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K J Clemetson

Publications and source records attributed to K J Clemetson.

At least 127 records · Page 7Linked to original sources
PubMed1981

Glycoproteins of platelet membranes from Glanzmann's thrombasthenia. A comparison with normal using carbohydrate-specific or protein-specific labelling techniques and high-resolution two-dimensional gel electrophoresis.

Platelets from Glanzmann's thrombasthenia patients and from normal donor were surface labeled by techniques specific for sugars (terminal sialic acid, penultimate galactose/N-acetylgalactosamine) and proteins (tyrosine-histidine residues). These labelled platelets were solubilized in sodium dodecyl sulphate and separated on a two-dimensional electrophoretic system [O'Farrell. P. H. (1975) J. Biol. Chem. 250, 4007--4021] first according to their isoelectric point (pI) and then according to their molecular weight. In addition, unlabelled sodium-dodecyl-sulphate-solubilized platelets were separated on a two-dimensional polyacrylamide gel and the glycoproteins were identified by binding of 125I-labelled Lens culinaris lectin (specific for mannose and glucose). In one Glanzmann's thrombasthenia patient glycoproteins IIbA1 and IIIaA1 were absent and in two others lower amounts of two glycoproteins were found in positions similar or close to these two membrane glycoproteins. The terminal sialic acid moieties of major glycoproteins (IbA1, IbB1 and IIIbA1) were more intensely labelled in Glanzmann's thrombasthenia than in normals and these glycoproteins had an altered pI. A glycoprotein tentatively designated as Ic/IIa(?) had an altered pI and was labelled more intensely in Glanzmann's thrombasthenia platelets than in normals. A number of low-molecular-weight glycoproteins (IVa, IVb, VII) and one high-molecular weight glycoprotein normally found in platelets of healthy donors were reproducibly not detected in Glanzmann's thrombasthenia platelets. These results obtained by a combination of highly sensitive techniques strongly indicate that in Glanzmann's thrombasthenia the absence or reduction of two major membrane glycoproteins (IIbA1, IIIaA1) is not the only defect but that there appears to be a profound perturbation of the platelet membrane surface.

Blood Platelet Disorders↗
PubMed1981

Relationship between glycocalicin and glycoprotein Ib of human platelets.

Asialoglycoprotein Ib and asialoglycocalicin have been isolated from the membranes and from the supernatant, respectively, after sonication of neuraminidase-treated platelets, by lectin affinity chromatography on peanut agglutinin. The isolated asialoglycoprotein Ib had an apparent molecular weight of 160,000 when not reduced and 150,000 when reduced, whereas the asialoglycocalicin had an apparent molecular weight of 150,000, both reduced and unreduced, on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Both preparations contained only trace amounts of impurities. The asialoglycoprotein Ib and asialoglycocalicin in both the unreduced and reduced states were separated by gel electrophoresis, radioiodinated in gel slices, and digested with trypsin, and the digests were analyzed by two-dimensional high-voltage electrophoresis and thin-layer chromatography followed by autoradiography. The tryptic peptide maps showed great similarities between glycocalicin and glycoprotein Ib, with the latter (both unreduced and reduced) containing additional peptides, supporting the idea that glycocalicin is derived from glycoprotein Ib. The unreduced glycoprotein Ib contained additional peptides compared to the reduced due to the disulfide-bond-linked beta component. There were also slight differences between unreduced and reduced glycocalicin, indicating that at least one intramolecular disulfide bond is present.

Blood Platelets↗
PubMed1981

Antibodies against platelet membrane glycoproteins. II. Influence on ADP- and collagen-induced platelet aggregation, crossed immunoelectrophoresis studies and relevance to Glanzmann's thrombasthenia.

In Glanzmann's thrombasthenia glycoproteins IIb and IIIa are missing or strongly reduced and aggregation to ADP, collagen and thrombin is impaired. Antibodies against glycoproteins IIb and IIIa did not entirely induce a thrombasthenia-like state in normal platelets. However, they did strongly inhibit collagen-induced aggregation and inhibited the second wave of aggregation induced by ADP. Crossed immunoelectrophoresis studies using Triton X-100 extracts of whole platelets with these antibodies gave a single immunoprecipitate. This immunoprecipitate was absent when similar studies were carried out with thrombasthenic platelets. Platelet antibodies gave a number of immunoprecipitates with normal platelets and differences were observed with thrombasthenic platelets, the most notable of which was a marked reduction in one of the major immunoprecipitates. These results provide further evidence that glycoproteins IIb and IIIa are involved in the latter stages of platelet aggregation.

