scholarly journals A murine monoclonal antibody that blocks fibrinogen binding to normal platelets also inhibits fibrinogen interactions with chymotrypsin- treated platelets

Blood ◽  
1984 ◽  
Vol 64 (1) ◽  
pp. 59-63 ◽  
Author(s):  
EI Peerschke ◽  
BS Coller
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Aggregation ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Membrane Glycoproteins ◽  
Molecular Weights ◽  
Fibrinogen Receptor ◽  
Fibrinogen Binding ◽  
Unstimulated Control ◽  
Lines Of Evidence

We recently described a monoclonal antibody, 10E5 , that completely blocks adenosine diphosphate (ADP) induced fibrinogen binding to platelets and aggregation induced by ADP, epinephrine, and thrombin. Multiple lines of evidence indicate that 10E5 binds to platelet membrane glycoproteins IIb and/or IIIa. Because it has been reported that platelets treated with chymotrypsin aggregate when fibrinogen is added, we tested the effect of 10E5 antibody on chymotrypsin-induced fibrinogen binding and platelet aggregation. Aspirin-treated human platelets were washed in modified Tyrode's buffer (pH 7.5), incubated for 5 minutes at 22 degrees C with 300 micrograms/mL chymotrypsin, and washed again. The amount of 10E5 antibody bound to these platelets (37,232 +/- 2,928 molecules/platelet; mean +/- SEM, N=9) was similar to that bound to unstimulated control platelets (36,910 +/- 2,669) and did not differ significantly from the amount of antibody bound to ADP- treated platelets (P less than .01, N = 5). The amount of 10E5 bound to chymotrypsin-treated platelets correlated directly with the amount of fibrinogen bound to separate aliquots of the same platelet samples (r = .876, P less than .001). The 10E5 antibody caused virtually complete inhibition of both the binding of fibrinogen to chymotrypsin-treated platelets and the aggregation induced by exogenous fibrinogen. Immunoprecipitation studies of 125I-labeled chymotrypsin-treated platelets revealed that the 10E5 antibody bound proteins with molecular weights characteristic of glycoproteins IIb and IIIa. These data suggest that the fibrinogen receptor on chymotrypsin-treated platelets is identical to that on ADP-treated platelets and that this receptor is either near to, or on, the glycoprotein IIb/IIIa complex.

scholarly journals A murine monoclonal antibody that blocks fibrinogen binding to normal platelets also inhibits fibrinogen interactions with chymotrypsin- treated platelets

Blood ◽  
1984 ◽  
Vol 64 (1) ◽  
pp. 59-63 ◽  
Author(s):  
EI Peerschke ◽  
BS Coller
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Aggregation ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Membrane Glycoproteins ◽  
Molecular Weights ◽  
Fibrinogen Receptor ◽  
Fibrinogen Binding ◽  
Unstimulated Control ◽  
Lines Of Evidence

Abstract We recently described a monoclonal antibody, 10E5 , that completely blocks adenosine diphosphate (ADP) induced fibrinogen binding to platelets and aggregation induced by ADP, epinephrine, and thrombin. Multiple lines of evidence indicate that 10E5 binds to platelet membrane glycoproteins IIb and/or IIIa. Because it has been reported that platelets treated with chymotrypsin aggregate when fibrinogen is added, we tested the effect of 10E5 antibody on chymotrypsin-induced fibrinogen binding and platelet aggregation. Aspirin-treated human platelets were washed in modified Tyrode's buffer (pH 7.5), incubated for 5 minutes at 22 degrees C with 300 micrograms/mL chymotrypsin, and washed again. The amount of 10E5 antibody bound to these platelets (37,232 +/- 2,928 molecules/platelet; mean +/- SEM, N=9) was similar to that bound to unstimulated control platelets (36,910 +/- 2,669) and did not differ significantly from the amount of antibody bound to ADP- treated platelets (P less than .01, N = 5). The amount of 10E5 bound to chymotrypsin-treated platelets correlated directly with the amount of fibrinogen bound to separate aliquots of the same platelet samples (r = .876, P less than .001). The 10E5 antibody caused virtually complete inhibition of both the binding of fibrinogen to chymotrypsin-treated platelets and the aggregation induced by exogenous fibrinogen. Immunoprecipitation studies of 125I-labeled chymotrypsin-treated platelets revealed that the 10E5 antibody bound proteins with molecular weights characteristic of glycoproteins IIb and IIIa. These data suggest that the fibrinogen receptor on chymotrypsin-treated platelets is identical to that on ADP-treated platelets and that this receptor is either near to, or on, the glycoprotein IIb/IIIa complex.

