A Novel Mechanism for Exposure of Fibrinogen Binding Sites on GPIIb-IIIa by a Monoclonal Antibody

1995 ◽  
Vol 73 (01) ◽  
pp. 138-143 ◽  
Author(s):  
Takaaki Hato ◽  
Akito Watanabe ◽  
Shingo Nakatani ◽  
Yoko Minamoto ◽  
Shigeru Fujita
Keyword(s):  
Monoclonal Antibody ◽  
Conformational Changes ◽  
Binding Sites ◽  
Platelet Membrane ◽  
Cross Linking ◽  
Membrane Glycoprotein ◽  
Cross Link ◽  
Specific Domain ◽  
Fibrinogen Binding ◽  
The Difference

SummaryConformational changes in platelet membrane glycoprotein (GP) IIb-IIIa, whose nature is not defined, lead to exposure of fibrinogen binding sites. We have reported previously that F(ab’)2fragments of a monoclonal antibody, PMA4, directed against the GPIIb-IIIa complex- specific domain, induced binding of fibrinogen to platelets without causing intracellular activation, whereas Fab did not. In this study, we examined the mechanism responsible for the difference in the ability of PMA4 F(ab’)2and Fab to expose fibrinogen binding sites. PMA4 Fab had affinity for GPIIb-IIIa similar to that of PMA4 F(ab’)2. Addition of F(ab’)2goat anti-mouse Fab antibody to cross-link PMA4 Fab-bound GPIIb-IIIa molecules induced fibrinogen binding. There was a direct correlation between the number of molecules of PMA4 F(ab’)2and the amount of fibrinogen bound. PMA4 did not recognize ligand-induced binding sites (LIBS). These results suggest that the cross-linking of special sites on the GPIIb-IIIa complex-specific domain by bivalent antibody alters the conformation of GPIIb-IIIa to a state competent to bind soluble fibrinogen and that conformational changes in non-LIBS are involved in the mechanism for exposing fibrinogen binding sites on GPIIb-IIIa.

scholarly journals Platelet membrane alterations induced by the local anesthetic dibucaine

Blood ◽  
1986 ◽  
Vol 68 (2) ◽  
pp. 463-471 ◽  
Author(s):  
EI Peerschke
Keyword(s):  
Monoclonal Antibody ◽  
Binding Sites ◽  
Periodic Acid ◽  
Platelet Membrane ◽  
Actin Binding ◽  
Scatchard Analysis ◽  
Affinity Binding ◽  
High Affinity ◽  
Fibrinogen Binding ◽  
Induced Aggregation

Abstract Tertiary amine local anesthetics modify a variety of platelet membrane- related functions. The present study explored dibucaine (DB)-induced inhibition of platelet cohesion by examining structural and functional alterations of the human platelet membrane glycoprotein IIb-IIIa complex (GPIIb-IIIa) and platelet Ca2+ homeostasis. Complete inhibition of ADP-induced aggregation was achieved five minutes after platelet exposure to 0.10 to 0.25 mmol/L of DB when fibrinogen binding was reduced by 50%. At higher concentrations of DB (approximately 1 mmol/L), ADP-induced fibrinogen binding was completely blocked. Scatchard analysis revealed loss of high-affinity binding sites in addition to reduction in Bmax. In contrast, chymotrypsin-treated platelets sustained 50% inhibition of fibrinogen binding when incubated with 0.4 to 0.5 mmol/L DB, and kinetic analysis showed that the high- affinity platelet-fibrinogen interactions were reduced but not absent. Fibrinogen binding to chymotrypsin-treated platelets could not be completely inhibited even at high DB concentrations (1 mmol/L). The inhibition of fibrinogen binding to chymotrypsin-treated platelets correlated with changes in binding of a monoclonal antibody (10E5) specific for an epitope on the GPIIb-IIIa complex. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and radioelectroimmunoassay of DB-treated platelets, however, showed no evidence of a reduction or degradation of GP IIb or IIIa. Platelet incubation with DB (five minutes, 0.1 to 1.0 mmol/L) was also accompanied by: increased platelet membrane-associated Ca2+ involving low-affinity binding sites [Kd = 5 X 10(-5) mol/L-]; increased 45Ca2+ uptake which correlated with degradation of actin-binding protein (ABP) and digestion of GPIb as visualized on periodic-acid Schiff (PAS)- stained SDS gels and as inferred from decreased binding of a monoclonal antibody (6D1) directed against this glycoprotein; and enhanced Ca2+ exchange. Thus, exposure of platelets to DB results in membrane-related alterations that may contribute to inhibition of platelet cohesion: Decreased fibrinogen receptor exposure by traditional agonists and diminished accessibility of the GPIIb-IIIa complex to extracellular ligands correlate with DB-induced inhibition of platelet aggregation; and increased calcium uptake and exchange across the platelet membrane likely leads to activation of the calcium-dependent protease(s) which was previously shown to correlate with DB-induced inhibition of ristocetin-induced platelet agglutination.