Adenosine Diphosphate↗
PubMed1981

Antibodies against platelet membrane glycoproteins. I. Crossed immunoelectrophoresis studies with antibodies that inhibit ristocetin-induced platelet aggregation.

Platelet membrane glycoproteins have been isolated by lectin-affinity chromatography and antibodies prepared against them. Platelets that have lost glycocalicin no longer respond to ristocetin-human VIIIR:WF, bovine VIIIR:WF, or to glycocalicin or glycoproteins Ia and Ib antibodies but are still agglutinated by glycoproteins IIb and IIIa antibodies. Glycoproteins Ia and Ib and glycocalicin antibodies, IgG and Fab' fragments, inhibited ristocetin-human VIIIR:WF-induced aggregation of fixed, washed platelets and of platelets in plasma while glycoproteins IIb and IIIa antibodies were without effect. Cross immunoelectrophoretic studies showed that glycocalicin was present on whole platelets in only trace amounts. Glycocalicin antibodies, however, recognized a slower migrating component. Platelets incubated in an EDTA-free medium no longer respond to ristocetin-human VIIIR:WF. Membranes isolated from such platelets contained glycocalicin which cross-reacted with a remnant of the slower migrating component. Glycoproteins Ia and Ib antibodies gave more complex patterns but it was possible to identify the slower moving component recognized by the glycocalicin antibodies. These results show that glycocalicin is not normally found as such on whole platelets but is present as a precursor which is most likely glycoprotein Ib. On degradation of this precursor, glycocalicin is released from the membrane and VIIIR:WF-receptor activity is lost.

Animals↗
PubMed1980

Human blood platelet membrane glycoproteins. Resolution in different polyacrylamide gel electrophoretic systems.

The separation of the major platelet membrane glycoproteins of normal subjects and subjects with well defined platelet membrane glycoprotein abnormalities have been examined using four different polyacrylamide gel electrophoretic techniques (continuous and discontinuous). The mobilities of the resolved glycoprotein bands have been correlated with the glycoprotein nomenclature proposed for the conventional sodium dodecyl sulphate-phosphate buffer system. Since the glycoprotein distribution varies depending on the system used, the merits of each method should be considered before application to a specific problem.

Acetates↗
PubMed1980

Characterization of human blood platelet membrane proteins and glycorproteins by their isoelectric point (pI) and apparent molecular weight using two-dimensional electrophoresis and surface-labelling techniques.

Intact human blood platelets were radioactively labelled at the surface by techniques specific for proteins or glycoproteins. Labelled platelet samples were analyzed by a high-resolution two-demensional separation system involving isoelectric focusing in the first dimension and discontinuous sodium dodecyl sulphate-polyacrylamide gel electrophoresis in the second. The major platelet membrane glycoprotein (GP) bands (Ib, IIb, IIIa and IIIb) were found to be highly heterogeneous even after removal of terminal sialic acid residues. Lactoperoxidase-catalyzed iodination of platelets showed that the major labelled proteins (Ib, IIb, IIIa and IIIb) had altered isoelectric points (pI) and molecular weights after neuraminidase treatment. A number of membrane glycoproteins previously undetected by one-dimensional gel electrophoresis were demonstrated and good evidence provided that the major platelet surface proteins are glycosylated.

Blood Platelets↗
PubMed1980

Studies on platelet glycoproteins in Glanzmann's thrombasthenia using 125I-labelled lectins.

Glycoproteins (GP) of Glanzmann's thrombasthenia (GT) and normal platelets were separated on sodium dodecyl sulphate (SDS) polyacrylamide slab gels and identified by their binding of 125I-labelled lectins having different sugar specificities. This highly sensitive technique showed that in thrombasthenic platelets, glycoproteins in the positions of IIb and III (IIIa) were greatly reduced when compared to normal platelets. After incubation with neuraminidase, IIb became undectable. GT platelet glycoproteins I, IV (IIIb), and high molecular weight GPs bound 125I-lectins more strongly than those in normal platelets, showing that there are drastic changes in the carbohydrate moieties of these major GPs.

Blood Platelets↗
PubMed1979

High resolution two-dimensional gel electrophoresis of the proteins and glycoproteins of human blood platelets and platelet membranes.

The proteins and glycoproteins of human blood platelets and platelet membranes in both the reduced and the unreduced states have been analysed by isoelectric focusing and sodium dodecyl sulphate-discontinuosus polyacrylamide gel electrophoresis in a two-dimensional technique. Gels which had been stained with periodic acid-Schiff's reagent could be counter-stained with Coomassie Brilliant Blue, simplifying the recognition of components which stain with both reagents. The major glycoproteins and some of the proteins have been identified and the characteristics of the membrance and of the whole platelet components established in this system.

