A Novel Regulatory Epitope Defined by a Murine Monoclonal Antibody to the Platelet GPIIb-IIIa Complex (αIIbβ3 Integrin)

SummaryWe characterized a murine monoclonal antibody, PT25-2 (IgG1), raised against washed human platelets. The antibody and its Fab fragments were both capable of inducing platelet aggregation in a fibrinogen-dependent manner and induced 125I-fibrinogen binding to unstimulated platelets (120,000 molecules/platelet at a 100 nM IgG concentration). The antibody immunoprecipitated the αIIbβ3 complex from lysates of iodinated platelets but did not react with the respective subunits when complex formation was disrupted by treatment with 5 mM EDTA at 37°C for 30 min. However, simply removing the extracellular divalent cation with EDTA had no effect on antibody binding indicating that the antibody’s epitope depends upon a conformational structure maintained by αβ subunit association. Antibody binding to unstimulated, washed platelets yielded binding parameters (Kd = 40 nM, Bmax = 100,000 molecules/platelet), which were found to be virtually unchanged when binding was performed using thrombin or RGDS-peptide-stimulated platelets. Thus, the PT25-2 antibody defines a novel regulatory epitope expressed by the αIIbβ3 integrin on unstimulated, quiescent platelets.

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

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648166 ◽

1991 ◽

Vol 65 (04) ◽

pp. 432-437 ◽

Cited By ~ 9

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

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Partial Inhibition of Platelet Aggregation and Fibrinogen Binding by a Murine Monoclonal Antibody to GPIIIa: Requirement for Antibody Bivalency

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648991 ◽

1994 ◽

Vol 72 (06) ◽

pp. 964-972 ◽

Cited By ~ 16

Author(s):

Jeffery L Kutok ◽

Barry S Coller

Keyword(s):

Monoclonal Antibody ◽

Platelet Aggregation ◽

Murine Monoclonal Antibody ◽

Platelet Glycoprotein ◽

Igg Antibody ◽

Surface Receptors ◽

Partial Inhibition ◽

Fibrinogen Binding ◽

Igg Binding ◽

Agonist Concentration

SummaryWe produced a murine monoclonal antibody, 7H2, and localized its epitope to one or more small regions on platelet glycoprotein (GP) Ilia. 7H2-IgG and 7H2-F(ab’)2 completely inhibit platelet aggregation and fibrinogen binding at low agonist concentrations, but only partially inhibit aggregation and fibrinogen binding at high agonist concentrations; 7H2-Fab has no effect on aggregation or fibrinogen binding at any agonist concentration. 7H2-IgG binds to the entire platelet population as judged by flow cytometry. At near saturating concentrations, ∼40,000 7H2-IgG antibody molecules bind per platelet. In contrast, ∼80,000 7H2 Fab molecules bind per platelet, suggesting that 7H2-IgG binding is bivalent. 7H2 was unable to inhibit fibrinogen binding to purified, immobilized GPIIb/IIIa. These data indicate that the bivalent binding of 7H2 to GPIIIa is required for its partial inhibition of fibrinogen binding to platelets, perhaps through dimerization of GPIIb/IIIa surface receptors (or more complex GPIIb/IIIa redistribution triggered by 7H2 binding) resulting in limited accessibility of fibrinogen to its binding site(s).

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Human Anti-Streptokinase Antibodies Induce Platelet Aggregation in an Fc Receptor (CD32) Dependent Manner

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649851 ◽

1995 ◽

Vol 74 (03) ◽

pp. 938-942 ◽

Cited By ~ 13

Author(s):

Jamal Lebrazi ◽

Gérard Helft ◽

Mustapha Abdelouahed ◽

Ismaïl Elalamy ◽

Massoud Mirshahi ◽

...

Keyword(s):

Platelet Aggregation ◽

Response Curve ◽

Human Platelets ◽

Antibody Concentration ◽

Dependent Manner ◽

Platelet Response ◽

Induce Platelet Aggregation ◽

Fibrinogen Binding ◽

Two Factors ◽

Unimodal Response

SummaryExposure to streptokinase (SK) elicits anti-SK antibodies (Abs), which inhibit fibrinolysis and induce platelet aggregation. The mechanism of the latter is not fully understood, although it seems to involve platelet binding by a plasminogen streptokinase and anti-SK ternary complex. Anti-SK Abs were purified by affinity chromatography from serum of patients having received SK for acute myocardial infarction (AMI), and were shown to be of the IgG type. Their effects were studied with (i) human platelets in citrated plasma in the presence of SK or acetylated plasminogen-SK activator complex (APSAC), and (ii) in washed platelets, resuspended in Tyrode buffer after lowering the ionic strength, in the presence of APSAC (which provides both SK and plasminogen). An antibody concentration-response curve was obtained, showing a plateau in the presence of 0.1 mg/ml IgG. By increasing the concentration of APSAC, we obtained a unimodal response curve, the optimal concentration of APSAC being 0.05 U/ml. Aggregation was suppressed by chelating calcium with EDTA, blocking fibrinogen binding by the synthetic peptide Arg-Gly-Asp-Ser (RGDS), and raising intraplatelet cAMP with Iloprost (a prostacyclin analogue). Aggregation required the interaction of the anti-SK Ab Fc domain with the platelet Fc-gamma receptor type II, also known as CD32, since: (i) it was blocked by the monoclonal antibody IV-3 directed against CD32, (ii) it did not occur with F(ab)’2 fragments, which block the response to the intact IgG. The clinical relevance of these platelet-activating anti-SK antibodies remains to be determined. Two factors might influence clinical outcome: (i) the amount and type of pre-existing anti-SK Abs; (ii) the known interindividual variability of the platelet response to binding and activation by IgG involving the CD32 molecule.

