Biomedical subjects

D R Phillips

Publications and source records attributed to D R Phillips.

At least 181 records · Page 10Linked to original sources

PubMed1987

Choice in residential care: myths and realities.

This paper examines the proposition that the welfare policies enacted and legitimized in the name of 'choice' have the effect of increasing the effective choice of service by consumers of welfare. The example of residential care of the elderly is used to illustrate the ways in which privatisation has influenced choices of care for elderly people in Devon. The paper reviews empirical material from two survey of private homes in Devon in order to throw light on the 'myths' and realities of choice in residential accommodation and in other welfare provision available to the elderly in Devon. The paper identifies as the key debate the question of priorities: whether resources should be concentrated on providing suitable residential places for the increasing number of the very disabled elderly or used to increase the alternatives for the larger, more able groups. The latter option can perhaps be reasonably achieved through market mechanisms, whilst the former is more likely to require the specialised skills of social workers and other caring professionals. Although it is possible to argue that the growth in numbers of private old people's homes represents an increase in choice for the elderly, on closer examination this appears too simple an assertion. The expansion of the private sector has not necessarily expanded the range of alternative forms of care. In particular, there seems a major flaw in current policies by which the operation of the DHSS subsidy increases the availability of residential rather than domiciliary care.

Aged↗

PubMed1987

The platelet fibrinogen receptor: an immunogold-surface replica study of agonist-induced ligand binding and receptor clustering.

Platelet aggregation requires the binding of fibrinogen to its receptor, a heterodimer consisting of the plasma-membrane glycoproteins (GP) IIb and IIIa. Although the GPIIb-IIIa complex is present on the surface of unstimulated platelets, it binds fibrinogen only after platelet activation. We have used an immunogold-surface replica technique to study the distribution of GPIIb-IIIa and bound fibrinogen over broad areas of surface membranes in unstimulated, as well as thrombin-activated and ADP-activated human platelets. We found that the immunogold-labeled GPIIb-IIIa was monodispersed over the surface of unstimulated platelets, although the cell surface lacked immunoreactive fibrinogen. On thrombin-stimulated platelets, approximately 65% of the GPIIb-IIIa molecules were in clusters within the plane of the membrane. Fibrinogen, which had been released from the alpha-granules of these cells, bound to GPIIb-IIIa on the cell surface and was similarly clustered. To determine whether the receptors clustered before ligand binding, or as a consequence thereof, we studied the surface distribution of GPIIb-IIIa after stimulation with ADP, which causes activation of the fibrinogen receptor function of GPIIb-IIIa without inducing the release of fibrinogen. In the absence of added fibrinogen, the unoccupied, yet binding-competent receptors on ADP-stimulated platelets were monodispersed. The addition of fibrinogen caused the GPIIb-IIIa molecules to cluster on the cell surface. Clustering was also induced by the addition of the GPIIb-IIIa-binding domains of fibrinogen, namely the tetrapeptide Arg-Gly-Asp-Ser on the alpha-chain or the gamma-chain decapeptide gamma 402-411. These results show that receptor occupancy causes clustering of GPIIb-IIIa in activated platelets.

Adenosine Diphosphate↗

PubMed1987

The platelet membrane glycoprotein IIb/IIIa complex. Structure, function, and relationship to adhesive protein receptors in nucleated cells.

The GPIIb/IIIa complex functions as the aggregation site on the platelet membrane surface. This complex has been purified, characterized biochemically and morphologically, and reconstituted into phospholipid vesicles. Fibrinogen and fibronectin bind to reconstituted GPIIb/IIIa with many of the properties that characterize their binding to intact platelets. The GPIIb/IIIa complex appears to be a member of a widely distributed family of cell-surface glycoproteins that mediate cellular interactions. The terms cytoadhesins30 and integrins39 have been suggested for the members of this family of two-subunit molecules. The aminotermini of the alpha subunits of these molecules have been sequenced and appear to be homologous. Three beta subunits have been identified for this family of receptors, indicating that many alpha subunits have a common beta subunit. The three beta subunits have been sequenced, and there is about a 40 to 50% identity among their amino acid sequences. It thus appears that the receptors mediating cellular interactions have evolved from a common ancestral gene.

