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N O Solum

Publications and source records attributed to N O Solum.

At least 55 records · Page 3Linked to original sources
PubMed1987

The N-DSK gamma-chain binds to immunoprecipitated GP IIb-IIIa.

The CNBr-split N-terminal disulphide knot of the fibrinogen molecule (N-DSK) binds to ADP-stimulated gel-filtered platelets and immunoprecipitated fibrinogen receptor. To investigate which part of the N-DSK molecule that is involved in this binding, the glycoprotein IIb-IIIa complex (the fibrinogen receptor) was immunoprecipitated in crossed immunoelectrophoresis of Triton X-100 extracts of platelets against rabbit antibodies to whole platelet proteins. The immunoelectrophoresis plates were incubated with solubilized, carboxymethylated 125I-labelled A alpha -, B beta - or gamma-chains of N-DSK, and investigated for binding by autoradiography. The N-DSK gamma-chain, but not the A alpha - or B beta -chains demonstrated binding to the GP IIb-IIIa complex. These results show that the fibrinogen molecule contains a third sequence of amino acids, in addition to the two previously reported ones that can be involved in binding of fibrinogen to the fibrinogen receptor on the platelets.

Binding Sites↗
PubMed1987

Identification of platelet antigens by monoclonal antibodies using crossed immunoelectrophoresis with immunoblotting of the monoclonal antibody.

A method is described for the identification of antigens by monoclonal antibodies. This is applicable whenever precipitating antibodies to the same antigens from a different species are available. The method is based upon: Separation and immunoprecipitation of cellular proteins with a polyspecific antiserum in crossed immunoelectrophoresis in the presence of the non-denaturing detergent Triton X-100 and the monoclonal antibody. Coprecipitation of the monoclonal antibody with its antigen. Subsequent passive transfer of the monoclonal antibody in the antibody-antigen complex onto a nitrocellulose membrane. Visualization of the blotted antibody using an enzyme-linked secondary antibody and a chromogenic substrate. Identification of the corresponding antigen by comparisons to the immunoprecipitate pattern of the original immunoplate. To test this method we have analyzed the detection of the antigens recognized by six previously described monoclonal antibodies against platelet membrane proteins and von Willebrand factor. Specific immunoblots were obtained in each case using small amounts of monoclonal antibodies. Thus, the technique provides an alternative when epitopes are denatured by SDS, and avoids the use of radioactively labelled monoclonal antibodies.

Antibodies, Monoclonal↗
PubMed1986

Competitions between fibrinogen with its degradation products for interactions with the platelet-fibrinogen receptor.

Direct binding of 125-I-labelled plasmic and CNBr-derived fibrin (ogen) fragments (pre-X, X, Y, D, Degta, Efg, E1, N-DSK, N-dsk) to gel-filtered platelets was compared to their ability to support or inhibit ADP-induced aggregation, and to compete with fibrinogen for binding to ADP-stimulated platelets. Pre-X was the only fragment that supported aggregation. All fragments tested except for E derived from fibrinogen (Efg) and Degta bound specifically to the platelets and inhibited ADP-induced aggregation in the presence of fibrinogen. Competitive binding studies with fibrinogen and fragments labelled with different isotopes of iodine, or inhibition of binding of labelled fibrinogen with unlabelled fragments showed that all of the fragments except Efg and Degta were able to compete with fibrinogen for binding. When simultaneous binding of N-dsk and fibrinogen was studied, an increased binding of both ligands was observed probably due to complex formation. The results fully agree with previous findings of binding to immunoprecipitated glycoprotein IIb-IIIa after crossed immunoelectrophoresis. We conclude that the fibrinogen molecule contains at least six sequences responsible for platelet interaction, two in the E domain and two in each of the C-terminal parts of the fibrinogen molecule.

Adenosine Diphosphate↗
PubMed1986

Binding of 125I-labelled fibrin(ogen) fragments to platelets and to immunoprecipitated glycoprotein IIb-IIIa complex.