scholarly journals Platelet membrane alterations induced by the local anesthetic dibucaine

Blood ◽  
1986 ◽  
Vol 68 (2) ◽  
pp. 463-471
Author(s):  
EI Peerschke
Keyword(s):  
Monoclonal Antibody ◽  
Binding Sites ◽  
Periodic Acid ◽  
Platelet Membrane ◽  
Actin Binding ◽  
Scatchard Analysis ◽  
Affinity Binding ◽  
High Affinity ◽  
Fibrinogen Binding ◽  
Induced Aggregation

Tertiary amine local anesthetics modify a variety of platelet membrane- related functions. The present study explored dibucaine (DB)-induced inhibition of platelet cohesion by examining structural and functional alterations of the human platelet membrane glycoprotein IIb-IIIa complex (GPIIb-IIIa) and platelet Ca2+ homeostasis. Complete inhibition of ADP-induced aggregation was achieved five minutes after platelet exposure to 0.10 to 0.25 mmol/L of DB when fibrinogen binding was reduced by 50%. At higher concentrations of DB (approximately 1 mmol/L), ADP-induced fibrinogen binding was completely blocked. Scatchard analysis revealed loss of high-affinity binding sites in addition to reduction in Bmax. In contrast, chymotrypsin-treated platelets sustained 50% inhibition of fibrinogen binding when incubated with 0.4 to 0.5 mmol/L DB, and kinetic analysis showed that the high- affinity platelet-fibrinogen interactions were reduced but not absent. Fibrinogen binding to chymotrypsin-treated platelets could not be completely inhibited even at high DB concentrations (1 mmol/L). The inhibition of fibrinogen binding to chymotrypsin-treated platelets correlated with changes in binding of a monoclonal antibody (10E5) specific for an epitope on the GPIIb-IIIa complex. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and radioelectroimmunoassay of DB-treated platelets, however, showed no evidence of a reduction or degradation of GP IIb or IIIa. Platelet incubation with DB (five minutes, 0.1 to 1.0 mmol/L) was also accompanied by: increased platelet membrane-associated Ca2+ involving low-affinity binding sites [Kd = 5 X 10(-5) mol/L-]; increased 45Ca2+ uptake which correlated with degradation of actin-binding protein (ABP) and digestion of GPIb as visualized on periodic-acid Schiff (PAS)- stained SDS gels and as inferred from decreased binding of a monoclonal antibody (6D1) directed against this glycoprotein; and enhanced Ca2+ exchange. Thus, exposure of platelets to DB results in membrane-related alterations that may contribute to inhibition of platelet cohesion: Decreased fibrinogen receptor exposure by traditional agonists and diminished accessibility of the GPIIb-IIIa complex to extracellular ligands correlate with DB-induced inhibition of platelet aggregation; and increased calcium uptake and exchange across the platelet membrane likely leads to activation of the calcium-dependent protease(s) which was previously shown to correlate with DB-induced inhibition of ristocetin-induced platelet agglutination.

scholarly journals Reversible conformational changes induced in glycoprotein IIb-IIIa by a potent and selective peptidomimetic inhibitor

Blood ◽  
1992 ◽  
Vol 80 (10) ◽  
pp. 2539-2547 ◽  
Author(s):  
WC Kouns ◽  
D Kirchhofer ◽  
P Hadvary ◽  
A Edenhofer ◽  
T Weller ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibody ◽  
Cell Adhesion ◽  
Platelet Aggregation ◽  
Conformational Changes ◽  
Platelet Membrane ◽  
Platelet Glycoprotein ◽  
Low Molecular Weight ◽  
Antithrombotic Agents ◽  
Fibrinogen Binding