Amino Acid Sequence↗

PubMed1987

Spectrin is associated with membrane-bound actin filaments in platelets and is hydrolyzed by the Ca2+-dependent protease during platelet activation.

We recently showed that platelets contain submembranous actin filaments that are linked to glycoprotein (GP) Ib on the plasma membrane. In the present study, experiments were performed to determine whether spectrin was associated with these filaments. The membrane-bound filaments were isolated from Triton X-100 (Sigma, St Louis) lysates of unstimulated platelets by differential centrifugation. Platelet spectrin was detected immunologically by using antibodies against human brain and RBC spectrin. Immunoblots showed that platelet spectrin consisted of two polypeptides (mol wt 240,000 and 235,000) that were similar in apparent mol wt to those of the alpha and beta chains of brain spectrin but differed slightly from those of RBC spectrin (mol wt 240,000 and 220,000). Immunoprecipitation experiments identified platelet spectrin as two minor polypeptides migrating on sodium dodecyl sulfate (SDS)-polyacrylamide gels between actin-binding protein (mol wt 250,000) and the platelet polypeptide P235 (mol wt 235,000). Immunoblots of fractions isolated from Triton X-100-lysed platelets revealed that the alpha and beta chains of platelet spectrin were associated almost entirely with the actin filaments that were linked to the plasma membrane. Little spectrin was recovered in the Triton X-100-soluble fraction or with the actin filaments that were not membrane bound. During activation of platelets with thrombin or ionophore A23187, the alpha and beta chains of spectrin were hydrolyzed, generating a major degradation product of mol wt 160,000 and a minor one of mol wt 170,000. These two hydrolytic products were also generated in Triton X-100 lysates incubated in the presence of Ca2+ but were not produced when lysates were treated with leupeptin, ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), or N-ethylmaleimide, known inhibitors of the Ca2+-dependent protease. These experiments show that spectrin is a previously unidentified component of the membrane-bound actin filament network and that hydrolysis of spectrin by the Ca2+-dependent protease may regulate the interactions of the filaments during platelet activation.

Adult↗

PubMed1986

Kinetics and sequence specificity of drug-DNA interactions: an in vitro transcription assay.

An assay has been developed to detect the DNA sequence specificity of drug binding sites, and the kinetics of dissociation of drug from those sites, under conditions involving active transcription of the DNA. Specific transcriptional blockage sites were detected in the presence of actinomycin D and a bisintercalator, bis(anthracycline); the rate of RNA chain growth past the drug binding sites yields the rate of dissociation of drug from these sites. Rate constants for dissociation from the whole promoter fragment measured by the detergent sequestration method were found to be significantly faster than the rate determined for dissociation from the specific transcriptional blockage site. However, the absence of significant blockage at other drug binding sites implies much more rapid drug dissociation from those sites in the transcriptional complex. We conclude that transcriptional blockage results from a DNA sequence-dependent interaction of the drug-DNA complex with RNA polymerase; the sequences that are effective for blockage appear to be GpC for actinomycin and (CpA)3 for a bis(daunomycin) compound. Transcriptional inhibition may in general show greater sequence specificity than is exhibited by simple binding.

Base Sequence↗

PubMed1986

Fibronectin-binding properties of the purified platelet glycoprotein IIb-IIIa complex.