Clustering of Integrin αIIb-β3Differently Regulates Tyrosine Phosphorylation of pp72syk, PLCγ2 and pp125FAKin Concanavalin A-stimulated Platelets

1999 ◽  
Vol 81 (01) ◽  
pp. 124-130 ◽  
Author(s):  
Enrico Festetics ◽  
Alessandra Bertoni ◽  
Fabiola Sinigaglia ◽  
Cesare Balduini ◽  
Mauro Torti
Keyword(s):  
Platelet Aggregation ◽  
Tyrosine Phosphorylation ◽  
Concanavalin A ◽  
Tyrosine Kinases ◽  
Human Platelets ◽  
Membrane Glycoproteins ◽  
Platelet Surface ◽  
Fibrinogen Receptor ◽  
Natural Ligand ◽  
Fibrinogen Binding

SummaryTyrosine phosphorylation of the non-receptor tyrosine kinases pp72sykand pp125FAKand of the γ2 isoform of phospholipase C (PLCγ2) in human platelets stimulated with the lectin Concanavalin A was investigated. Concanavalin A induced the rapid tyrosine phosphorylation of pp72sykand PLCγ2 with a similar kinetics, while tyrosine phosphorylation of pp125FAKoccurred in a later phase of platelet activation. When compared with other platelet agonists, Concanavalin A revealed to be at least as potent as collagen in inducing tyrosine phosphorylation of PLCγ2 and pp125FAK, while tyrosine phosphorylation of pp72sykinduced by the lectin was much stronger than that induced by thrombin or collagen. Concanavalin A-induced tyrosine phosphorylation of pp72syk, PLCγ2 and pp125FAKwas not dependent on platelet aggregation as it occurred normally even in the absence of sample stirring and when fibrinogen binding to integrin αIIb-β3was inhibited by the peptide RGDS. Tyrosine phosphorylation of pp72syk, PLCγ2 and pp125FAKrequired the binding of the lectin to the platelet surface, but was not observed in platelets treated with succinyl-Concanavalin A, a derivative of the lectin that interacts with the same receptors but does not promote clustering of membrane glycoproteins. Moreover, the aggregation-independent tyrosine phosphorylation of pp125FAKand pp72sykinduced by Concanavalin A required the expression of integrin αIIb-β3on the platelet surface as it was strongly inhibited in platelets from patients affected by Glanzmann thrombasthenia. By contrast, tyrosine phosphorylation of PLCγ2 occurred normally also in thrombasthenic platelets stimulated with Concanavalin A. These results demonstrate that, even in the absence of aggregation, the clustering of integrin αIIb-β3induced by Concanavalin A on the platelet surface directly promotes tyrosine phosphorylation of pp72sykand pp125FAKand provide further evidence that the oligomerization of the fibrinogen receptor promoted by its natural ligand during platelet aggregation may be responsible for the tyrosine phosphorylation of these proteins induced by physiological agonists.

scholarly journals Thrombospondin promotes platelet aggregation

Blood ◽  
1988 ◽  
Vol 72 (1) ◽  
pp. 109-115
Author(s):  
GP Tuszynski ◽  
VL Rothman ◽  
A Murphy ◽  
K Siegler ◽  
KA Knudsen
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Aggregation ◽  
Human Platelets ◽  
Platelet Glycoprotein ◽  
Receptor System ◽  
Platelet Surface ◽  
Fibrinogen Receptor ◽  
Surface Receptor ◽  
Lines Of Evidence