Blood Platelets↗
PubMed1979

The stimulation of a T cell response in the syngeneic host by the P-815 mastocytoma cell and the isolation of a tumor-associated antigen which has some H-2 antigen characteristics.

Although not obviously immunogenic when developing intraperitoneally or subcutaneously, the P-815 mastocytoma cell induces a significant immune response when injected intradermally into the syngeneic host. In some animals the immune reaction leads to spontaneous tumor regression and survival of the animals, but in most cases the effectively initiated immune-reaction is abrogated by as yet unknown mechanisms. The T cell is identified as the killer cell both in vivo and in vitro during the described period of immune reactivity. By means of two antibodies from two different species, rendered specific for the P-815 tumor cell by in vivo absorption in DBA/2 mice, two distinct antigens have been identified, isolated and partially purified from P-815 cell membranes. One of them is obviously different from H-2 antigens, the other one shows striking similarities with H-2 antigens (molecular weight, lectin binding properties), but does not coprecipitate with H-2 antigens in an indirect precipitation assay, using Staph. aureus Cowan I as the Fc-binding agent.

Animals↗
PubMed1979

Human platelet membrane receptor for bovine von Willebrand factor (platelet aggregating factor): an integral membrane glycoprotein.

The platelet membrane receptor for bovine von Willebrand factor, platelet aggregating factor, has been reported to be a property of a soluble glycoprotein, glycocalicin, that is loosely attached to the platelet surface and represents one of the major glycoproteins of the platelet glycocalyx. The studies reported here, however, demonstrate that fractions from human platelets containing glycocalicin have no bovine von Willebrand factor receptor activity. Instead, only fractions containing platelet membranes have receptor activity. By using a nonionic detergent, Brij 99, active receptor can be solubilized from the membrane. Some quantitation of the intact or solubilized receptor activity is possible because the aggregation curves produced by mixtures of various dilutions of membranes and a constant concentration of standard normal bovine plasma are linear when plotted against the logarithm of the concentration of receptor. The dose-response curve obtained with Brij 99-solubilized membranes is not parallel to that obtained with intact membranes. Lectin-specificity studies of the bovine von Willebrand factor receptor, soluble in Brij 99, demonstrate binding to a wheat germ agglutinin-Sepharose 4B affinity gel but little or no binding to similar affinity gels of concanavalin A or Lens culinaris lectin. By using wheat germ agglutinin-Sepharose 4B as a lectin affinity column, partial purification of the receptor is possible. Stability studies of the receptor in intact membranes show essentially no loss of activity for at least 6 days when membranes are stored at 4 degrees C in buffers containing 1 mM EDTA. One freezing and thawing cycle results in minimal loss of initial activity but the receptor activity of the thawed material is less stable over time than is fresh material. Repeated freezing and thawing destroys the activity and, once lost, it can not be recovered, even with detergents.

Blood Coagulation Factors↗
PubMed1979

Studies on the mechanism of ristocetin-induced platelet agglutination: binding of ristocetin to platelets.

Ristocetin was trace-labeled with [3H] by the reductive methylation method. It was shown to agglutinate human platelets in the presence of VIIIR:WF in a manner indistinguishable from unlabeled ristocetin. The binding of the labeled ristocetin to normal and enzyme-modified human platelets was studied both in the presence and absence of VIIIR:WF and at nonagglutinating and agglutinating concentrations of ristocetin. Virtually no difference in [3H]ristocetin binding was seen whether VIIIR:WF was present or not. Platelets treated with chymotrypsin, which destroys their ability to agglutinate to VIIIR:WF and ristocetin, did not bind less ristocetin than did control platelets. A pronounced, direct relationship was found between [3H]ristocetin bound by normal platelets and total ristocetin concentration. This implies that at the higher (agglutinating) concentrations of ristocetin either more binding sites are exposed or, more probably, aggregation of ristocetin occurs.

Alkylation↗
PubMed1977

Isolation of the membrane glycoproteins of human blood platelets by lectin affinity chromatography.

The major platelet membrane glycoproteins have been solubilized in 1.0% sodium deoxycholate and subjected to affinity chromatography on the lectins from Lens culinaris, wheat germ and Abrus precatorius. Polyacrylamide gel electrophoresis in the presence and absence of a reducing agent together with the differential binding of the lectins to the glycoproteins permitted the distinction of at least seven separate glycoprotein entities. A new nomenclature for the glycoproteins is proposed to accomodate the additional data. Using combinations of lectin columns, glycoproteins Ia and Ib could be prepared in a pure state and IIb and IIIa could be greatly purified. The binding of lectins to glycoprotein Ib has been strongly implicated as a necessary step in the aggregation response of platelets to lectins.

Blood Platelets↗