caused GMP-140 to be redistributed to the plasma membrane surface, as in normal platelets. In agreement with previous studies, an endogenously synthesized secretory protein, platelet factor 4, was undetectable in gray platelets. However, the alpha-granule proteins albumin and IgG, which are thought to be derived from endocytosis of plasma proteins into megakaryocytes, were present in substantial quantities and were secreted efficiently from gray platelets. Therefore, the fundamental defect in the gray platelet syndrome may be in the targeting of endogenously synthesized secretory proteins to developing alpha granules in megakaryocytes.

Blood Platelet Disorders↗
PubMed1987

Heterogeneity of platelet secretion in response to thrombin demonstrated by fluorescence flow cytometry.

Platelet membrane changes that accompany in vivo activation may be difficult to detect if only a small fraction of circulating platelets has undergone secretion. This study describes an approach to that problem by using a method to measure the number of molecules of fluorescein-labeled antibody bound to individual platelets by flow cytometry. The platelet response to different concentrations of thrombin was determined by measuring the binding of a monoclonal antibody (S12) to GMP-140, an alpha-granule membrane protein that becomes exposed on the platelet surface during alpha-granule secretion. Unstimulated platelets bound a mean of 1,120 molecules of S12 per cell, and 93% of platelets bound less than 2,000 molecules. Platelet stimulation by 0.25 U/mL thrombin caused maximum S12 binding with a mean of 7,529 molecules per cell. Even at low concentrations of thrombin (0.025 U/mL), 5% of platelets were maximally activated, binding over 7,000 molecules of S12 per cell. Conversely, at 0.25 U/mL thrombin, 13% of platelets continued to bind less than 2,000 molecules of S12 per cell. A mixture of as little as 5% thrombin-activated platelets with unstimulated platelets could be detected by this method. Therefore flow cytometry offers an important tool for investigating patients who may have circulating activated platelets as part of a disorder predisposing to thrombosis or hemorrhage.

Antibodies, Monoclonal↗
PubMed1986

Phosphorylation of thymus myosin increases its apparent affinity for actin but not its maximum adenosinetriphosphatase rate.

Vertebrate nonmuscle myosins contain two phosphorylatable light chains. The maximum rate, Vmax, of the actin-activated adenosinetriphosphatase (ATPase) of unphosphorylated calf thymus myosin was found to be about 100 nmol/(min X mg), the same as that of thymus myosin with two phosphorylated light chains. However, the Kapp (actin concentration required to achieve 1/2 Vmax) of the unphosphorylated myosin was 15-20-fold greater than that of the phosphorylated myosin. When actin complexed with either skeletal muscle tropomyosin or calf thymus tropomyosin was used, the values for Vmax were about the same as those obtained with F-actin. In the presence of skeletal muscle tropomyosin, the Kapp of the unphosphorylated myosin was only 2-3-fold greater than that of the phosphorylated myosin, and in the presence of thymus tropomyosin, there was about a 5-fold difference in their Kapp values. Thus, light chain phosphorylation regulates the actin-activated ATPase of thymus myosin not by increasing Vmax but rather by decreasing the Kapp of this myosin for actin. These rather small differences in Kapp suggest that other proteins may be involved in the regulation of the actin-activated ATPase of thymus myosin. Regulated actin (actin plus skeletal muscle troponin-tropomyosin) was used to examine possible effects of thin-filament regulatory proteins. In the presence of calcium, phosphorylation caused only a slight increase in Vmax and a 2-fold decrease in Kapp of the regulated actin-activated ATPase of thymus myosin.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1986

Platelet surface glycoproteins. Studies on resting and activated platelets and platelet membrane microparticles in normal subjects, and observations in patients during adult respiratory distress syndrome and cardiac surgery.

The accurate definition of surface glycoprotein abnormalities in circulating platelets may provide better understanding of bleeding and thrombotic disorders. Platelet surface glycoproteins were measured on intact platelets in whole blood and platelet membrane microparticles were assayed in cell-free plasma using 125I-monoclonal antibodies. The glycoproteins (GP) studied were: GP Ib and GP IIb-IIIa, two of the major intrinsic plasma membrane glycoproteins; GMP-140, an alpha-granule membrane glycoprotein that becomes exposed on the platelet surface following secretion; and thrombospondin (TSP), an alpha-granule secreted glycoprotein that rebinds to the platelet surface. Thrombin-induced secretion in normal platelets caused the appearance of GMP-140 and TSP on the platelet surface, increased exposure of GP IIb-IIIa, and decreased antibody binding to GP Ib. Patients with adult respiratory distress syndrome had an increased concentration of GMP-140 and TSP on the surface of their platelets, demonstrating in vivo platelet secretion, but had no increase of platelet microparticles in their plasma. In contrast, patients after cardiac surgery with cardiopulmonary bypass demonstrated changes consistent with membrane fragmentation without secretion: a decreased platelet surface concentration of GP Ib and GP IIb with no increase of GMP-140 and TSP, and an increased plasma concentration of platelet membrane microparticles. These methods will help to define acquired abnormalities of platelet surface glycoproteins.

Antibody Specificity↗
PubMed1985

Platelet activation and secretion associated with emotional stress.