Thrombospondin (TSP), isolated from human platelets, promotes aggregation of both nonstimulated platelets and platelets stimulated with thrombin or ADP. The TSP-promoted aggregation is specific since a monoclonal antibody against TSP inhibits the effect of exogenously added TSP and inhibits thrombin-induced platelet aggregation in the absence of added TSP. Several lines of evidence suggest that TSP mediates its effect on aggregation of nonstimulated and stimulated platelets through different platelet-surface receptor systems. The TSP- promoted aggregation of nonstimulated platelets was inhibited by a monoclonal antibody to platelet glycoprotein IV (GPIV), but not by a monoclonal antibody to the fibrinogen receptor, GPIIb-IIIa. In contrast, the antibody to GPIIb-IIIa totally inhibited the TSP- potentiated aggregation of thrombin-stimulated platelets, whereas the antibody to GPIV has no effect. Thus, these studies suggest that TSP promotes platelet aggregation by at least two mechanisms--one dependent on and one independent of the platelet fibrinogen receptor system.

scholarly journals Thrombospondin promotes platelet aggregation

Blood ◽  
1988 ◽  
Vol 72 (1) ◽  
pp. 109-115 ◽  
Author(s):  
GP Tuszynski ◽  
VL Rothman ◽  
A Murphy ◽  
K Siegler ◽  
KA Knudsen
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Aggregation ◽  
Human Platelets ◽  
Platelet Glycoprotein ◽  
Receptor System ◽  
Platelet Surface ◽  
Fibrinogen Receptor ◽  
Surface Receptor ◽  
Lines Of Evidence

Abstract Thrombospondin (TSP), isolated from human platelets, promotes aggregation of both nonstimulated platelets and platelets stimulated with thrombin or ADP. The TSP-promoted aggregation is specific since a monoclonal antibody against TSP inhibits the effect of exogenously added TSP and inhibits thrombin-induced platelet aggregation in the absence of added TSP. Several lines of evidence suggest that TSP mediates its effect on aggregation of nonstimulated and stimulated platelets through different platelet-surface receptor systems. The TSP- promoted aggregation of nonstimulated platelets was inhibited by a monoclonal antibody to platelet glycoprotein IV (GPIV), but not by a monoclonal antibody to the fibrinogen receptor, GPIIb-IIIa. In contrast, the antibody to GPIIb-IIIa totally inhibited the TSP- potentiated aggregation of thrombin-stimulated platelets, whereas the antibody to GPIV has no effect. Thus, these studies suggest that TSP promotes platelet aggregation by at least two mechanisms--one dependent on and one independent of the platelet fibrinogen receptor system.

Effects of a Monoclonal Antibody to Glycoprotein IIb/IIIa (P256) and of Enzymically Derived Fragments of P256 on Human Platelets

1991 ◽  
Vol 65 (04) ◽  
pp. 432-437 ◽  
Author(s):  
A W J Stuttle ◽  
M J Powling ◽  
J M Ritter ◽  
R M Hardisty
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Platelet Aggregation ◽  
Calcium Ions ◽  
Human Platelet ◽  
Human Platelets ◽  
In Vivo Detection ◽  
Fibrinogen Binding ◽  
Specific Antagonist

SummaryThe anti-platelet monoclonal antibody P256 is currently undergoing development for in vivo detection of thrombus. We have examined the actions of P256 and two fragments on human platelet function. P256, and its divalent fragment, caused aggregation at concentrations of 10−9−3 × 10−8 M. A monovalent fragment of P256 did not cause aggregation at concentrations up to 10−7 M. P256–induced platelet aggregation was dependent upon extracellular calcium ions as assessed by quin2 fluorescence. Indomethacin partially inhibited platelet aggregation and completely inhibited intracellular calcium mobilisation. Apyrase caused partial inhibition of aggregation. Aggregation induced by the divalent fragment was dependent upon fibrinogen and was inhibited by prostacyclin. Aggregation induced by the whole antibody was only partially dependent upon fibrinogen, but was also inhibited by prostacyclin. P256 whole antibody was shown, by flow cytometry, to induce fibrinogen binding to indomethacin treated platelets. Monovalent P256 was shown to be a specific antagonist for aggregation induced by the divalent forms. In–111–labelled monovalent fragment bound to gel-filtered platelets in a saturable and displaceable manner. Monovalent P256 represents a safer form for in vivo applications

scholarly journals Analysis of platelet aggregation disorders based on flow cytometric analysis of membrane glycoprotein IIb-IIIa with conformation-specific monoclonal antibodies

