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Biomedical subjects

D R Phillips

Publications and source records attributed to D R Phillips.

At least 217 records · Page 12Linked to original sources
PubMed1981

Changes in the cytoskeletal structure of human platelets following thrombin activation.

The temporal changes in human platelet actin polymerization, cytoskeletal morphology, and protein content that occur during thrombin-induced platelet activation were investigated by analysis of Triton-extracted platelets. Measurement of the DNase inhibitory activity of control platelets immediately after adding an equal volume of 2% Triton X-100, 10 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, and 0.1 M Tris, pH 7.4, showed that approximately 50% of the actin in unstimulated platelets was filamentous and unable to inhibit DNase-catalyzed hydrolysis of DNA. Activation of platelets with thrombin for 15 s caused the amount of actin in the filamentous form to increase to approximately 65%. Examination of the morphology and protein composition of these filamentous structures showed that the cytoskeletal structures from control platelets consisted of a random array of filaments which contained 14% of the total platelet myosin and 6% of the total actin-binding protein. In contrast, the cytoskeletal structures of thrombin-activated platelets appeared as cytoskeletal structures of individual platelets. The composition of these cytoskeletons varied depending on the time of thrombin activation. Those from platelets activated with thrombin for 15 to 30 s contained 90% of the platelet myosin and 20% of the platelets with thrombin before Triton addition resulted in a decreased association of myosin to 60% with no change in either the actin or actin-binding protein content of the cytoskeletal structures. Since these changes are rapid and precede serotonin secretion, it is suggested that they are involved in the physiological response of the platelet.

Actins↗
PubMed1981

Purification and preliminary physicochemical characterization of human platelet membrane glycoprotein V.

Treatment of human platelets with alpha-thrombin leads to the selective hydrolysis of only one membrane protein, glycoprotein V. To determine whether glycoprotein V was directly cleaved by alpha-thrombin and to permit further characterization of this glycoprotein as the potential functional thrombin receptor, glycoprotein V was purified to > 98% homogeneity. Washed platelets were prepared from concentrates within 18 h of venipuncture, since clinically expired platelets (> 72 h from venipuncture) were shown to contain little or no detectable glycoprotein V. Glycoprotein V was eluted from the platelet membrane by equilibrating the platelets (4 X 10(9)/ml) at 37 degrees C for 24 h in 0.01 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes), pH 7.6 (1 mM in EDTA, 0.3 M in NaCl). Purification was achieved by initially performing ammonium sulfate fractionation, followed by chromatography on Sephacryl S-200, hydroxylapatite, and DEAE- and CM-cellulose, and yielded 0.5 to 1.0 mg of purified glycoprotein per 100 units of platelet concentrate (approximately 6 X 10(12) platelets). Purified glycoprotein V (Mr = 82,000) was a thrombin substrate, and on hydrolysis yielded a major fragment, GPVf1 (Mr = 69,500), identical in molecular weight with that observed previously in the supernatant of thrombin-treated, periodate-labeled platelets. Glycoprotein V existed as at least eight distinct isoelectric forms with pI values ranging from 5.85 +/- 0.05 to 6.55 +/- 0.05. The purified glycoprotein contained approximately 48% carbohydrate by weight, consisting of neutral hexose, hexosamine, and sialic acid in a molar ratio of approximately 8:2:1. Rabbit antiglycoprotein V antibody gave a single precipitin line against purified glycoprotein V, which showed a line of complete identity with Triton-solubilized washed platelets. The availability of purified glycoprotein V and antiglycoprotein V antibody will be useful in delineating the role of this glycoprotein in thrombin activation of platelets.

Blood Platelets↗
PubMed1981

A thermodynamic view of polymer degradation, and its application to extensive sonication of DNA.