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

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615656 ◽

2001 ◽

Vol 85 (04) ◽

pp. 702-709 ◽

Cited By ~ 3

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.

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

Blood ◽

10.1182/blood.v64.1.59.bloodjournal64159 ◽

1984 ◽

Vol 64 (1) ◽

pp. 59-63 ◽

Cited By ~ 1

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.

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Exposure of platelet fibrinogen receptors by a monoclonal antibody to CD9 antigen

Blood ◽

10.1182/blood.v72.1.224.224 ◽

1988 ◽

Vol 72 (1) ◽

pp. 224-229 ◽

Cited By ~ 39

Author(s):

T Hato ◽

K Ikeda ◽

M Yasukawa ◽

A Watanabe ◽

Y Kobayashi

Keyword(s):

Monoclonal Antibody ◽

Platelet Aggregation ◽

Lymphoblastic Leukemia ◽

Combined Treatment ◽

Creatine Phosphate ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Fibrinogen Binding ◽

Atp Secretion

Abstract We found that a monoclonal antibody to CD9 antigen, PMA2, induces fibrinogen binding to platelets and examined the mechanism for this. That PMA2 recognized the CD9 antigen was confirmed by its immunoblot- reactivity with a 24,000-dalton protein, reactivity with platelets and common acute lymphoblastic leukemia (ALL) cells, and competitive binding with the ALB6 antibody known as the CD9 antibody. At saturation, PMA2 bound to approximately 46,000 sites per platelet. The binding of 125I-fibrinogen to platelets occurred in a PMA2 concentration-dependent manner and was blocked by EDTA or an anti- glycoprotein (GP)IIb-IIIa monoclonal antibody. PMA2-stimulated platelets caused ATP secretion and thromboxane B2 synthesis under non- stirred conditions. The role of secreted ADP and thromboxane in fibrinogen-binding and subsequent platelet aggregation was studied using creatine phosphate/creatine phosphokinase (CP/CPK) and aspirin. CP/CPK or aspirin alone reduced fibrinogen binding to 20% to 30%; however, this binding was sufficient to support full platelet aggregation. Combined treatment with CP/CPK and aspirin abolished fibrinogen binding and aggregation. These results demonstrate that the binding of IgG molecules to the CD9 antigen exposes fibrinogen receptors through both secreted ADP and thromboxane and that either one of both can expose the receptors to an extent sufficient to aggregate platelets.

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

Blood ◽

10.1182/blood.v64.1.59.59 ◽

1984 ◽

Vol 64 (1) ◽

pp. 59-63 ◽

Cited By ~ 19

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.

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Exposure of platelet fibrinogen receptors by a monoclonal antibody to CD9 antigen

Blood ◽

10.1182/blood.v72.1.224.bloodjournal721224 ◽

1988 ◽

Vol 72 (1) ◽

pp. 224-229

Author(s):

T Hato ◽

K Ikeda ◽

M Yasukawa ◽

A Watanabe ◽

Y Kobayashi

Keyword(s):

Monoclonal Antibody ◽

Platelet Aggregation ◽

Lymphoblastic Leukemia ◽

Combined Treatment ◽

Creatine Phosphate ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Fibrinogen Binding ◽

Atp Secretion

We found that a monoclonal antibody to CD9 antigen, PMA2, induces fibrinogen binding to platelets and examined the mechanism for this. That PMA2 recognized the CD9 antigen was confirmed by its immunoblot- reactivity with a 24,000-dalton protein, reactivity with platelets and common acute lymphoblastic leukemia (ALL) cells, and competitive binding with the ALB6 antibody known as the CD9 antibody. At saturation, PMA2 bound to approximately 46,000 sites per platelet. The binding of 125I-fibrinogen to platelets occurred in a PMA2 concentration-dependent manner and was blocked by EDTA or an anti- glycoprotein (GP)IIb-IIIa monoclonal antibody. PMA2-stimulated platelets caused ATP secretion and thromboxane B2 synthesis under non- stirred conditions. The role of secreted ADP and thromboxane in fibrinogen-binding and subsequent platelet aggregation was studied using creatine phosphate/creatine phosphokinase (CP/CPK) and aspirin. CP/CPK or aspirin alone reduced fibrinogen binding to 20% to 30%; however, this binding was sufficient to support full platelet aggregation. Combined treatment with CP/CPK and aspirin abolished fibrinogen binding and aggregation. These results demonstrate that the binding of IgG molecules to the CD9 antigen exposes fibrinogen receptors through both secreted ADP and thromboxane and that either one of both can expose the receptors to an extent sufficient to aggregate platelets.