To further investigate which parts of the fibrinogen molecule that are responsible for its binding to the fibrinogen receptor on human platelets, the following approaches were made: The glycoprotein IIb-IIIa complex (the putative fibrinogen receptor) was immunoprecipitated in crossed immunoelectrophoresis of Triton X-100-extracts of platelets against antibodies to whole platelet proteins. Subsequently, the immunoplates were incubated with 125I-labelled, plasmin- or CNBr-cleaved fibrinogen fragments (pre-X,X,Y,D,Degta,Efg,N-DSK) or fibrin fragments (E1,N-dsk), characterized by partial sequenation. The immunoplates were exposed to X-ray films, and binding of the fragments to the glycoprotein IIb-IIIa complex was examined. The findings were compared to the results obtained from studies on binding of the same fragments to intact gel-filtered platelets after ADP-stimulation. The following conclusions were made: All fragments except Efg and Degta bound to the immunoprecipitated GPIIb-IIIa complex as well as to ADP-stimulated platelets suggesting that at least two sequences in the E domain and one in each of the D domains of fibrinogen are involved in binding to the platelet receptor. The GPIIb-IIIa complex is the only surface-located platelet antigen that binds fibrinogen and the aforementioned fragments. The binding of the fragments to the receptor is dependent on divalent cations.

Antibodies↗
PubMed1986

Increased binding to ADP-stimulated platelets and aggregation effect of the dysfibrinogen Oslo I as compared with normal fibrinogen.

Interactions of the dysfibrinogen Oslo I with platelets were investigated. This fibrinogen is a B beta-chain variant with faster than normal fibrin monomer polymerization. Fibrinogen Oslo I acted more efficiently in ADP-induced platelet aggregation, and bound to gel-filtered platelets with a higher affinity constant than did normal fibrinogen. At all concentrations more fibrinogen molecules became bound per platelet with the dysfibrinogen than with normal fibrinogen, both when the fibrinogens were tested separately or as a mixture using 125I or 131I to label the two types. At high concentrations this was probably due to ligand polymerization of the dysfibrinogen. These observations indicate that the increased cofactor function in platelet aggregation may be related to the increased affinity of the dysfibrinogen for the platelets.

Adenosine Diphosphate↗
PubMed1985

Effects of diamide and dibucaine on platelet glycoprotein Ib, actin-binding protein and cytoskeleton.

During extraction of platelets by 1% Triton X-100, the actin-binding protein (platelet filamin) and a 230 kDa protein are degraded by a calcium-activated thiol protease. Occurrence of degradation products of Mr 190 000 (HF-1) and 90 000 (HF-2) is a sensitive indicator of this proteolysis, and can be used to decide whether reduced amounts of the actin-binding protein in extracts are due to proteolysis or to incorporation in the Triton-insoluble (cytoskeletal) fraction. Diamide, which is a sulfhydryl-oxidizing protein cross-linker, inhibits the calcium-activated protease, polymerizes the actin-binding protein and the 230 kDa protein, increases the incorporation of glycoprotein Ib into the cytoskeletal fraction, and inhibits platelet agglutination induced by bovine von Willebrand factor. Inhibition of platelet agglutination by pretreatment with diamide is partly reversed by dibucaine which activates the calcium-activated protease. These observations are in accordance with a working hypothesis that interactions of glycoprotein Ib with cytoskeleton affect, and possibly regulate, its receptor function in the intact platelet.

Animals↗
PubMed1985

Platelet--von Willebrand factor interactions in type IIB von Willebrand's disease.

Type IIB von Willebrand's disease (vWD) is a distinct form of this disorder in which the largest multimers of the von Willebrand factor (vWF) are lacking in plasma but present in platelets. When the vasopressin analogue, 1-deamino-8-D-arginine vasopressin (DDAVP), is given to patients with type IIB vWD, an abnormal vWF is released to plasma. This vWF causes thrombocytopenia in vivo and platelet aggregation in vitro. Aggregation occurs in the plasma milieu and thus at physiological fibrinogen concentration. In this study we demonstrate that IIB post-DDAVP vWF aggregated only metabolically active platelets. The platelet aggregation was completely inhibited by EDTA and PGE1, and either inhibited or greatly weakened by ASA, demonstrating the role of divalent cations and thromboxane A2 formation. In spite of inhibiting platelet aggregation, EDTA, PGE1 and ASA did not prevent platelet binding of IIB post-DDAVP vWF. An antiserum against GP Ib made normal platelets less responsive to the IIB vWF although neither platelet aggregation nor vWF binding were completely prevented. The aggregation was fibrinogen-dependent and platelets from patients with Glanzmann's thrombasthenia were unresponsive. The studies provide evidence that IIB post-DDAVP vWF is bound to unstimulated platelets and that the interaction between vWF and platelets in type IIB vWD is different from ristocetin-induced as well as thrombin- and epinephrine-induced binding to platelets of normal vWF.