A simple thermodynamic theory is developed, which predicts (in agreement with a wide variety of other theoretical approaches and experimental results) that for simple polymers the most probable Schulz distribution of fragments will be obtained in a polymer degradation process that is allowed to proceed to a dynamic equilibrium. When the same method is applied to a double-stranded polymer like DNA, however, it predicts that some narrowing of this distribution may occur in conjunction with a limited amount of base unpairing at the fragment termini. The compatibility of this prediction with the experimental results of long-time sonication of DNA is considered.

DNA↗
PubMed1981

Interaction of platelet plasma membranes with thrombin-activated platelets.

This study described the binding of platelet plasma membranes to either control or thrombin-activated platelets. Glycoproteins in plasma membranes isolated from human platelets were labeled by oxidation with periodate followed by reduction with [3H]NaBH4. Labeled membranes were incubated with either control or thrombin-activated platelets. The amount of membranes bound was measured by separating platelets with bound membranes from solution by rapid centrifugation through 27% sucrose and determining the amount of radioactivity associated with platelets. Five- to sevenfold more membranes bound to thrombin-activated platelets than to control platelets. This enhanced binding of labeled membranes was completely inhibited by an excess of unlabeled platelet membranes. Human erythrocyte membranes had little affinity for either washed or thrombin-activated platelets and therefore did not compete for platelet-membrane binding. Binding of platelet membranes to thrombin-treated platelets was inhibited by prior incubation of the platelets with PGI2 suggesting that the enhanced binding of membranes was to activated platelets. This study demonstrates that the purified platelet membranes have functional sites that can mediate membrane binding to platelets and that quantitation of membrane binding appears to reflect the increased aggregation capability of activated platelets.

Blood Platelets↗
PubMed1980

Ca2+-mediated association of glycoprotein G (thrombinsensitive protein, thrombospondin) with human platelets.

Washed human platelets suspended in buffers containing either 1.8 mM Ca2+ and 0.49 mM Mg2+ or 1 mM EDTA were treated with human alpha-thrombin to induce secretion. Glycoprotein G, a major glycoprotein in alpha-granules, was quantitatively secreted from platelets activated in the EDTA-containing buffer but remained with the platelet in the presence of Ca2+ and Mg2+. Addition of Ca2+ to the platelets that were activated in the presence of EDTA caused glycoprotein G to bind to platelets. To determine if glycoprotein G is expressed on the membrane surface of the activated platelet, platelets were rapidly labeled by a method employing lactoperoxidase-catalyzed iodination. Although glycoprotein G was barely detected on the surface of unstimulated platelets, labveling 1 min after thrombin treatment showed that glycoprotein G rapidly became one of the prominent surface proteins. These findings show that an alpha-granule protein, glycoprotein G, is one of the major glycoproteins on the membrane surface of thrombin-activated platelets and that its binding is dependent on divalent cations.

Adult↗
PubMed1980

Proton nuclear magnetic resonance study of the self-complementary hexanucleotide d(pTpA)3 and its interaction with daunomycin.

The helix-coil transition of the self-complementary hexanucleotide d(pTpA)3 has been studied in 1 M NaCl by high-resolution proton nuclear magnetic resonance spectroscopy. Almost all of the 12 resonances deriving from the three environments of the four nucleotide protons have been assigned to the central, internal, or terminal nucleotides. At 5 degrees C, the effect of extensive fraying is evident since the central base pairs exhibit of extensive fraying is evident since the central base pairs exhibit only 20% of the chemical shifts observed for poly(dA-dT) . poly(dA-dT) accompanying denaturation. Daunomycin interacts with the hexanucleotide duplex at 5 degrees C and stabilizes it by 21 degrees C at a drug/nucleotide ratio of 0.063 (i.e., drug/hexanucleotide duplex ratio of 0.75). The chemical shifts of the drug protons suggest that ring D of daunomycin does not overlap significantly with the central base pairs of the hexanucleotide and that it extends out from the "helix". This information, together with studies of space-filling models of the complex, suggests that rings B and C of daunomycin overlap with adjacent base pairs and are skewed with respect to the base pairs.