Abstract Platelet glycoprotein (GP) IIb-IIIa inhibitors may become useful antithrombotic agents. Ro 4–5054 is a low molecular weight, noncyclic, peptidomimetic inhibitor that is three orders of magnitude more potent than RGDS in inhibiting fibrinogen binding to purified GPIIb-IIIa and in preventing platelet aggregation. Comparisons of RGDS and Ro 4–5054 in cell adhesion assays showed that, in contrast to RGDS, Ro 4–5054 was highly selective GPIIb-IIIa inhibitor. Effects of RGDV and Ro 4– 5054 on the conformation and activation state of GPIIb-IIIa were also examined. RGDV and Ro 4–5054 induced conformational changes in purified inactive GPIIb-IIIa as determined by binding of the monoclonal antibody D3GP3 (D3). These conformational alterations were not reversed after inhibitor removal, as indicated by the continued exposure of the D3 epitope and a newly acquired ability to bind fibrinogen. Similarly, RGDV and Ro 4–5054 induced conformational changes in GPIIb-IIIa on the intact platelet. However, after removal of the inhibitors, exposure of the D3 epitope was fully reversed and the platelets did not aggregate in the absence of agonist. Thus, while RGD(X) peptides and Ro 4–5054 transformed purified inactive GPIIb-IIIa into an irreversibly activated conformer, the effects of these inhibitors were reversible on the intact platelet. This suggests that factors present in the platelet membrane or cytoplasm may regulate in part the ability of the complex to shift between active and inactive conformers.

scholarly journals Reversible conformational changes induced in glycoprotein IIb-IIIa by a potent and selective peptidomimetic inhibitor

Blood ◽  
1992 ◽  
Vol 80 (10) ◽  
pp. 2539-2547
Author(s):  
WC Kouns ◽  
D Kirchhofer ◽  
P Hadvary ◽  
A Edenhofer ◽  
T Weller ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibody ◽  
Cell Adhesion ◽  
Platelet Aggregation ◽  
Conformational Changes ◽  
Platelet Membrane ◽  
Platelet Glycoprotein ◽  
Low Molecular Weight ◽  
Antithrombotic Agents ◽  
Fibrinogen Binding

Platelet glycoprotein (GP) IIb-IIIa inhibitors may become useful antithrombotic agents. Ro 4–5054 is a low molecular weight, noncyclic, peptidomimetic inhibitor that is three orders of magnitude more potent than RGDS in inhibiting fibrinogen binding to purified GPIIb-IIIa and in preventing platelet aggregation. Comparisons of RGDS and Ro 4–5054 in cell adhesion assays showed that, in contrast to RGDS, Ro 4–5054 was highly selective GPIIb-IIIa inhibitor. Effects of RGDV and Ro 4– 5054 on the conformation and activation state of GPIIb-IIIa were also examined. RGDV and Ro 4–5054 induced conformational changes in purified inactive GPIIb-IIIa as determined by binding of the monoclonal antibody D3GP3 (D3). These conformational alterations were not reversed after inhibitor removal, as indicated by the continued exposure of the D3 epitope and a newly acquired ability to bind fibrinogen. Similarly, RGDV and Ro 4–5054 induced conformational changes in GPIIb-IIIa on the intact platelet. However, after removal of the inhibitors, exposure of the D3 epitope was fully reversed and the platelets did not aggregate in the absence of agonist. Thus, while RGD(X) peptides and Ro 4–5054 transformed purified inactive GPIIb-IIIa into an irreversibly activated conformer, the effects of these inhibitors were reversible on the intact platelet. This suggests that factors present in the platelet membrane or cytoplasm may regulate in part the ability of the complex to shift between active and inactive conformers.

scholarly journals Further studies on the topography of the N-terminal region of human platelet glycoprotein IIIa. Localization of monoclonal antibody epitopes and the putative fibrinogen-binding sites

1991 ◽  
Vol 274 (2) ◽  
pp. 457-463 ◽  
Author(s):  
J J Calvete ◽  
J Arias ◽  
M V Alvarez ◽  
M M Lopez ◽  
A Henschen ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Binding Sites ◽  
Human Platelet ◽  
Binding Domain ◽  
Terminal Region ◽  
Platelet Glycoprotein ◽  
Gamma Chain ◽  
Fibrinogen Binding ◽  
Glycoprotein Iiia ◽  
Membrane Bound