Fibronectin binds to specific receptors on the surface of washed, thrombin-activated platelets. Evidence suggests that these receptors are closely associated with the platelet glycoprotein IIb-IIIa complex (GP IIb-IIIa). To determine whether GP IIb-IIIa itself can form a platelet receptor for fibronectin, we used a filtration assay to examine the interaction of purified fibronectin with purified GP IIb-IIIa incorporated into phospholipid vesicles. 125I-Fibronectin binding to the phospholipid vesicles required the presence of incorporated GP IIb-IIIa and was specific, time-dependent, reversible, saturable, and divalent cation-dependent (Mg2+ greater than Ca2+). The dissociation constant for 125I-fibronectin binding to the GP IIb-IIIa-containing vesicles in the presence of 2 mM MgCl2 was 87 nM. Proteins or peptides that inhibit 125I-fibronectin binding to whole platelets also inhibited 125I-fibronectin binding to the GP IIb-IIIa vesicles. Thus, specific 125I-fibronectin binding was inhibited by excess unlabeled fibrinogen or fibronectin, the anti-GP IIb-IIIa monoclonal antibody 10E5, the decapeptide from the carboxyl terminus of the fibrinogen gamma-chain, and the tetrapeptide Arg-Gly-Asp-Ser from the cell-binding domain of fibronectin. In contrast to results obtained using whole platelets, unlabeled fibronectin inhibited 125I-fibronectin binding to the GP IIb-IIIa vesicles. These results show that 125I-fibronectin binds directly to purified GP IIb-IIIa with most of the previously reported properties of 125I-fibronectin binding to washed, thrombin-stimulated platelets. Thus, GP IIb-IIIa has the potential to function as a platelet receptor for fibronectin as well as for fibrinogen.

Amino Acid Sequence↗

PubMed1986

Primary health care in the Philippines: banking on the barangays?

Primary health care has been hailed by some countries as the only practical means of providing any form of health care for expanding populations in poor economies. This is particularly true in Third World countries where the cost explosion of technology-oriented health care has been a major problem in extending services. Therefore, the PHC package of education, nutrition, preventive medicine and treatment of the most common diseases and injuries is sometimes regarded as the most beneficial application of scarce resources. The Philippines claims to be one of the first (perhaps the first) countries to have adopted PHC as a national strategy for health care and, since 1981, impressive achievements have been attained in this sector by contrast with reversals in many other sectors of the economy. PHC has not challenged the pre-eminence of Metro-Manila in the provision of hospital and specialist facilities but it has extended some basic care particularly to rural regions of the country. This paper reviews the background to health care in the Philippines and it then examines the implementation of PHC in Negros Oriental, where PHC has taken on the additional feature of special use of indigenous materials and resources. The administrative, financial and legal bases and some geographical facets of PHC are highlighted in this province. The campaign relies heavily on local (barangay) initiatives and community participation, in part to minimise resources which have to be devoted to health in a very troubled national economy. In spite of local skills and enthusiasm, this arguably still involves the abrogation of a degree of government responsibility for health care. As a result, the Philippines strategy may be said to be "banking on the barangays."

Community Health Services↗

PubMed1986

Platelet glycoproteins IIb and IIIa: evidence for a family of immunologically and structurally related glycoproteins in mammalian cells.

Human and bovine cultured cell lines and circulating leukocytes were examined for the presence of surface proteins similar to platelet glycoproteins IIb (GPIIb) and IIIa (GPIIIa). Human endothelial cells, smooth muscle cells, and MG-63 fibroblast-like cells were found to have surface proteins that cross-reacted with platelet GPIIb and GPIIIa antibodies, existed as complexes, and had molecular weights similar to those of the corresponding platelet glycoproteins. Bovine endothelial cells and smooth muscle cells also expressed GPIIb- and GPIIIa-like surface proteins. Metabolic labeling studies with [35S]methionine demonstrated that the cultured cells synthesized these glycoproteins. The GPIIIa-like protein in human endothelial and smooth muscle cells had the same isoelectric point as platelet GPIIIa, whereas their GPIIb alpha-like protein was slightly more acidic than platelet GPIIb alpha (pI = 5.2-5.3 versus 5.5). Platelet and endothelial cell GPIIb alpha (but not GPIIIa) showed an increased electrophoretic mobility in Ca2+ -containing versus EDTA-containing gels, implying a Ca2+ -GPIIb alpha interaction. The amino acid sequence of the amino termini of platelet GPIIb alpha and GPIIb beta and of the alpha chains of the leukocyte LFA-1 and Mac-1 glycoprotein complexes had significant sequence homology. These data indicate that glycoproteins that have either immunological cross-reactivity or amino-terminal sequence homology with the platelet GPIIb-IIIa complex are widely distributed in human and non-human adherent cells and circulating leukocytes and suggest that these proteins may be the products of a large gene family whose expression is cell specific.

