scholarly journals Molecular characterization of quinine/quinidine drug-dependent antibody platelet interaction using monoclonal antibodies

Blood ◽  
1985 ◽  
Vol 66 (6) ◽  
pp. 1292-1301 ◽  
Author(s):  
MC Berndt ◽  
BH Chong ◽  
HA Bull ◽  
H Zola ◽  
PA Castaldi
Keyword(s):  
Monoclonal Antibodies ◽  
Platelet Aggregation ◽  
Adenosine Diphosphate ◽  
Fc Receptor ◽  
Alpha Subunit ◽  
Antiplatelet Antibody ◽  
Glycoprotein Ib ◽  
Calcium Dependent ◽  
Antibody Recognition ◽  
Drug Dependent

Abstract Two murine monoclonal antibodies, FMC 25 and AN 51, directed against distinct epitopes on the glycoprotein Ib complex, have been used to further define the mechanism of quinine/quinidine drug-dependent antibody interaction with platelets. FMC 25, directed against an epitope on glycoprotein IX, had no effect on platelet aggregation induced by collagen or adenosine diphosphate and little, if any, effect on ristocetin-induced platelet agglutination. FMC 25 and its (Fab)2 fragment, however, were potent inhibitors of drug-dependent antibody- induced platelet aggregation and blocked binding of drug-dependent antibody to platelets as assessed by indirect platelet immunofluorescence. In contrast, AN 51, directed against an epitope on the alpha-subunit of glycoprotein Ib, blocked ristocetin-induced, factor VIII/von Willebrand factor (FVIII/vWF)-dependent platelet agglutination but not drug-dependent antibody-induced platelet aggregation or binding of drug-dependent antibody to platelets. Selective proteolytic removal of the majority of the alpha-subunit of glycoprotein Ib (glycocalicin) from platelets by treatment with calcium- dependent protease did not affect binding of drug-dependent antibody. In addition, a quinidine-dependent antiplatelet antibody immunoprecipitated glycoprotein Ib complex from normal platelets and the membrane-associated proteolytic remnant of the glycoprotein Ib complex from calcium-dependent protease-treated platelets. Preincubation of drug-dependent antibody with purified glycoprotein Ib complex inhibited subsequent binding of antibody to platelets, but the separated components, glycoprotein Ib and glycoprotein IX, were both ineffective, suggesting that the normal interaction between glycoprotein Ib and glycoprotein IX in the intact complex was necessary for drug-dependent antibody recognition. The functional response of platelets to drug-dependent antibody was not mediated by way of platelet Fc receptor, since aggregation of washed platelets by acetone- aggregated IgG was not inhibited by FMC 25 (Fab)2. FVIII/vWF was not required for drug-dependent antibody-induced platelet aggregation. The combined evidence is consistent with quinine/quinidine-dependent antibody-platelet interaction occurring by way of a FVIII/vWF- independent, Fc receptor-independent mechanism that probably involves binding of antibody to glycoprotein IX or the beta-subunit of glycoprotein Ib or both.

scholarly journals Molecular characterization of quinine/quinidine drug-dependent antibody platelet interaction using monoclonal antibodies

Blood ◽  
1985 ◽  
Vol 66 (6) ◽  
pp. 1292-1301 ◽  
Author(s):  
MC Berndt ◽  
BH Chong ◽  
HA Bull ◽  
H Zola ◽  
PA Castaldi
Keyword(s):  
Monoclonal Antibodies ◽  
Platelet Aggregation ◽  
Adenosine Diphosphate ◽  
Fc Receptor ◽  
Alpha Subunit ◽  
Antiplatelet Antibody ◽  
Glycoprotein Ib ◽  
Calcium Dependent ◽  
Antibody Recognition ◽  
Drug Dependent