Blood Platelets↗
PubMed1985

Platelet membrane proteins.

Studies on platelet membrane proteins during the past decade have defined a series of such components and their roles in key phenomena in hemostasis and thrombosis such as platelet adhesion and aggregation. Central among the membrane proteins is the glycoprotein group. At this time, several of these proteins have been characterized as to apparent molecular weights and subunit composition whereas sugar and amino acid composition and sequences await further studies for most of them. The absence of specific membrane glycoproteins in congenital bleeding diseases has helped to identify their functions as receptors for known ligands for binding reactions central to the hemostatic mechanism. Some of the membrane proteins may exist more or less loosely attached to each other on the surface of intact platelets, and this may be important for their functions.

Bernard-Soulier Syndrome↗
PubMed1984

Glycoprotein Ib in the Triton-insoluble (cytoskeletal) fraction of blood platelets.

Glycoprotein Ib could be demonstrated in the Triton-insoluble (cytoskeletal) fraction of platelets prepared with EGTA by SDS-polyacrylamide gel electrophoresis and staining with the periodic acid Schiff's reagent. Crossed immunoelectrophoresis showed that glycoprotein Ib could be extracted from such Triton-insoluble residues when the extraction solution contained 1% Triton X-100 plus 5 mM CaCl2, but not if it also contained leupeptin. This indicates that glycoprotein Ib was associated to structures in the cytoskeletal fraction in such a way that it could be extracted only after activation of a calcium-dependent protease, and degradation of the actin-binding protein was demonstrated. After crossed immunoelectrophoresis of platelet extracts prepared in the presence of leupeptin or EDTA, a glycoprotein Ib-related, rocket-shaped immunoprecipitate was seen originating from the application well. This was interpreted as being related to glycoprotein Ib associated to actin polymers which did not sediment at low-speed centrifugation. Incubation of platelets with 32P as sodium phosphate led to incorporation of phosphatase-sensitive 32P in all of the glycoprotein Ib-related immunoprecipitates except for that of glycocalicin. This supports the idea that glycoprotein Ib traverses the plasma membrane and can be phosphorylated at the inner surface whereas glycocalicin represents the terminal part of the glycoprotein Ib alpha-chain exposed at the outer surface.

Blood Platelets↗
PubMed1983

Glycoprotein IIb-IIIa complex in platelets of patients and heterozygotes of Glanzmann's thrombasthenia.

The concentration of the glycoprotein (GP) IIb-IIIa complex in thrombasthenic platelets of 8 patients of 6 families has been estimated. In the thrombasthenic platelets of 3 patients this complex is absent (thrombasthenia type I and subtype I). In 2 patients only traces are detectable and in 3 patients GP IIb-IIIa complex is strongly reduced (less than 5%). On the basis of the haemostatic data as well as the content of GP IIb-IIIa complex and platelet fibrinogen the classification of these types as subtypes of thrombasthenia type II is discussed. The diagnostic applicability of GP IIb-IIIa complex determination for heterozygote detection in types of thrombasthenia with absent or extremely reduced GP IIb-IIIa complex is shown.

Blood Platelet Disorders↗
PubMed1983

Monocytes and platelets share the glycoproteins IIb and IIIa that are absent from both cells in Glanzmann's thrombasthenia type I.

By means of an antiserum specific to the complex of the platelet membrane glycoproteins IIb and IIIa we demonstrate here that monocytes and purified monocyte membranes share these glycoproteins with platelets. The monocyte glycoprotein IIb-IIIa complex showed complete immunological identity with the platelet counterpart and, furthermore, dissociated after EDTA treatment exactly as did the platelet complex. In Glanzmann's thrombasthenia type I, monocytes as well as platelets lack this antigen completely.

