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Biomedical subjects

D R Phillips

Publications and source records attributed to D R Phillips.

At least 199 records · Page 11Linked to original sources
PubMed1984

Molecular defects in interactions of platelets with the vessel wall.

The objectives of this review have been to summarize the recent research on inherited defects involving abnormal platelet function and to illustrate how studies of hemorrhagic syndromes have led to an increased understanding of the molecular events involved in platelet adhesion and aggregation. Emphasis has been placed on the two primary hemostatic reactions: the interaction of platelets with von Willebrand factor to promote adhesion to the subendothelium, and the interaction of platelets with fibrinogen to promote platelet aggregation. Even as these events are more clearly defined, new concepts of molecular structure, function, and heterogeneity are emerging, and the variety of recognized genetic defects is becoming more complex.

Afibrinogenemia↗
PubMed1984

Application of analytical and semi-preparative high-performance liquid chromatography to anthracyclines and bis-anthracycline derivatives.

Efficient high-performance liquid chromatographic (HPLC) methods have been developed for routine analysis of a number of anthracycline derivatives using reversed-phase muBondapak C18 columns and an isocratic methanol-water-ammonium carbonate (or acetate) solvent system. The rates of formation of bis-daunomycin derivatives of alpha, omega-dicarboxylic acid hydrazides have been investigated using the analytical methods developed. It has been demonstrated that the reaction proceeds via an intermediate mono-hydrazone. Both the mono- and bis-hydrazones have been isolated by preparative HPLC.

Antibiotics, Antineoplastic↗
PubMed1984

Actin filament content and organization in unstimulated platelets.

The extent of actin polymerization in unstimulated, discoid platelets was measured by DNase I inhibition assay in Triton X-100 lysates of platelets washed at 37 degrees C by gel filtration, or in Triton X-100 lysates of platelets washed at ambient temperatures by centrifugation in the presence of prostacyclin. About 40% of the actin in the discoid platelets obtained by either method existed as filaments. These filaments could be visualized by electron microscopy of thin sections. Similar results were obtained when the actin filament content of discoid platelets was measured by sedimentation of filaments from Triton X-100 lysates at high g forces (145,000 g for 45 min). However, few of these filaments sedimented at the lower g forces often used to isolate networks of actin filaments from cell extracts. These results indicate that actin filaments in discoid cells are not highly crosslinked. Platelets isolated by centrifugation in the absence of prostacyclin were not discoid, but were instead irregular with one or more pseudopodia. These platelets also contained approximately 40-50% of their actin in a filamentous form; many of these filaments sedimented at low g forces, however, indicating that they were organized into networks. The discoid shape of these centrifuged platelets could be restored by incubating them for 1-3 h at 37 degrees C, which resulted in the reversal of filament organization. High g forces were then required for the sedimentation of the actin. Approximately 80-90% of the actin in platelets washed at 4 degrees C was filamentous; this high actin filament content could be attributed to actin polymerization during the preparation of the platelets at low temperatures. These studies show that platelet activation involves mechanisms for the structural reorganization of existing filaments, in addition to those previously described for mediating actin polymerization.

Actins↗
PubMed1983

Calcium cation regulation of glycoprotein IIb-IIIa complex formation in platelet plasma membranes.

The regulating effect of Ca2+ on the association and dissociation of the glycoprotein IIb-IIIa complex from human platelet membranes was determined both for detergent-solubilized and intact plasma membranes. Glycoproteins IIb and IIIa were solubilized from isolated membranes with 0.5% Triton X-100 and incubated in buffers containing ionized calcium, which resulted in the formation of the glycoprotein IIb-IIIa complex. With the addition of EGTA to reduce the ionized calcium content of the solution, the glycoprotein IIb-IIIa complex dissociated. This dissociation was measured by comparing the sedimentation properties of the glycoproteins and by observing the susceptibility of glycoprotein IIb to thrombin-catalyzed hydrolysis. With 10(-3) M Ca2+, glycoproteins IIb and IIIa were resistant to hydrolysis at thrombin concentrations up to 2.4 X 10(-5) M. When the Ca2+ concentration was decreased to less than 10(-4) M by chelation with EDTA or EGTA, glycoprotein IIb was cleaved by thrombin. This increased susceptibility to thrombin hydrolysis at decreasing Ca2+ levels correlated with the increased dissociation of the glycoprotein IIb-IIIa complex as determined by sucrose density centrifugation. Susceptibility to thrombin hydrolysis was also used as a probe to determine the extent to which Ca2+ regulates the formation of the glycoprotein IIb-IIIa complex within membranes. At more than micromolar levels of Ca2+, less than 10% of the membrane-bound glycoprotein IIb was cleaved by thrombin. Increased hydrolysis was observed at decreasing concentrations of Ca2+. Resistance to thrombin hydrolysis was partially regained upon the readdition of Ca2+ to dissociated glycoproteins. These data indicate that micromolar concentrations of Ca2+ exert a direct effect on platelet plasma membrane structure by regulating the intramembranous interactions of glycoprotein IIb.