Blood ◽  
1990 ◽  
Vol 76 (10) ◽  
pp. 2017-2023 ◽  
Author(s):  
MH Ginsberg ◽  
AL Frelinger ◽  
SC Lam ◽  
J Forsyth ◽  
R McMillan ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Ligand Binding ◽  
Cell Activation ◽  
Flow Cytometric Analysis ◽  
Adenosine Diphosphate ◽  
Receptor Activation ◽  
Affinity Column ◽  
Primary Defect ◽  
Fibrinogen Receptor ◽  
Fibrinogen Binding

Abstract Normal primary platelet aggregation requires agonist-mediated activation of membrane GPIIb-IIIa, binding of fibrinogen to GPIIb-IIIa, and cellular events after ligand binding. PAC1 monoclonal antibody distinguishes between resting and activated states of GPIIb-IIIa, and other antibodies preferentially recognize GPIIb (PMI-1) or IIIa (anti- LIBS1) after the binding of fibrinogen or fibrinogen-mimetic peptides, such as GRGDSP. Using these antibodies and platelet flow cytometry, we studied two distinct persistent platelet aggregation abnormalities. Platelets from a thrombasthenic variant, which contained near-normal amounts of GPIIb-IIIa, failed to aggregate or bind PAC1 in response to agonists. In addition, GRGDSP, which binds to normal GPIIb-IIIa without prior cell activation, failed to increase the binding of PMI-1 or anti- LIBS1 to the thrombasthenic platelets, suggesting a primary defect in ligand binding. Chromatography of detergent-solubilized platelets on a KYGRGDS affinity column confirmed that the patient's GPIIb-IIIa lacked the fibrinogen binding site. In another patient with myelofibrosis and defective aggregation, PAC1 failed to bind to adenosine diphosphate- stimulated platelets, but did bind when protein kinase C was directly activated with phorbol myristate acetate. Furthermore, the binding of PMI-1 and anti-LIBS1 increased in response to GRGDSP, confirming a defect in agonist-mediated fibrinogen receptor activation rather than in fibrinogen binding or events distal to binding. These studies indicate that this immunochemical approach is useful in classification of clinical abnormalities of platelet aggregation as defects in either (a) fibrinogen receptor activation, (b) fibrinogen binding, or (c) postoccupancy events.

Interspecies Comparison of Platelet Aggregation, LIBS Expression and Clot Retraction: Observed Differences in GPIIb-IIIa Functional Activity

1995 ◽  
Vol 74 (06) ◽  
pp. 1551-1556 ◽  
Author(s):  
Lisa K Jennings ◽  
Melanie M White ◽  
Timothy D Mandrell
Keyword(s):  
Platelet Aggregation ◽  
Conformational Changes ◽  
Low Dose ◽  
Species Differences ◽  
Receptor Occupancy ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Clot Retraction ◽  
Fibrinogen Receptor ◽  
Functional Aspects

SummaryWe examined interspecies differences in the function of the platelet fibrinogen receptor, GPIIb-IIIa, by comparing platelet aggregation responses to adenosine diphosphate (ADP) added alone or in combination with a GPIIIa specific monoclonal antibody (mAb), D3. D3 can activate the GPIIb-IIIa receptor in the absence of platelet activation, and it preferentially binds to a region on the GPIIIa subunit after the GPIIb-IIIa complex is occupied by ligand. Using human, monkey, dog, rabbit and pig platelets, we examined whether all species’ platelets bound the D3 mAb similarly, and if the binding of Arg-Gly-Asp-Ser (RGDS) peptides induced the exposure of the anti-LIBS (D3) epitope as previously described for human platelets. We also evaluated how blocking of this neoantigenic region by the D3 mAb affected clot retraction, a process that requires linkage of GPIIb-IIIa with fibrin(ogen) and the platelet cytoskeleton. We found that all species tested bound the D3 mAb. Only in human and monkey platelets did D3 cause aggregation as well as inhibit clot retraction. However, in all species tested, except for pig, D3 prevented disaggregation of platelets typically observed when platelets are treated with low dose ADP. With the exception of pig platelets, there was increased D3 binding to platelets in the presence of RGDS peptides. We propose that this region of GPIIIa is important in the conformational changes that GPIIb-IIIa undergoes during the binding of ligand in most species tested. Our studies suggest 1) there are measurable inter-species differences in GPIIb-IIIa mediated platelet aggregation and clot retraction, 2) LIBS expression due to receptor occupancy is a common but not all-inclusive response and 3) interspecies comparisons may be useful in understanding structural and functional aspects of platelet GPIIb-IIIa.