Two murine monoclonal antibodies, FMC 25 and AN 51, directed against distinct epitopes on the glycoprotein Ib complex, have been used to further define the mechanism of quinine/quinidine drug-dependent antibody interaction with platelets. FMC 25, directed against an epitope on glycoprotein IX, had no effect on platelet aggregation induced by collagen or adenosine diphosphate and little, if any, effect on ristocetin-induced platelet agglutination. FMC 25 and its (Fab)2 fragment, however, were potent inhibitors of drug-dependent antibody- induced platelet aggregation and blocked binding of drug-dependent antibody to platelets as assessed by indirect platelet immunofluorescence. In contrast, AN 51, directed against an epitope on the alpha-subunit of glycoprotein Ib, blocked ristocetin-induced, factor VIII/von Willebrand factor (FVIII/vWF)-dependent platelet agglutination but not drug-dependent antibody-induced platelet aggregation or binding of drug-dependent antibody to platelets. Selective proteolytic removal of the majority of the alpha-subunit of glycoprotein Ib (glycocalicin) from platelets by treatment with calcium- dependent protease did not affect binding of drug-dependent antibody. In addition, a quinidine-dependent antiplatelet antibody immunoprecipitated glycoprotein Ib complex from normal platelets and the membrane-associated proteolytic remnant of the glycoprotein Ib complex from calcium-dependent protease-treated platelets. Preincubation of drug-dependent antibody with purified glycoprotein Ib complex inhibited subsequent binding of antibody to platelets, but the separated components, glycoprotein Ib and glycoprotein IX, were both ineffective, suggesting that the normal interaction between glycoprotein Ib and glycoprotein IX in the intact complex was necessary for drug-dependent antibody recognition. The functional response of platelets to drug-dependent antibody was not mediated by way of platelet Fc receptor, since aggregation of washed platelets by acetone- aggregated IgG was not inhibited by FMC 25 (Fab)2. FVIII/vWF was not required for drug-dependent antibody-induced platelet aggregation. The combined evidence is consistent with quinine/quinidine-dependent antibody-platelet interaction occurring by way of a FVIII/vWF- independent, Fc receptor-independent mechanism that probably involves binding of antibody to glycoprotein IX or the beta-subunit of glycoprotein Ib or both.

scholarly journals Fibrinogen competes with von Willebrand factor for binding to the glycoprotein IIb/IIIa complex when platelets are stimulated with thrombin

Blood ◽  
1984 ◽  
Vol 64 (4) ◽  
pp. 797-800 ◽  
Author(s):  
HR Gralnick ◽  
SB Williams ◽  
BS Coller
Keyword(s):  
Monoclonal Antibodies ◽  
Von Willebrand Factor ◽  
Adenosine Diphosphate ◽  
Glycoprotein Ib ◽  
Human Fibrinogen ◽  
Factor Binding ◽  
Normal Plasma ◽  
Platelet Binding ◽  
Von Willebrand ◽  
Willebrand Factor

Two monoclonal antibodies--one that blocks ristocetin-induced platelet binding of von Willebrand factor to glycoprotein Ib and one that blocks adenosine diphosphate-induced binding of fibrinogen to the glycoprotein IIb/IIIa complex--were used to assess the binding site(s) for von Willebrand factor when platelets are stimulated with thrombin or adenosine diphosphate (ADP). Neither agonist induced binding of von Willebrand factor to glycoprotein Ib. ADP and thrombin induced von Willebrand factor binding exclusively to the glycoprotein IIb/IIIa complex. The results of the site of binding of von Willebrand factor with thrombasthenic platelets were consistent with the data obtained with the monoclonal antibodies and normal platelets. Human fibrinogen caused complete inhibition of thrombin-induced von Willebrand factor binding to normal platelets at concentrations considerably below that found in normal plasma. We conclude that thrombin induces very little binding of exogenous von Willebrand factor to platelets at normal plasma fibrinogen levels.

scholarly journals Pseudo-Bernard-Soulier syndrome: thrombocytopenia caused by autoantibody to platelet glycoprotein Ib