Blood Platelets↗
PubMed1983

Fibrinogen binding to human platelet plasma membranes. Identification of two steps requiring divalent cations.

Fibrinogen binding to platelet plasma membranes, which is a prerequisite for platelet aggregation, was determined by incubating 125I-labeled fibrinogen with isolated membranes and measuring the amount of radioactivity sedimenting with the membranes through 15% sucrose. Fibrinogen binding was optimal at 10(-3) M Ca2+. Scatchard analyses of the fibrinogen binding showed that the membrane capacity for fibrinogen was 1.6 X 10(-12) mol/mg of membrane protein, with a dissociation constant (Kd) = 1.2 X 10(-8) M. When Ca2+ levels were manipulated by the addition of varying amounts of EGTA at a fixed Mg2+ concentration of 3 X 10(-3) M, specific binding of fibrinogen to platelet membranes occurred only at Ca2+ concentrations greater than or equal to 10(-6) M. Membranes isolated from platelets of an individual with Glanzmann's thrombasthenia bound only 12% as much fibrinogen as control platelets. The data in the present study suggest that there are two divalent cation binding sites that must be occupied for fibrinogen to bind: one site is specific for calcium and is saturated at 10(-6) M Ca2+; the other site is less specific and is saturated at a 10(-3) M concentration of either Ca2+ or Mg2+. Fibrinogen binding to intact platelets and, consequently, platelet aggregation only required 10(-3) M extracellular divalent cation and was not specific for Ca2+. These data indicate that the cytoplasm is a potential source for the requirement of 10(-6) M Ca2+, and that changes in the intracellular concentration of Ca2+ may cause the expression of fibrinogen receptors during ADP-induced platelet activation.

Blood Platelet Disorders↗
PubMed1983

Calcium-dependent proteolysis occurs during platelet aggregation.

Control and stimulated platelets were analyzed by two-dimensional polyacrylamide gel electrophoresis to determine whether proteins are altered during platelet activation. Platelets were stimulated with thrombin, collagen, or the calcium ionophore A23187, and aggregation was brought about by stirring in the presence of Ca2+. These activated platelets contained at least three polypeptides not found in control platelets: 1) Mr = 200,000, pI between 6.2 and 6.4; 2) Mr = 100,000, pI = 6.3; and 3) Mr = 91,000, pI = 6.1. An additional polypeptide, polypeptide 4, with Mr = 97,000 and pI = 5.9, was present only in platelets activated by thrombin. When aggregation was prevented, either by adding 5 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) to the platelet suspension or by incubating the platelet suspension without stirring, polypeptides 1-3 were not formed. Partial hydrolysis of polypeptides 2 and 4 with Staphylococcus aureus V8 protease yielded distinct sets of peptide hydrolytic fragments. These differed from those produced by the hydrolysis of alpha-actinin, a major platelet protein, which has a molecular weight similar to polypeptides 2 and 4. Polypeptides 1-3 were also produced during incubation of platelet lysates in the presence of Ca2+. Generation of these polypeptides in lysates was prevented either by chelation of Ca2+ with EGTA or by the addition of N-ethylmaleimide, leupeptin, or mersalyl, inhibitors of the calcium-dependent protease. These data show that the calcium-dependent protease is activated during aggregation of platelets by physiological agents and suggest that this protease could have a role in platelet response to stimulation.

Blood Platelets↗
PubMed1983

Studies on the response of Lactobacillus casei to folate vitamin in foods.

1. Folate was measured microbiologically with Lactobacillus casei in extracts from a range of different foods at two incubation pH values, 6.1 and 6.8. 2. The values for folate content obtained at pH 6.2 were, in several instances, considerably higher than at pH 6.8. The 'positive drift' seen in the values for dilutions incubated at pH 6.8 were absent from results at pH 6.2. 3. A comparison was made of the ability of the two main sources (hog-kidney and chicken pancreas) of deconjugase enzyme to produce measurable folate and the hog-kidney enzyme was shown to produce consistently higher values. 4. The results presented here will have significance for studies of folate intake using calculations from food composition tables, in the light of the apparent discrepancy between calculated folate intake and the recommended daily allowances for the UK.

Animals↗
PubMed1983

Evidence of possible dose-dependent doxorubicin plasma kinetics in man.

The published plasma clearance profiles of doxorubicin in man (with differing liver status) have been analyzed further using a kinetic model instead of a linear regression model. At high (60 mg/m2 and 45 mg/m2) and intermediate (30 mg/m2) doxorubicin dose levels, a triphasic drug clearance pattern is apparent. However, at a low dose level (15 mg/m2), a biphasic profile can adequately describe the kinetics. This association of the kinetics with drug dose has been demonstrated in several ways, the simplest of which involves graphic overlaying of juxtaposed plasma clearances, and shows the emergence of a kinetic profile for low dose levels that is markedly different from that at high dose levels. At doses greater than about 45 mg/m2, a probable saturation of response mechanisms is observed. It is not possible to interpret these results in terms of pure dose-dependent responses because of the varying health status of the individual subjects between and within experimental treatment. However, in light of our findings further investigation into the possibility of such an effect should be undertaken, particularly in view of its clinical ramifications.