Amino Acid Sequence↗

PubMed1985

A method for purifying the platelet membrane glycoprotein IIb-IIIa complex.

A method has been developed for the rapid isolation of platelet membrane glycoproteins (GP) IIb and IIIa. This method produces an excellent yield and does not require the prior isolation of platelet membranes. Outdated platelets were washed and solubilized in Triton X-100. Concanavalin A affinity chromatography was used to purify a platelet glycoprotein fraction. The concanavalin A-retained glycoproteins were eluted and adsorbed with a heparin-Sepharose column to remove a major contaminant, thrombospondin. Sephacryl S-300 gel filtration was used as the final purification step to remove most fibrinogen and low-molecular-weight contaminants. Wheat germ agglutinin affinity chromatography was used to completely remove trace amounts of fibrinogen. The purified GP IIb and GP IIIa were analyzed by sucrose gradient sedimentation and found to consist of heterodimer complexes.

Blood Platelets↗

PubMed1985

Human and bovine endothelial cells synthesize membrane proteins similar to human platelet glycoproteins IIb and IIIa.

Human umbilical vein endothelial (HUVE) and bovine aortic endothelial (BAE) cells in culture were examined to determine whether membrane proteins similar to human platelet glycoproteins (GP) IIb and IIIa were present. The HUVE and BAE cells were either 125I-surface labeled or metabolically labeled. Triton X-100 lysates of labeled cells were immunoprecipitated with polyclonal antibodies prepared against purified human platelet GP IIb-IIIa complex. Two membrane proteins were detected on both HUVE (Mr = 130,000 and 110,000) and BAE (Mr = 135,000 and 105,000) cells, which were similar to human platelet GP IIb (Mr = 125,000) and GP IIIa (Mr = 108,000). The two membrane proteins from HUVE cells and the two from BAE cells cosedimented in sucrose gradients, indicating that they exist as a complex. Unlike the human platelet GP IIb-IIIa complex, the HUVE and BAE membrane protein complexes were not dissociated by chelation of Ca2+. Platelet GP IIb and GP IIIa and the related membrane proteins on both HUVE and BAE cells showed similar changes in electrophoretic mobility upon disulfide reduction. These data demonstrate that human and bovine endothelial cells synthesize membrane proteins that have properties similar to the platelet membrane GP IIb-IIIa complex.

Animals↗

PubMed1985

Calcium regulation of the platelet membrane glycoprotein IIb-IIIa complex.

Platelet membrane glycoproteins (GP) IIb and IIIa form a Ca2+-dependent, heterodimer complex (GP IIb-IIIa) in detergent solution. To determine whether these glycoproteins were complexed or dissociated in intact human platelets, 125I-labeled whole platelets were lysed with an EDTA/Triton X-100 buffer, which stabilized both the complexed and dissociated forms of GP IIb and GP IIIa. The percentage of the heterodimer complex present after lysis was determined by quantitating the sedimentation of GP IIIa in sucrose gradients. The distribution of GP IIIa in sucrose gradient fractions from unlabeled platelets was detected by incubation of 125I-concanavalin A with Western blots. It was determined that GP IIb and GP IIIa existed predominantly as the GP IIb-IIIa complex in either unstimulated (92% complexed) or thrombin-stimulated (90% complexed) platelets. The GP IIb-IIIa complex in unstimulated platelets could be reversibly dissociated by brief (up to 10-min) incubations at 37 degrees C with EDTA, while longer (30-min) incubations with EDTA resulted in irreversible polymerization of GP IIb and GP IIIa. The dissociation of the GP IIb-IIIa complex by EDTA in intact cells was temperature-dependent, and the critical extracellular Ca2+ concentration for dissociation was 10(-5) to 10(-6) M. These results demonstrate that GP IIb and GP IIIa exist as a Ca2+-dependent, heterodimer complex in intact platelets and that treatment of platelets with EDTA can have multiple effects on this complex.

Blood Platelets↗

PubMed1985

Reconstitution of the purified platelet fibrinogen receptor. Fibrinogen binding properties of the glycoprotein IIb-IIIa complex.