Blood ◽  
1987 ◽  
Vol 70 (2) ◽  
pp. 428-431
Author(s):  
DV Devine ◽  
MS Currie ◽  
WF Rosse ◽  
CS Greenberg
Keyword(s):  
Immunoglobulin G ◽  
Adenosine Diphosphate ◽  
Platelet Glycoprotein ◽  
Laboratory Studies ◽  
Antiplatelet Antibody ◽  
Glycoprotein Ib ◽  
Immune Mediated ◽  
Induced Aggregation ◽  
Willebrand Factor ◽  
Bernard Soulier Syndrome

The Bernard-Soulier syndrome is an inherited bleeding disorder that is due to a deficiency in platelet glycoprotein Ib. Bernard-Soulier platelets fail to agglutinate in response to ristocetin despite normal levels of factor VIII:von Willebrand factor. We report a patient who developed severe refractory thrombocytopenia postsurgically while receiving procainamide therapy. Thrombocytopenia was immune mediated since the patient's platelets bore high levels of antiplatelet antibody. Radioimmunoprecipitation studies demonstrated that the autoantibodies had specificity for platelet glycoproteins Ib and V as well as platelet HLA. The patient's plasma as well as purified immunoglobulin G completely inhibited the ristocetin-induced aggregation of normal platelets but did not inhibit adenosine diphosphate-induced aggregation. The laboratory studies revealed that this patient suffered from antibody-mediated thrombocytopenia with unusual characteristics that we have called pseudo-Bernard-Soulier syndrome.

scholarly journals Synergistic action of two murine monoclonal antibodies that inhibit ADP- induced platelet aggregation without blocking fibrinogen binding

Blood ◽  
1987 ◽  
Vol 69 (2) ◽  
pp. 668-676
Author(s):  
PJ Newman ◽  
RP McEver ◽  
MP Doers ◽  
TJ Kunicki
Keyword(s):  
Monoclonal Antibodies ◽  
Platelet Aggregation ◽  
Adenosine Diphosphate ◽  
Platelet Surface ◽  
Synergistic Inhibition ◽  
Activated Platelets ◽  
Fibrinogen Binding ◽  
Complete Restoration ◽  
Binding Event ◽  
Murine Monoclonal Antibodies

We have used two murine monoclonal antibodies, each directed against one component of the human platelet membrane glycoprotein (GP) IIb-IIIa complex, to examine further the molecular requirements for fibrinogen binding to the platelet surface and subsequent platelet-platelet cohesion (aggregation). Although neither AP3, which is directed against GPIIIa, nor Tab, which is specific for GPIIb, were individually able to inhibit adenosine diphosphate (ADP)-induced fibrinogen binding, platelet aggregation, or secretion, the combination of AP3 and Tab completely abolished platelet aggregation and the release reaction. Unexpectedly, this synergistic inhibition of platelet-platelet cohesion occurred in the presence of apparently normal fibrinogen binding. Both the number of fibrinogen molecules bound and the dissociation constant for fibrinogen binding remained essentially unchanged in the presence of these two antibodies. Inhibition of aggregation was dependent upon the divalency of both AP3 and Tab because substitution of Fab fragments of either antibody for the intact IgG resulted in a complete restoration of both aggregation and secretion. In contrast to ADP induction, thrombin-activated platelets neither aggregated nor bound fibrinogen in the presence of AP3 plus Tab but were fully capable of secretion, which illustrated the multiple mechanisms by which the platelet surface can respond to different agonists. These data demonstrate that fibrinogen binding to the platelet surface alone is not sufficient to support platelet-platelet cohesion and that an additional post-fibrinogen-binding event(s) that is inhibitable by these two monoclonal antibodies may be required.

scholarly journals Pseudo-Bernard-Soulier syndrome: thrombocytopenia caused by autoantibody to platelet glycoprotein Ib