Platelets are believed to play a role in the pathogenesis of atherosclerosis and of the vascular obstruction that causes the acute complications of coronary artery disease. Since specific behavioral patterns appear to be related to the development of coronary artery disease and since emotional stress may predispose an individual to acute cardiovascular ischemia, it was hypothesized that platelet activation by catecholamines might be involved in these events. To study emotional stress, plasma samples were obtained from 61 senior medical residents immediately before they were to speak in public. There were significant increases in the plasma concentrations of the platelet-secreted proteins platelet factor 4 and beta-thromboglobulin and epinephrine and norepinephrine immediately before speaking, which demonstrates that platelet activation and secretion occur in association with this type of emotional stress. Four trials were carried out to study the mechanism for this observed platelet secretion: (1) phenoxybenzamine, (2) propranolol, (3) 650 mg aspirin, and (4) 80 mg aspirin were given several hours before the public speaking engagement. Neither phenoxybenzamine nor propranolol in doses that blocked the hemodynamic effects of alpha 1- and beta 1-adrenergic stimulation modified platelet secretion. Aspirin also did not block platelet secretion, which suggests that platelets were not being stimulated through a cyclooxygenase-dependent pathway. This study provides direct evidence of platelet secretion in vivo in association with emotional stress, and underscores the potential importance of platelet activation and secretion in the acute events that occur in patients with vascular disease.

Adult↗
PubMed1985

Immunoglobulin G is a platelet alpha granule-secreted protein.

It has been known for 27 yr that blood platelets contain IgG, yet its subcellular location and significance have never been clearly determined. In these studies, the location of IgG within human platelets was investigated by immunocytochemical techniques and by the response of platelet IgG to agents that cause platelet secretion. Using frozen thin-sections of platelets and an immunogold probe, IgG was located within the alpha-granules. Thrombin stimulation caused parallel secretion of platelet IgG and two known alpha-granule proteins, platelet factor 4 and beta-thromboglobulin, beginning at 0.02 U/ml and reaching 100% at 0.5 U/ml. Thrombin-induced secretion of all three proteins was inhibited by prostaglandin E1 and dibutyryl-cyclic AMP. Calcium ionophore A23187 also caused parallel secretion of all three proteins, whereas ADP caused virtually no secretion of any of the three. From these data and a review of the literature, we hypothesize that plasma IgG is taken up by megakaryocytes and delivered to the alpha-granules, where it is stored for later secretion by mature platelets.

Adenosine Diphosphate↗
PubMed1978

Further characterization of calcium-accumulating vesicles from human blood platelets.

Human blood platelets are capable of removing Ca2+ from the cytoplasm by means of an active, ATP-dependent and cyclic AMP-stimulated transport system. Calcium-accumulating vesicles are obtained by sonicating platelets. On density gradient centrifugation, this activity is found in the heavier of two membrane fractions. Concentrated in this fraction are also the Ca2+-stimulated Mg2+-ATPase and glucose-6-phosphatase, believed to be a marker for internal membrane systems. When the isolated vesicles are loaded with Ca2+, a third band separates from the two vesicular fractions in the density gradient. This band C contains virtually all the Ca2+-accumulating activity. Evidence that this activity is due to an active uptake and not to surface binding or adsorption is presented. Whereas electron microscopy does not reveal striking differences between active and inactive fractions, differences in protein composition are revealed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Furthermore, this band contains an enzyme system which converts arachidonic acid to malondialdehyde and therefore this fraction must be the site of prostaglandin synthesis. Membranes prepared by loading platelets with glycerol, followed by osmotic lysis are unable to accumulate calcium. In sodium dodecyl sulphate-polyacrylamide gel electrophoresis such membranes show significant differences in their protein pattern as compared to the actively Ca2+-accumulating vesicular membranes of band C. All preparations with Ca2+-accumulating activity also contain markers for plasma membranes and the question whether this activity is due exclusively to an intracellular structural element equivalent to the sarcoplasmic reticulum of muscle or whether an "extrusion pump" expelling Ca2+ to the outside of the cell is also involved, cannot yet be ;nswered.

Biological Transport, Active↗
PubMed1978

Platelet behavior and aging in the circulation.

This review of the development of knowledge concerning the fate and function of circulating platelets and our recent experiments with a radioisotope label for platelet surface glycoproteins support the hypothesis that platelets are continualy involved in reversible contact interactions for the support of endothelial integrity. These encounters result in the loss of pieces of the platelet surface membrane and could contribute to the structural and functional changes of platelet senescence. The concepts of continuous involvement of platelets in hemostatic encounters and a fixed circulating lifespan are not mutually exclusive phenomena: platelet hemostatic encounters could preferentially involve younger platelets and be mostly reversible while the metabolic machinery which determines platelet survival deteriorates in a manner unrelated to these platelet-surface interactions. Obviously the nature of platelet behavior and aging in the circulation requires much more precise definition.

Animals↗
PubMed1978

Studies on platelet plasma membranes. III. Membrane glycoprotein loss from circulating platelets in rabbits: inhibition by aspirin-dipyridamole and acceleration by thrombin.

DD125ISA, which binds to platelet surface glycoproteins, is lost more rapidly from double-labeled circulating rabbit platelets than is 51Cr, a label of platelet cytoplasm. Treatment of rabbits with aspirin and dipyridamole to diminish platelet function did not affect 51Cr survival but inhibited DD125ISA loss so that it disappeared from the circulation at the same rate as 51Cr . Intravenous thrombin infusion increased DD125ISA loss relative to 51Cr loss. Therefore surface glycoprotein loss was prevented by agents which inhibit platelet function and was accelerated by intravascular coagulation. These observations support the hypothesis that platelet surface glycoprotein loss is a continuous process in the normal circulation which may occur during reversible contact interactions in the process of hemostasis and thrombosis.

Animals↗