Antigens, Surface↗
PubMed1983

Comparison of protein and lipid composition of the human platelet alpha-granule membranes and glycerol lysis membranes.

Platelet glycerol lysis membranes and alpha-granule membranes were compared with respect to protein and lipid composition. Crossed immunoelectrophoresis using antibodies against whole platelets, and sodium dodecyl sulphate polyacrylamide gel electrophoresis, revealed the presence of the glycoproteins IIb and IIIa, myosin and an antigen termed G4 in both membrane fractions. The glycoproteins Ia, Ib and IIIb, in addition to beta 2-microglobulin and actin, appeared specific for the glycerol lysis membranes, whereas two antigens, termed G8 and G18, were observed only in the alpha-granule membranes. The localization of glycoprotein IIa was inconclusive. Comparison with the surface-located proteins revealed that the glycerol lysis membranes represented a reasonable approximation to a plasma membrane preparation. Radioactively labelled immunoprecipitates obtained after crossed immunoelectrophoresis of 125I-labelled platelets were cut out and applied to sodium dodecyl sulphate electrophoresis on polyacrylamide slab gels. Autoradiography of the dried gels revealed that antigen G4 represented a protein with an average molecular weight of 146 000 in its unreduced state and 132 000 in its reduced state. Antigen G18 represented a protein of molecular weight 130 000-135 000 in the reduced as well as unreduced state. Quantitation of protein and lipids showed that the alpha-granule membranes contained about one-third as much cholesterol and 2-times as much protein in relation to phospholipids as compared to the glycerol lysis membranes. No significant difference between the two membrane preparations was found as regards the composition of their phospholipids.

Antigens↗
PubMed1983

Demonstration of 125I-labelled thrombin binding platelet proteins by use of crossed immunoelectrophoresis and autoradiography.

A possible receptor for thrombin on the platelet membrane has been identified. Whole platelets were treated with 125I-labelled thrombin followed by washing of the platelets, solubilization in Triton X-100, crossed immunoelectrophoresis and autoradiography. A heavily labelled antigen which migrated slightly more slowly than albumin was observed. No corresponding arc was seen on the same immunoplate when stained with Coomassie brilliant blue, indicating that the antigen possessed weak antigenic properties and/or was present in very small amounts. When 125I-labelled thrombin that had been inactivated by phenylmethylsulphonyl fluoride was used, no such labelled arc was seen. The radiolabelled immunoprecipitate does not represent any of the antigens identified hitherto in the immunoelectrophoretic patterns obtained with platelets or platelet material. The electrophoretic mobility of the antigen was influenced neither by neuraminidase treatment of the platelets prior to the 125I-labelled thrombin exposure nor by inclusion of concanavalin A, wheat-germ lectin or lentil lectin in the gel during the first-dimension electrophoresis. This suggests that the antigen does not represent a glycoprotein. Upon subcellular fractionation the radioactively labelled arc was observed in the cytosol fraction following crossed immunoelectrophoresis and autoradiography. Analysis of the secreted proteins after induction of the release reaction with 125I-labelled thrombin revealed labelling of immunoprecipitates representing thrombospondin, albumin and the 'line' form of platelet factor 4. This confirms that stable complexes of 125I-labelled thrombin and platelet proteins can exist in the presence of Triton X-100 and during electrophoresis.

Animals↗
PubMed1983

Calcium-binding proteins from human platelets. A study using crossed immunoelectrophoresis and 45Ca2+.