Body Fluid Compartments↗
PubMed1982

Purification of glycoproteins IIb and III from human platelet plasma membranes and characterization of a calcium-dependent glycoprotein IIb-III complex.

Human platelet membrane glycoproteins IIb and III are two major integral membrane components that have been identified as sites mediating thrombin-induced aggregation. For purposes of our study, glycoproteins IIb and III were solubilized by extracting platelet plasma membranes with a buffer containing 0.1% Triton X-100 and were separated by gel filtration chromatography on Sephacryl S-300, employing Triton X-100-containing column buffers with or without urea or guanidine hydrochloride. The physical properties of the purified glycoproteins were: for glycoprotein IIb, Rs = 61 A, s20.w = 4.7, f/f0 = 1.7, Mr = 125,000 (hydrodynamic values), Mr = 136,000 (sodium dodecyl sulfate gels); for glycoprotein III, Rs = 67 A, s20,w = 3.2 f/f0 = 2.1, Mr = 93,000 (hydrodynamic values), Mr = 95,000 (sodium dodecyl sulfate gels). Although the amino acid compositions of the two glycoproteins were similar, antibodies raised against glycoprotein IIb did not crossreact with glycoprotein III. If divalent cations were not chelated in the Triton extract, glycoproteins IIb and III coeluted during gel filtration chromatography (apparent Stokes radius of 71 A) and co-sedimented on sucrose gradients (apparent s20.w of 8.6), from which Mr = 265,000 was calculated. Glycoproteins IIb and III were coprecipitated by an antibody monospecific for glycoprotein IIb. The two glycoproteins dissociated into monomers when EDTA was added to Triton lysates. Readdition of Ca2+ caused them to reassociate into a complex with physical properties similar to those of the complex in the original Triton lysate. The data show that glycoproteins IIb and III are a heterodimer complex, that complex formation depends upon the presence of Ca2+, and that chelation of Ca2+ causes dissociation into monomeric glycoproteins.

Blood Platelets↗
PubMed1982

Role of phosphorylation in mediating the association of myosin with the cytoskeletal structures of human platelets.

The effect of myosin light chain phosphorylation on the association of myosin with the cytoskeletal structures of platelets was quantitated. In unstimulated platelets, little myosin light chain was phosphorylated and myosin remained in solution when cytoskeletons from Triton X-100 lysates of platelets were sedimented by centrifugation. In platelets activated by thrombin, the calcium ionophore A23187, or collagen, the rate and extent of myosin light chain phosphorylation paralleled the association of myosin with platelet cytoskeletal structures. Dephosphorylation of myosin light chain and myosin dissociation from the cytoskeleton occurred at comparable rates at longer times after addition of the stimulating agents to platelets. Quantitation of radioactive phosphate in the cytoskeleton-associated myosin and in the soluble myosin showed that the phosphorylated myosin light chain was selectively isolated with the Triton-insoluble cytoskeletons, whereas nonphosphorylated myosin was not associated. Inhibition of the light chain kinase with the calmodulin antagonist trifluoperazine inhibited myosin light chain phosphorylation and incorporation of myosin into the platelet cytoskeletons. Inhibition of light chain phosphorylation by prostaglandin E1 and prostacyclin produced similar effects. Thus, phosphorylation of the myosin light chain stabilizes the association of myosin with the contractile structures within platelets.

Alprostadil↗
PubMed1982

Studies on the response of Lactobacillus casei to different folate monoglutamates.

1. The response of Lactobacillus casei was measured for a number of the monoglutamyl forms of folate derivatives. 2. At the concentrations of folate commonly used in the assay of folate vitamin in foods the response of L. casei to folic acid, (pteroylglutamic acid) and 5-formyl-tetrahydrofolic acid was similar, but 5-methyl-tetrahydrofolic acid gave as little as half the response of folic acid. 3. The response was modified by altering pH but not by concentration of ascorbate. 4. These results have implications for the assays of foods for folate where mixtures of folate derivatives are present. 5. A modified procedure is suggested in which the monoglutamates give similar responses.

Ascorbic Acid↗
PubMed1982

A possible role of Cu2+ ions in bovine antibody-antigen interactions.

A preparation of purified bovine IgG1 was found to contain 0.5 moles of Cu2+ ions per mole of protein. Scatchard analysis of the binding of Cu2+ ions to bovine IgG1 indicated the presence of 4 +/- 1 binding sites with an affinity of 2 x 10(5) M-1. Calculations indicate that in bovine plasma, IgG1 will not compete significantly with albumin for Cu2, whereas a proportion of Cu2+ may well be bound to IgG1 in colostrum. Physiological levels of Cu2+ were found to enhance the interaction between IgG1 antibody and Brucella abortus in the presence of albumin. Irrespective of whether IgG1 binds Cu2+ in vivo, it is possible that Cu2+ can influence the results of serological tests.

Animals↗