Blood ◽  
1987 ◽  
Vol 70 (2) ◽  
pp. 428-431 ◽  
Author(s):  
DV Devine ◽  
MS Currie ◽  
WF Rosse ◽  
CS Greenberg
Keyword(s):  
Immunoglobulin G ◽  
Adenosine Diphosphate ◽  
Platelet Glycoprotein ◽  
Laboratory Studies ◽  
Antiplatelet Antibody ◽  
Glycoprotein Ib ◽  
Immune Mediated ◽  
Induced Aggregation ◽  
Willebrand Factor ◽  
Bernard Soulier Syndrome

Abstract The Bernard-Soulier syndrome is an inherited bleeding disorder that is due to a deficiency in platelet glycoprotein Ib. Bernard-Soulier platelets fail to agglutinate in response to ristocetin despite normal levels of factor VIII:von Willebrand factor. We report a patient who developed severe refractory thrombocytopenia postsurgically while receiving procainamide therapy. Thrombocytopenia was immune mediated since the patient's platelets bore high levels of antiplatelet antibody. Radioimmunoprecipitation studies demonstrated that the autoantibodies had specificity for platelet glycoproteins Ib and V as well as platelet HLA. The patient's plasma as well as purified immunoglobulin G completely inhibited the ristocetin-induced aggregation of normal platelets but did not inhibit adenosine diphosphate-induced aggregation. The laboratory studies revealed that this patient suffered from antibody-mediated thrombocytopenia with unusual characteristics that we have called pseudo-Bernard-Soulier syndrome.

scholarly journals Monoclonal antibodies bound to subunits of the integrin GPIIb-IIIa are internalized and interfere with filopodia formation and platelet aggregation

Blood ◽  
1990 ◽  
Vol 76 (8) ◽  
pp. 1564-1571 ◽  
Author(s):  
WM Isenberg ◽  
DF Bainton ◽  
PJ Newman
Keyword(s):  
Monoclonal Antibodies ◽  
Platelet Aggregation ◽  
Adenosine Diphosphate ◽  
Membrane Flow ◽  
Thin Sections ◽  
Fibrinogen Receptor ◽  
Platelet Morphology ◽  
Fibrinogen Binding ◽  
Transmission Electron ◽  
Cytoskeletal Rearrangements

Abstract The monoclonal antibodies Tab and AP3 are directed, respectively, against GPIIb and GPIIIa, the subunits of the platelet fibrinogen receptor. When added together to platelets, these antibodies prevent adenosine diphosphate (ADP)-induced platelet aggregation, despite normal fibrinogen binding (Newman et al, Blood 69:668, 1987). To explore the cellular requirements of aggregation after fibrinogen binding, we used several techniques to study platelets treated with Tab and AP3, then stimulated with ADP. We used scanning and transmission electron microscopy to evaluate platelet morphology, immunolabel- surface replication to determine whether individual GPIIb-IIIa complexes clustered, immunocytochemistry on frozen thin sections to study the subcellular distribution of the integrin GPIIb-IIIa and fibrinogen, and biochemical methods to assess the activation of the platelet cytoskeleton. We found that the treated cells had short, blunted projections instead of normal filopodia. Other morphologic abnormalities, apparent in thin section, were aberrantly placed alpha- granules and microtubules, and a prominent, worm-like, fibrinogen- filled surface-connected canalicular system. Biochemical analysis suggested that such platelets undergo massive actomyosin-controlled membrane flow, which serves to sequester GPIIb-IIIa and makes the platelets refractory to aggregation. We conclude that aggregation requires the formation of long, slender filopodia, probably directed by cytoskeletal rearrangements after activation, and that the transmembrane GPIIb-IIIa complex may play a role in signaling these events.

scholarly journals Expression of the leukocyte functional molecule (LFA-1) on mouse platelets