Daunorubicin↗
PubMed1980

Identification of membrane proteins mediating the interaction of human platelets.

Membrane glycoproteins that mediate platelet-platelet interactions were investigated by identifying those associated with the cytoskeletal structures from aggregated platelets. The cytoskeletal structures from washed platelets, thrombin-activated platelets (platelets incubated with thrombin in the presence of mM EDTA to prevent aggregation) and thrombin- aggregated platelets (platelets activated in the presence of mM Ca(++) were prepared by first treating platelet suspensions with 1 percent Triton X-100 and 5 mM EGTA and then isolating the insoluble residue by centrifugation. The readily identifiable structures in electron micrographs of the residue from washed platelets had the shape and dimensions of actin filaments. Analysis of this residue from washed platelets had the shape and dimensions of actin filaments. Analysis of this residue by SDS gel electrophoresis showed that it consisted primarily of three proteins: actin (mol wt = 43,000), myosin (mol wt = 200,000) and a high molecular weight polypeptide (mol wt = 255,000) which had properties indentical to actin-binding protein (filamin). When platelets are activated with thrombin in the presence of EDTA to prevent aggregation, there was a marked increase in the amount of insoluble precipitate in the subsequent Triton extraction. Transmission electron microscopy showed that this residue not only contained the random array of actin filaments as seen above, but also organized structures from individual platelets which appeared as balls of electron-dense filamentous material approximately 1mum in diameter. SDS polyacrylamide gel analysis of the Triton residue of activated platelets showed that this preparation contained more actin, myosin and actin-binding protein than that from washed platelets plus polypeptides with mol wt of 56,000 and 90,000 and other minor polypeptides. Thus, thrombin activation appeared to increase polymerization of actin in association with other cytoskeletal proteins into structures that are observable after Triton extraction. The cytoskeletal structures from thrombin-aggregated platelets were similar to those from thrombin-activated platelets, except that the structural elements from individual platelets remained aggregated rather than randomly dispersed in the actin filaments. This suggested that the membrane components that mediate the direct interaction of platelets were in Triton residue from aggregated platelets. Only a small percentage of the membrane surface proteins and glycoproteins were found in the cytoskeletal structures from either washed platelets or thrombin-activated platelets. In contrast, the aggregated cytoskeletal structures from thrombin-aggregated platelets contained membrane glycoproteins IIb (26 percent of the total in pre-extracted platelets) and III (14 percent), suggesting that one or both of these glycoproteins participate in the direct interaction of platelets during aggregation.

Actins↗
PubMed1980

Purification and characterization of two high-affinity (adenosine 3',5'-monophosphate)-binding proteins from yeast. Identification as multiple forms of glyceraldehyde-3-phosphate dehydrogenase.

Two high-affinity cAMP-binding proteins (I and II) have been purified to homogeneity from baker's yeast by a procedure avoiding proteolytic damage. These proteins have been identified as multiple forms of glyceraldehyde-3-phosphate dehydrogenase. The two cAMP-binding proteins are similar in affinities for cAMP, have identical elution volumes on gel filtration, and contain one type of subunit (Mr 37 500). The form II of glyceraldehyde-3-phosphate dehydrogenase is free of NAD+ and has a Kd of 1.3 X 10(-6) M with respect to cAMP. A marked concentration-dependent self-association of the subunits of the form-II protein was revealed by Yphantis sedimentation equilibrium studies. Significant monomer concentrations are present at total concentrations less than 0.02 mg/ml. Conventional sedimentation equilibrium analyses indicated a tetramer Mr of 170 000. The high-affinity binding of cAMP to glyceraldehyde-3-phosphate dehydrogenase may significantly reduce intracellular cAMP levels and is also discussed in relation to the nature of eukaryote cAMP-binding proteins with similar native or subunit Mr values which are at present functionally undefined.

3',5'-Cyclic-AMP Phosphodiesterases↗