Calcium-binding platelet proteins were examined by crossed immunoelectrophoresis of solubilized platelets against antibodies to whole platelets followed by incubation of the immunoplates with 45Ca2+ and autoradiography. When the immunoplates had been pretreated with EDTA at pH 9.0 in order to remove divalent cations, three immunoprecipitates were markedly labelled with 45Ca2+. These corresponded to the glycoprotein IIb-IIIa complex, glycoprotein Ia and a presently unidentified antigen termed G18. These antigens were membrane-bound and surface-oriented. When an excess of EDTA was introduced in the incubation media the results revealed that the glycoprotein IIb-IIIa complex and antigen G18, but not glycoprotein Ia, contained sites with a stronger affinity for calcium than has EDTA at pH 7.4. Immunoprecipitates of the separate glycoproteins IIb and IIIa both bound calcium in the same manner as the glycoprotein IIb-IIIa complex. As another approach, platelet-rich plasma was incubated with 45Ca2+ prior to crossed immunoelectrophoresis of the solubilized platelets. A single immunoprecipitate was weakly labelled. This did not correspond to any of the immunoprecipitates which were visible after staining with Coomassie blue. The labelling of this antigen was markedly increased when the platelet-rich plasma had been preincubated with EDTA and in this case a weak labelling of the glycoprotein IIb-IIIa precipitate also became apparent. No increased incorporation of calcium occurred in any of these immunoprecipitates when the platelets were aggregated with ADP in the presence of 45Ca2+.

Annexin A6↗
PubMed1983

Demonstration of a new glycoprotein Ib-related component in platelet extracts prepared in the presence of leupeptin.

The water-soluble protein glycocalicin is generated during platelet lysis by a proteolytic attack on the integral membrane glycoprotein GP Ib. However, only small amounts of glycocalicin are formed when platelets are solubilized by 1% Triton X-100. Crossed immunoelectrophoresis of such extracts using an antiserum to glycocalicin, shows a continuous immunoprecipitate consisting of two peaks, one representing glycocalicin and the other GP Ib. When leupeptin was present during solubilization, subsequent immunoelectrophoresis revealed yet another GP Ib-related component represented by a third, slow-migrating peak of the immunoprecipitate. During incubation of platelets with dibucaine followed by solubilization in the presence of leupeptin, a gradual transformation of this new form of GP Ib into the previously defined one took place prior to the formation of glycocalicin. An increase followed by a decrease in the agglutination response of the platelets to bovine von Willebrand factor occurred concomitant with these transformations. SDS-polyacrylamide gel electrophoresis of Triton X-100 extracts of platelets did not reveal any difference in the size of GP Ib whether or not leupeptin had been present during the solubilization.

Animals↗
PubMed1983

A new bleeding disorder: lack of platelet aggregatory response to adrenaline and lack of secondary aggregation to ADP and platelet activating factor (PAF).

A bleeding disorder, probably familial, with absent adrenaline-induced platelet aggregation and lack of secondary aggregation response to ADP and platelet activating factor (PAF), is described. The laboratory findings do not fit any hitherto recognized hemorrhagic disease. The disorder was not caused by alpha-adrenergic receptor deficiency, but the ultimate defect has not yet been unraveled. This patient illustrates that a normal response to more than one aggregating stimulus is necessary for normal hemostasis, and indicates a physiopathological role for adrenaline not hitherto recognized. Whether this also applies to PAF remains to be proven.

Adenosine Diphosphate↗
PubMed1983

Heparin-binding platelet proteins demonstrated by crossed affinity immunoelectrophoresis.

Platelet proteins that interact with heparin were studied using crossed affinity immunoelectrophoresis. Platelet proteins solubilized in Triton X-100 were applied to crossed immunoelectrophoresis against anti-platelet antibodies, and an intermediate gel containing heparin covalently linked to Sepharose 4B was inserted. Six immunoprecipitates were absent or showed an altered position compared to control immunoplates, indicating that the corresponding antigens were bound to the immobilized heparin. These precipitates represented platelet factor 4, thrombospondin, glycoprotein Ib, and three antigens termed G4, 17 and 25. The subcellular location of the heparin-binding proteins was either in the surface membrane (glycoprotein Ib and the antigens 17 and 25), or in the alpha-granules (platelet factor 4, thrombospondin and G4). both forms of platelet factor 4 appearing after crossed immunoelectrophoresis, i.e. a line-form and a peak-form, bound strongly to the heparin. Glycoprotein Ib showed a weak binding whereas its proteolytic split product glycocalicin did not significantly bind to the heparin in the present system. It is concluded that the platelets contain at least six heparin-binding proteins which are present on the cellular surface or are able to be exposed to the extracellular medium after the release-reaction has occurred.

Blood Platelets↗