Blood ◽  
1986 ◽  
Vol 67 (6) ◽  
pp. 1757-1764 ◽  
Author(s):  
PJ McCaffery ◽  
MV Berridge
Keyword(s):  
Monoclonal Antibodies ◽  
Cell Surface ◽  
Peptide Mapping ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Alpha Subunit ◽  
Cell Surface Molecules ◽  
Surface Molecules ◽  
Alpha Subunits ◽  
Immune Adherence

Platelet involvement in adhesion, hemostasis, and immune adherence is mediated by functionally associated cell surface molecules. Platelets are also involved in cytolytic reactions, but little is known about the mechanisms or biologic significance of these processes. To further investigate cell surface molecules concerned with platelet function, antisera against mouse platelets, thymocytes, and macrophages and monoclonal antibodies against Mac-1 (complement receptor type 3) and leukocyte function-associated glycoprotein type 1 (LFA-1) were used to demonstrate LFA-1--like molecules on mouse platelets. The alpha subunits of platelet and thymocyte LFA-1 showed identical electrophoretic mobility but differed significantly from the alpha subunit of macrophage Mac-1. Peptide mapping demonstrated the identity of the beta subunits of these three molecules but showed that the alpha subunit of Mac-1 was distinct from the alpha subunits of platelet and thymocyte LFA-1. Platelet LFA-1, as demonstrated by surface iodination with lactoperoxidase and by labeling sialic acid residues with sodium borohydride, was not a major component of the platelet membrane. The functional significance of LFA-1 on mouse platelets has yet to be demonstrated, monoclonal antibodies against LFA-1 having little effect on adenosine diphosphate-induced platelet aggregation and immune adherence. In contrast, although Mac-1 could not be demonstrated on mouse platelets in immunoprecipitation studies, its presence was clearly demonstrated by low levels of antibody binding in enzyme-linked immunosorbent assays and by the ability of M1/70 monoclonal antibody to inhibit platelet immune adherence. Human platelets, which are inactive in immune adherence assays, are shown to lack LFA-1 and Mac-1.

scholarly journals Monoclonal antibodies bound to subunits of the integrin GPIIb-IIIa are internalized and interfere with filopodia formation and platelet aggregation

Blood ◽  
1990 ◽  
Vol 76 (8) ◽  
pp. 1564-1571
Author(s):  
WM Isenberg ◽  
DF Bainton ◽  
PJ Newman
Keyword(s):  
Monoclonal Antibodies ◽  
Platelet Aggregation ◽  
Adenosine Diphosphate ◽  
Membrane Flow ◽  
Thin Sections ◽  
Fibrinogen Receptor ◽  
Platelet Morphology ◽  
Fibrinogen Binding ◽  
Transmission Electron ◽  
Cytoskeletal Rearrangements

The monoclonal antibodies Tab and AP3 are directed, respectively, against GPIIb and GPIIIa, the subunits of the platelet fibrinogen receptor. When added together to platelets, these antibodies prevent adenosine diphosphate (ADP)-induced platelet aggregation, despite normal fibrinogen binding (Newman et al, Blood 69:668, 1987). To explore the cellular requirements of aggregation after fibrinogen binding, we used several techniques to study platelets treated with Tab and AP3, then stimulated with ADP. We used scanning and transmission electron microscopy to evaluate platelet morphology, immunolabel- surface replication to determine whether individual GPIIb-IIIa complexes clustered, immunocytochemistry on frozen thin sections to study the subcellular distribution of the integrin GPIIb-IIIa and fibrinogen, and biochemical methods to assess the activation of the platelet cytoskeleton. We found that the treated cells had short, blunted projections instead of normal filopodia. Other morphologic abnormalities, apparent in thin section, were aberrantly placed alpha- granules and microtubules, and a prominent, worm-like, fibrinogen- filled surface-connected canalicular system. Biochemical analysis suggested that such platelets undergo massive actomyosin-controlled membrane flow, which serves to sequester GPIIb-IIIa and makes the platelets refractory to aggregation. We conclude that aggregation requires the formation of long, slender filopodia, probably directed by cytoskeletal rearrangements after activation, and that the transmembrane GPIIb-IIIa complex may play a role in signaling these events.