Several lines of evidence indicate that the platelet membrane glycoprotein IIb-IIIa complex (GP IIb-IIIa) is necessary for the expression of platelet fibrinogen receptors. The purpose of the present study was to determine whether purified GP IIb-IIIa retains the properties of the fibrinogen receptor on platelets. Glycoprotein IIb-IIIa was incorporated by detergent dialysis into phospholipid vesicles composed of 30% phosphatidylcholine and 70% phosphatidylserine. 125I-Fibrinogen binding to the GP IIb-IIIa vesicles, as measured by filtration, had many of the characteristics of 125I-fibrinogen binding to whole platelets or isolated platelet plasma membranes: binding was specific, saturable, reversible, time dependent, and Ca2+ dependent. The apparent dissociation constant for 125I-fibrinogen binding to GP IIb-IIIa vesicles was 15 nM, and the maximal binding capacity was 0.1 mol of 125I-fibrinogen/mol of GP IIb-IIIa. 125I-Fibrinogen binding was inhibited by amino sugars, the GP IIb and/or IIIa monoclonal antibody 10E5, and the decapeptide from the carboxyl terminus of the fibrinogen gamma chain. Furthermore, little or no 125I-fibrinogen bound to phospholipid vesicles lacking protein or containing proteins other than GP IIb-IIIa (i.e. bacteriorhodopsin, apolipoprotein A-I, or glycophorin). Also, other 125I-labeled plasma proteins (transferrin, orosomucoid) did not bind to the GP IIb-IIIa vesicles. These results demonstrate that GP IIb-IIIa contains the platelet fibrinogen receptor.

Amino Acids↗

PubMed1985

Structure of human platelet membrane glycoproteins IIb and IIIa as determined by electron microscopy.

The glycoprotein (GP) IIb-IIIa complex was isolated from human platelet membranes and examined for glycoprotein stoichiometry and morphology. To determine the ratio of glycoproteins in the complex, the isolated glycoproteins were solubilized with sodium dodecyl sulfate and separated by high-performance liquid chromatography. Quantitative amino acid analysis of individual glycoproteins showed that the ratio of GP IIb to GP IIIa in the Ca2+-dependent complex was 0.93:1. Morphology was determined by electron microscopy of rotary-shadowed and negatively stained specimens. Individual complexes consisted of two domains: an oblong head of approximately 8 X 10 nm with two rodlike tails extending approximately 14-17 nm from one side of the head. Treatment of the isolated complex with EDTA resulted in the appearance of a mixture of oblong and filamentous structures, which could be separated by a sucrose gradient sedimentation in Triton X-100. As seen by rotary and unidirectional shadowing, GP IIb was a compact structure, approximately 8 X 10 nm in size. Isolated GP IIIa was more heterogeneous but was most often observed in an elongated form, varying in length from 20 to 30 nm and in width from 2 to 3 nm. By comparing these structures to that of the heterodimer complex, it was determined that the oblong domain was GP IIb and the rodlike tails were GP IIIa. Each milligram of isolated GP IIb-IIIa complex bound 0.30 mg of [3H]Triton X-100, indicating that the glycoprotein complex contained limited hydrophobic domains. Upon removal of detergent, GP IIb-IIIa complexes formed aggregates that sedimented in sucrose gradients as a diffuse peak ranging from 14 to 32 s. Examination of these aggregates by electron microscopy showed that they were composed of clusters or "rosettes" of 2 to 20 or more of the GP IIb-IIIa complexes. The orientation of these rosettes was such that the tails were joined in the center, with the head portions directed away from the interacting tails. It thus appears that the primary hydrophobic domains of the GP IIb-IIIa complex exist at the tips of the GP IIIa tails. Because the GP IIb-IIIa complex is an intrinsic membrane glycoprotein, these findings indicate a potential membrane attachment site for the GP IIb-IIIa complexes.

Amino Acids↗

PubMed1985

Platelet membrane glycoprotein IIb-IIIa complex incorporated into phospholipid vesicles. Preparation and morphology.