The precise localization of the epitopes for six monoclonal antibodies specific for the N-terminal region of human platelet glycoprotein IIIa (GPIIIa) was determined. The epitope for P37, a monoclonal antibody that inhibits platelet aggregation, was found at GPIIIa 101-109, flanked by the epitopes for P23-3 (GPIIIa 16-28), P23-4 (GPIIIa 83-91), P23-5 (GPIIIa 67-73), P23-7 (GPIIIa 114-122) and P40 (GPIIIa 262-302), and very close to the early chymotryptic cleavage site of GPIIIa in whole platelets (Phe-100). When the amino acid sequence of GPIIIa was searched for peptide sequences hydropathically complementary to the fibrinogen gamma-chain C-terminal (gamma 400-411) and A alpha-chain RGD-containing peptides, none was found for the gamma 400-411, two (GPIIIa 128-132 and 380-384) were found complementary to fibrinogen A alpha 571-575 and two (GPIIIa 109-113 and 129-133) were found for A alpha 94-99. Two of these putative fibrinogen-binding sites overlap with each other, and a third one overlaps with the epitope for P37. These findings reinforce the earlier suggestion that the N-terminal region of GPIIIa is involved in fibrinogen binding, and suggest the existence in GPIIIa of either multiple or alternative RGD-binding sites or one RGD-binding domain with several moieties. Finally, early chymotryptic cleavage of GPIIIa in whole platelets liberates to the soluble fraction the peptide stretch Ser-101-Tyr-348, which carries the epitope for P37 and the putative binding sites for fibrinogen. The rest of the molecule, together with the GPIIb-resistant moiety, remains membrane-bound. This leads us to propose that the fibrinogen-binding domain of GPIIIa is not involved in the binding to GPIIb to form the Ca2(+)-dependent GPIIb-GPIIIa complex.

scholarly journals Factor XIIIa binding to activated platelets is mediated through activation of glycoprotein IIb-IIIa

Blood ◽  
1994 ◽  
Vol 83 (4) ◽  
pp. 1006-1016 ◽  
Author(s):  
AD Cox ◽  
DV Devine
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Platelet Activation ◽  
Factor Xiiia ◽  
Cross Linking ◽  
Platelet Surface ◽  
Activated Platelets ◽  
Fibrinogen Binding ◽  
A Chain ◽  
Functional Receptor

Abstract Stabilization of a clot is dependent on fibrin cross-linking mediated by the transglutaminase, factor XIIIa (FXIIIa). In addition to fibrin stabilization, FXIIIa acts on a number of platelet-reactive proteins, including fibronectin and vitronectin, as well as the platelet proteins, glycoprotein (GP) IIb-IIIa, myosin, and actin. However, conditions inducing the platelet-activation dependent binding of FXIIIa have not been characterized nor have the sites mediating FXIIIa binding been identified. The generation of FXIIIa and consequent detection of FXIIIa on the platelet surface were compared with other thrombin- induced activation events; the rate at which FXIIIa bound to activated platelets was much slower than platelet degranulation or fibrin(ogen) binding. Whereas platelets could be rapidly induced to express a functional receptor for FXIIIa, the rate of FXIIIa binding to platelets is limited by the rate of conversion of FXIII to FXIIIa. Immunoprecipitation of radiolabeled platelets using polyclonal anti- FXIII A-chain antibody identified two proteins corresponding to GPIIb and GPIIIa. Preincubation of intact platelets with 7E3, a monoclonal antibody that blocks the fibrinogen binding site, or GRGDSP peptide inhibited FXIIIa binding by about 95% when measured by flow cytometry; FXIIIa binding to purified GPIIb-IIIa was also inhibited by 7E3. The binding of FXIIIa to purified GPIIb-IIIa was enhanced by the addition of fibrinogen, but not by that of fibronectin or thrombospondin, suggesting that FXIIIa also binds to fibrinogen associated with the complex. These observations suggest that activated platelets bearing FXIIIa may enhance stabilization of platelet-rich thrombi through surface-localized cross-linking events.