scholarly journals Fibrinogen competes with von Willebrand factor for binding to the glycoprotein IIb/IIIa complex when platelets are stimulated with thrombin

Blood ◽  
1984 ◽  
Vol 64 (4) ◽  
pp. 797-800 ◽  
Author(s):  
HR Gralnick ◽  
SB Williams ◽  
BS Coller
Keyword(s):  
Monoclonal Antibodies ◽  
Von Willebrand Factor ◽  
Adenosine Diphosphate ◽  
Glycoprotein Ib ◽  
Human Fibrinogen ◽  
Factor Binding ◽  
Normal Plasma ◽  
Platelet Binding ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Two monoclonal antibodies--one that blocks ristocetin-induced platelet binding of von Willebrand factor to glycoprotein Ib and one that blocks adenosine diphosphate-induced binding of fibrinogen to the glycoprotein IIb/IIIa complex--were used to assess the binding site(s) for von Willebrand factor when platelets are stimulated with thrombin or adenosine diphosphate (ADP). Neither agonist induced binding of von Willebrand factor to glycoprotein Ib. ADP and thrombin induced von Willebrand factor binding exclusively to the glycoprotein IIb/IIIa complex. The results of the site of binding of von Willebrand factor with thrombasthenic platelets were consistent with the data obtained with the monoclonal antibodies and normal platelets. Human fibrinogen caused complete inhibition of thrombin-induced von Willebrand factor binding to normal platelets at concentrations considerably below that found in normal plasma. We conclude that thrombin induces very little binding of exogenous von Willebrand factor to platelets at normal plasma fibrinogen levels.

scholarly journals Expression of the leukocyte functional molecule (LFA-1) on mouse platelets

Blood ◽  
1986 ◽  
Vol 67 (6) ◽  
pp. 1757-1764 ◽  
Author(s):  
PJ McCaffery ◽  
MV Berridge
Keyword(s):  
Monoclonal Antibodies ◽  
Cell Surface ◽  
Peptide Mapping ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Alpha Subunit ◽  
Cell Surface Molecules ◽  
Surface Molecules ◽  
Alpha Subunits ◽  
Immune Adherence

Abstract Platelet involvement in adhesion, hemostasis, and immune adherence is mediated by functionally associated cell surface molecules. Platelets are also involved in cytolytic reactions, but little is known about the mechanisms or biologic significance of these processes. To further investigate cell surface molecules concerned with platelet function, antisera against mouse platelets, thymocytes, and macrophages and monoclonal antibodies against Mac-1 (complement receptor type 3) and leukocyte function-associated glycoprotein type 1 (LFA-1) were used to demonstrate LFA-1--like molecules on mouse platelets. The alpha subunits of platelet and thymocyte LFA-1 showed identical electrophoretic mobility but differed significantly from the alpha subunit of macrophage Mac-1. Peptide mapping demonstrated the identity of the beta subunits of these three molecules but showed that the alpha subunit of Mac-1 was distinct from the alpha subunits of platelet and thymocyte LFA-1. Platelet LFA-1, as demonstrated by surface iodination with lactoperoxidase and by labeling sialic acid residues with sodium borohydride, was not a major component of the platelet membrane. The functional significance of LFA-1 on mouse platelets has yet to be demonstrated, monoclonal antibodies against LFA-1 having little effect on adenosine diphosphate-induced platelet aggregation and immune adherence. In contrast, although Mac-1 could not be demonstrated on mouse platelets in immunoprecipitation studies, its presence was clearly demonstrated by low levels of antibody binding in enzyme-linked immunosorbent assays and by the ability of M1/70 monoclonal antibody to inhibit platelet immune adherence. Human platelets, which are inactive in immune adherence assays, are shown to lack LFA-1 and Mac-1.

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