scholarly journals Effects of OKM5, a monoclonal antibody to glycoprotein IV, on platelet aggregation and thrombospondin surface expression [see comments]

Blood ◽  
1990 ◽  
Vol 76 (12) ◽  
pp. 2501-2509 ◽  
Author(s):  
ML Aiken ◽  
MH Ginsberg ◽  
V Byers-Ward ◽  
EF Plow
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Aggregation ◽  
Membrane Protein ◽  
Adenosine Diphosphate ◽  
Surface Expression ◽  
Inhibitory Effect ◽  
Target Antigen ◽  
Functional Responses ◽  
Fibrinogen Binding ◽  
Intact Antibody

The monoclonal antibody, OKM5, recognizes an 88-Kd monocyte membrane protein and also binds to the platelet membrane protein, GPIV (GPIIIb, CD36). In this study, we have found that the OKM5 target epitope is present at approximately 12,000 copies per platelet and that interaction with the antibody has both stimulatory and inhibitory effects on platelet function. In the absence of other stimuli, OKM5 induced platelet aggregation, secretion, and expression of fibrinogen receptors. These stimulatory responses required intact antibody as F(ab')2 fragments were not active but blocked the stimulatory activity of the intact antibody. In contrast, exposure of platelets to OKM5 followed by another strong stimulus such as thrombin resulted in a marked suppression of fibrinogen, fibronectin, and von Willebrand factor binding to the cells. This effect was not noted when a weak stimulus, adenosine diphosphate, was the second agonist. At OKM5 concentrations that interfered with fibrinogen binding to thrombin- stimulated platelets by 80% to 90%, platelet binding of exogenous thrombospondin, or surface expression of endogenous thrombospondin was not affected. The inhibitory effect of OKM5 on fibrinogen binding to thrombin-stimulated platelets was related to the formation of massive platelet aggregates in the samples. These results show that interaction of OKM5 with its target antigen on platelets can elicit diverse functional responses from the cells.

scholarly journals Biochemical and functional consequences of dissociation of the platelet membrane glycoprotein IIb-IIIa complex

Blood ◽  
1985 ◽  
Vol 66 (1) ◽  
pp. 92-98 ◽  
Author(s):  
SJ Shattil ◽  
LF Brass ◽  
JS Bennett ◽  
P Pandhi
Keyword(s):  
Platelet Aggregation ◽  
Surface Membrane ◽  
Broad Band ◽  
Adenosine Diphosphate ◽  
Platelet Membrane ◽  
Membrane Glycoproteins ◽  
Fibrinogen Receptor ◽  
Activated Platelets ◽  
Discrete Band ◽  
Functional Consequences