Platelet membrane glycoproteins (GP) IIb and IIIa have been identified as platelet aggregation sites. These glycoproteins form a heterodimer complex (GP IIb-IIIa) in the presence of Ca2+. To study the morphology of this glycoprotein complex in membranes, we incorporated GP IIb-IIIa into artificial phospholipid vesicles using a detergent (octyl glucoside) dialysis procedure. Phosphatidylserine-enriched vesicles (70% phosphatidylserine, 30% phosphatidylcholine) incorporated approximately 90% of the GP IIb-IIIa as determined by sucrose flotation. Glycoprotein IIb-IIIa incorporation into the vesicles was unaffected by ionic strength, suggesting a hydrophobic interaction between the glycoprotein and the phospholipid. In both intact platelets or phospholipid vesicles, GP IIb was susceptible to neuraminidase hydrolysis, indicating that most of the glycoprotein complexes were oriented toward the outside of the platelets or vesicles. The morphology of GP IIb-IIIa in the phospholipid vesicles was observed by negative staining electron microscopy. Individual GP IIb-IIIa complexes appeared as spikes protruding as much as 20 nm from the vesicle surface. Each spike consisted of a GP IIb "head," which was distal to the vesicle and was supported by the GP IIIa "tails." The GP IIb-IIIa complex appeared to be attached to the vesicle membrane by the tips of the GP IIIa tails. Treatment of vesicles with EGTA dissociated the GP IIb-IIIa complex. The dissociated glycoproteins remained attached to the phospholipid vesicles, indicating that both GP IIb and GP IIIa contain membrane-attachment sites. These data suggest a possible structural arrangement of the GP IIb-IIIa complex in whole platelets.

Blood Platelets↗

PubMed1985

Identification of two proteins (actin-binding protein and P235) that are hydrolyzed by endogenous Ca2+-dependent protease during platelet aggregation.

Our previous research has shown that the Ca2+-dependent protease within platelets is activated when platelets aggregate, resulting in the production of three polypeptides (Mr = 200,000, 100,000, and 91,000). We have now shown that these three polypeptides arise from the hydrolysis of actin-binding protein. An antibody against actin-binding protein raised in rabbits was shown to be specific for actin-binding protein on immunoblots of total platelet proteins. This antibody reacted with additional polypeptides of Mr = 200,000, 100,000, and 91,000 on immunoblots of the proteins of thrombin-activated platelets. Actin-binding protein was purified from fresh, human platelet concentrate and hydrolyzed with platelet-derived Ca2+-dependent protease; hydrolysis resulted in the appearance of three polypeptides with molecular weights and isoelectric points identical to those of the three polypeptides generated within intact, aggregating platelets. Production of these polypeptides was inhibited by leupeptin and by the chelation of Ca2+. Hydrolysis of actin-binding protein was observed at micromolar Ca2+ concentrations, demonstrating that the level of Ca2+ in aggregated platelets is sufficient to account for the hydrolysis of actin-binding protein by the Ca2+-dependent protease. P235 was also purified and tested for its susceptibility to the protease. It was hydrolyzed by the Ca2+-dependent protease, and two polypeptides (Mr = 200,000 and 46,000) were produced. Antibodies against P235 raised in rabbits reacted only with P235 on immunoblots of total platelet proteins. These antibodies also reacted with polypeptides of Mr = 200,000 and 46,000 on immunoblots of thrombin-activated platelets. These data show that both actin-binding protein and P235 are cleaved during thrombin-induced platelet aggregation and suggest that the activation of the Ca2+-dependent protease may permit reorganization of the platelet cytoskeleton in aggregating platelets.

Antibody Specificity↗

PubMed1985

The threshold growth response of Lactobacillus casei to 5-methyl-tetrahydrofolic acid: implications for folate assays.