Anti-fibrinogen antibody mediates fibrinogen binding to platelet membrane glycoprotein IIb-IIIa

1993 ◽  
Vol 85 (2) ◽  
pp. 341-347 ◽  
Author(s):  
Hiroshi Mohri ◽  
Juichi Tanabe ◽  
Hiroyuki Fujita ◽  
Heiwa Kanamori ◽  
Takao Ohkubo
Keyword(s):  
Platelet Membrane ◽  
Membrane Glycoprotein ◽  
Platelet Membrane Glycoprotein ◽  
Fibrinogen Binding

scholarly journals Fc-independent cross-linking of a novel platelet membrane protein by a monoclonal antibody causes platelet activation

Blood ◽  
1994 ◽  
Vol 84 (2) ◽  
pp. 547-555 ◽  
Author(s):  
S De Reys ◽  
MF Hoylaerts ◽  
M De Ley ◽  
J Vermylen ◽  
H Deckmyn
Keyword(s):  
Monoclonal Antibody ◽  
Membrane Protein ◽  
Platelet Activation ◽  
Cell Types ◽  
Serotonin Release ◽  
Platelet Membrane ◽  
Cross Linking ◽  
Antiplatelet Antibody ◽  
Affinity Isolation ◽  
Isoelectric Ph

Abstract A monoclonal antiplatelet antibody (MA-13G8E1) is described that dose- dependently induces platelet aggregation and serotonin release in an Fc- independent fashion. Whereas platelets were equally aggregated by F(ab')2 fragments of this monoclonal antibody (MoAb), its Fab fragments, on the other hand, were inactive, indicating that divalent interaction is an essential requirement to induce platelet activation by MA-13G8E1. In addition, we could show that platelet epitope cross- linking by MA-13G8E1 occurred on the same platelet. MA-13G8E1 stimulated platelet phospholipase C (PLC) and induced activation of protein kinase C (PKC), both of which were almost unaffected by aspirin pretreatment. Furthermore, PLC activation appeared to be a direct antibody-mediated effect, since intracellular Ca2+ rises were not inhibited by EGTA, cytochalasin B, or aggregation-blocking MA-16N7C2 (antiglycoprotein [anti-GP]IIb/IIa). The MA-13G8E1 antigen is constitutively expressed on resting platelets of different species (7,100 +/- 800 molecules per human platelet), but not on other cell types tested. Both immunoprecipitation and affinity isolation by MA- 13G8E1 showed two low-molecular weight proteins (45 and 36 kD), having slightly acidic isoelectric pH levels (4.5 to 5.5) and forming multimolecular complexes. In conclusion, we found an MoAb that is able to induce platelet activation in an Fc-independent fashion. The mechanism involves cross-linking of a hitherto undescribed platelet membrane protein, leading to PLC and PKC stimulation.

Immunochemical characterization of a new platelet specific monoclonal antibody and its use to demonstrate the cytoskeletal association of the platelet glycoprotein IIb-IIIa complex

1986 ◽  
Vol 6 (3) ◽  
pp. 323-333 ◽  
Author(s):  
Christopher Bird ◽  
Marion Callus ◽  
Lynne Trickett ◽  
Robin Thorpe
Keyword(s):  
Monoclonal Antibody ◽  
Fibroblast Cell ◽  
Cytoskeletal Proteins ◽  
Platelet Membrane ◽  
Platelet Glycoprotein ◽  
Membrane Glycoprotein ◽  
Polymorphonuclear Leucocytes ◽  
Microfilament System ◽  
Fibroblast Cell Lines

We describe the production and characterization of a monoclonal antibody specific for platelets. This antibody reacts strongly with human and primate platelets, but does not recognise human monocytes, polymorphonuclear leucocytes, lymphocytes, erythrocytes, leukaemic nor fibroblast cell lines, nor rodent platelets. Immunoprecipitation studies using radiolabelled platelet membrane proteins showed that the monoclonal antibody binds to the platelet membrane glycoprotein IIb-IIIa complex. Affinity chromatography using immobilized monoclonal antibody allows purification of the antigen, but also co-purifies the cytoskeletal proteins actin and myosin.Our results demonstrate immunochemically that although the GP IIb-IIIa complex is an external structure, it is connected through the cell membrane to the microfilament system.

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