Abstract The platelet membrane glycoproteins, IIb and IIIa, form a Ca2+- dependent heterodimer complex that functions as the fibrinogen receptor in activated platelets to mediate platelet aggregation. Little is known about factors that affect the IIb-IIIa complex within the platelet membrane. It has been observed that platelets incubated with ethylene glycol tetra-acetic acid (EGTA) at 37 degrees C are unable to aggregate or to bind monoclonal antibodies specific for the IIb-IIIa complex. To determine whether this is due to a dissociation of IIb from IIIa, we developed a method for quantitating the complex on nondenaturing, polyacrylamide gradient gels. Platelets were surface-labeled with 125I and then solubilized and electrophoresed in 0.2% Triton and 10 mmol/L CHAPS. Under these conditions and in the presence of 1 mmol/L Ca2+, glycoproteins IIb and IIIa migrated on the gels as a discrete band at Rf = 0.33. Protein that was eluted from this band bound to an immunoaffinity column specific for the IIb-IIIa complex. In contrast, when the IIb-IIIa complex was solubilized and then dissociated with EGTA, the discrete band at Rf = 0.33 was no longer present, and IIb and IIIa were now found in a broad band at Rf = 0.45 to 0.50. To study IIb and IIIa within the surface membrane, the 125I-labeled platelets were first incubated with 0.5 mmol/L EGTA (1 nmol/L free Ca2+) at 22 degrees C and then solubilized in the absence of EGTA. The IIb and IIIa from these platelets migrated at Rf = 0.33, indicating the presence of the intact IIb-IIIa complex. In contrast, when the platelets were incubated at 37 degrees C for one hour with the EGTA, the discrete band at Rf = 0.33 representing the IIb-IIIa complex gradually disappeared. This phenomenon could not be reversed by adding Ca2+ back to the platelets before solubilization and electrophoresis. This loss of the IIb-IIIa complex from intact platelets was accompanied by (a) a progressive and irreversible decrease in adenosine diphosphate (ADP)-induced platelet aggregation and (b) decreased binding of a complex-dependent monoclonal antibody to the platelets. These studies demonstrate that when platelets are exposed to low Ca2+ at 37 degrees C, the IIb-IIIa heterodimer complexes in their surface membranes are irreversibly disrupted. Because intact IIb-IIIa complexes are required for platelet aggregation, the loss of these complexes may account for the failure of these platelets to aggregate in response to ADP.

Studies on the Binding of 3H-SR121566, an Inhibitor of Gp IIb-IIIa Activation

2001 ◽  
Vol 85 (04) ◽  
pp. 702-709 ◽  
Author(s):  
P. Savi ◽  
G. Zamboni ◽  
O. Rescanières ◽  
J. M. Herbert
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Platelet Aggregation ◽  
Binding Sites ◽  
Human Platelets ◽  
Gamma Chain ◽  
Activated Platelets ◽  
Fibrinogen Binding ◽  
Direct Competition ◽  
Stimulation Of

SummarySR121566 is a new synthetic agent which inhibits the binding of fibrinogen to activated platelets, and platelet aggregation. 3H-SR121566 bound with nanomolar affinity (KD ranging from 45 to 72 nM) to Gp IIb-IIIa expressing cells only. On activated human platelets, this ligand allowed the detection of a maximal number of 100-140,000 binding sites. The binding of SR121566 to platelets, was displaced by several agents including RGD-containing peptides and synthetic RGD mimetics, but not by ReoPro®, a humanised monoclonal antibody which inhibits the binding of fibrinogen to the Gp IIb-IIIa complex. Neither the fibrinogen dodecapeptide nor fibrinogen itself were able to compete with SR121566 whether platelets were activated or not.Flow cytometry studies indicated that SR121566 which did not activate Gp IIb-IIIa by itself, dose-dependently prevented the detection of activation-induced binding sites on TRAP-stimulated platelets in the presence or absence of exogenous fibrinogen, indicating a direct effect on the activation state of the Gp IIb-IIIa complex. Moreover, SR121566 was able to reverse the activation of Gp IIb-IIIa and to displace the binding of fibrinogen when added up to 5 min after TRAP stimulation of platelets. When added at later times (15 to 30 min), SR121566 failed to displace fibrinogen binding, even if SR121566 binding sites were still accessible and the Gp IIb-IIIa complex not activated.In conclusion, our study is in accordance with the finding that fibrinogen is recognised by the activated Gp IIb-IIIa complex through the dodecapeptide sequence present on its gamma chain, and that this interaction is inhibited by SR121566 by preventing and reversing the activated conformation of Gp IIb-IIIa and not by direct competition with fibrinogen.

Sign in / Sign up

Export Citation Format

Share Document