The comparative and absolute growth response of Lactobacillus casei was measured nephelometrically for time periods of 17-23 h in the microbiological assay of folic acid and 5-methyl-tetrahydrofolic acid at concentrations of 0-8 ng/10 ml basal medium at pH 6.8. At concentrations of 0-1 ng/10 ml the comparative growth response to 5-methyl-tetrahydrofolic acid was markedly depressed whereas growth was the same at 2 ng/10 ml and above. Comparative growth was unaffected by the length of assay incubation, depressed growth being due to differences in log-phase growth rates with the rate-plot for 5-methyl-tetrahydrofolic acid being sigmoidal and for folic acid being a rectangular hyperbola with linearity only in the 0-1 ng/10 ml range. The reciprocal rate-plot for folic acid was linear whereas that for 5-methyl-tetrahydrofolic acid was coincidental only in part, giving rise to the same estimate of maximum velocity and substrate concentration for half-maximum velocity, with the exhibition of a strong threshold at low concentration. A previous observation (Phillips & Wright, 1982) that the L. casei growth response to 5-methyl-tetrahydrofolic acid may be significantly less than that to folic acid is confirmed as is the long-established view that the response to both folates may be equal. In the light of current knowledge regarding folate-binding, transport and metabolism by L. casei, it is argued that the intracellular oxidation of 5-methyl-tetrahydrofolic acid to 5, 10-methylene-tetrahydrofolic acid is a rate-limiting step at low substrate concentrations, subsequently giving rise to a threshold growth response peculiar to 5-methyl-tetrahydrofolic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Biological Assay↗

PubMed1985

Organization of the cytoskeleton in resting, discoid platelets: preservation of actin filaments by a modified fixation that prevents osmium damage.

This study evaluates the structural organization of the cytoskeleton within unactivated, discoid platelets. Previously, such studies have been difficult to interpret because of the ease with which platelets are stimulated, the sensitivity of actin filaments to cell extraction buffers, and the general problem of preserving actin filaments with conventional fixatives, compounded by the density of the cytoplasm in the platelet. In this study we have employed a new fixative containing lysine, which protects actin filaments against damage during fixation and thin-section processing. We used thick (0.25-micron) sections and conventional thin sections of extracted cells (fixed and lysed simultaneously by the addition of 1% Triton X-100 to the initial fixative) as well as thin sections of whole cells to examine three preparations of human platelets: discoid platelets washed by sedimentation; discoid platelets isolated by gel filtration; and circulating platelets collected by dripping blood directly from a vein into fixative. In all of these preparations, long, interwoven actin filaments were observed within the platelet and were particularly concentrated beneath the plasma membrane. These filaments appeared to be linked at irregular intervals to the membrane and to each other via short, approximately 20- to 50-nm-long cross-links of variable width. Although most filaments were outside the circumferential band of microtubules and the cisternae of the open canalicular system, individual filaments dipped down into the cytoplasm and were found between the microtubules and in association with other membranes. The ease with which single actin filaments can be seen in the dense cytoplasm of the human platelet after lysine/aldehyde fixation suggests the great potential of this new fixative for other cells.

Actin Cytoskeleton↗

PubMed1985

Monoclonal antibodies to human platelet glycoprotein IIb beta that initiate distinct platelet responses.

Hybridomas secreting monoclonal antibodies (MoAbs) to human platelet membrane glycoprotein IIb (GPIIb) were prepared by fusing cells of a mouse myeloma line to spleen cells from a BALB/c mouse immunized with purified GPIIb. Six of the hybridomas secreted MoAbs that recognized epitopes on the 23,000-dalton, disulfide-linked subunit of GPIIb, GPIIb beta. All six of these MoAbs agglutinated platelets in the absence of calcium. The agglutination titers of three of the MoAbs, however, were enhanced between 2 and 6 log2 dilutions when titrated in the presence of mmol/L of calcium. The enhancement in titer was the result of MoAb-induced platelet activation followed by platelet aggregation, a reaction that could also be initiated by the monovalent Fab fragments prepared from one of the MoAbs. The MoAbs did not significantly agglutinate platelets from patients with Glanzmann's thrombasthenia, confirming biochemical evidence that there is a paucity of GPIIb beta in the membranes of these cells. Our results show that MoAbs to epitopes on GPIIb beta initiate distinct platelet responses; therefore, they should be useful for studying the ways in which regions of surface glycoproteins are involved in platelet-platelet interactions. In addition, these reagents may prove of value in diagnosing and typing patients with Glanzmann's thrombasthenia.

Antibodies, Monoclonal↗