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Functional Characterization of PM6/13, a β3-specific (GPIIIa/CD61) Monoclonal Antibody that Shows Preferential Inhibition of Fibrinogen Binding over Fibronectin Binding to Activated Human Platelets

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614247 ◽

1998 ◽

Vol 79 (01) ◽

pp. 177-185 ◽

Cited By ~ 10

Author(s):

Ashia Siddiqua ◽

Michael Wilkinson ◽

Vijay Kakkar ◽

Yatin Patel ◽

Salman Rahman ◽

...

Keyword(s):

Monoclonal Antibody ◽

Platelet Aggregation ◽

Platelet Rich Plasma ◽

Functional Characterization ◽

Human Platelets ◽

Western Blots ◽

Activated Platelets ◽

Reducing Conditions ◽

Fibronectin Binding

SummaryWe report the characterization of a monoclonal antibody (MAb) PM6/13 which recognises glycoprotein IIIa (GPIIIa) on platelet membranes and in functional studies inhibits platelet aggregation induced by all agonists examined. In platelet-rich plasma, inhibition of aggregation induced by ADP or low concentrations of collagen was accompanied by inhibition of 5-hydroxytryptamine secretion. EC50 values were 10 and 9 [H9262]g/ml antibody against ADP and collagen induced responses respectively. In washed platelets treated with the cyclooxygenase inhibitor, indomethacin, PM6/13 inhibited platelet aggregation induced by thrombin (0.2 U/ml), collagen (10 [H9262]g/ml) and U46619 (3 [H9262]M) with EC50 = 4, 8 and 4 [H9262]g/ml respectively, without affecting [14C]5-hydroxytryptamine secretion or [3H]arachidonate release in appropriately labelled cells. Studies in Fura 2-labelled platelets revealed that elevation of intracellular calcium by ADP, thrombin or U46619 was unaffected by PM6/13 suggesting that the epitope recognised by the antibody did not influence Ca2+ regulation. In agreement with the results from the platelet aggregation studies, PM6/13 was found to potently inhibit binding of 125I-fibrinogen to ADP activated platelets. Binding of this ligand was also inhibited by two other MAbs tested, namely SZ-21 (also to GPIIIa) and PM6/248 (to the GPIIb-IIIa complex). However when tested against binding of 125I-fibronectin to thrombin stimulated platelets, PM6/13 was ineffective in contrast with SZ-21 and PM6/248, that were both potent inhibitors. This suggested that the epitopes recognised by PM6/13 and SZ-21 on GPIIIa were distinct. Studies employing proteolytic dissection of 125I-labelled GPIIIa by trypsin followed by immunoprecipitation with PM6/13 and analysis by SDS-PAGE, revealed the presence of four fragments at 70, 55, 30 and 28 kDa. PM6/13 did not recognize any protein bands on Western blots performed under reducing conditions. However Western blotting analysis with PM6/13 under non-reducing conditions revealed strong detection of the parent GP IIIa molecule, of trypsin treated samples revealed recognition of an 80 kDa fragment at 1 min, faint recognition of a 60 kDa fragment at 60 min and no recognition of any product at 18 h treatment. Under similar conditions, SZ-21 recognized fragments at 80, 75 and 55 kDa with the 55kDa species persisting even after 18 h trypsin treatment. These studies confirm the epitopes recognised by PM6/13 and SZ-21 to be distinct and that PM6/13 represents a useful tool to differentiate the characteristics of fibrinogen and fibronectin binding to the GPIIb-IIIa complex on activated platelets.

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Porcine von Willebrand Factor Binding to Human Platelet GPIb Induces Transmembrane Calcium Influx

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650338 ◽

1996 ◽

Vol 75 (04) ◽

pp. 655-660 ◽

Cited By ~ 59

Author(s):

Mario Mazzucato ◽

Luigi De Marco ◽

Paola Pradella ◽

Adriana Masotti ◽

Francesco I Pareti

Keyword(s):

Monoclonal Antibody ◽

Shear Stress ◽

Platelet Aggregation ◽

Von Willebrand Factor ◽

Human Platelet ◽

Platelet Glycoprotein ◽

Dependent Manner ◽

Transmembrane Flux ◽

Von Willebrand ◽

Willebrand Factor

SummaryPorcine von Willebrand factor (P-vWF) binds to human platelet glycoprotein (GP) lb and, upon stirring (1500 rpm/min) at 37° C, induces, in a dose-dependent manner, a transmembrane flux of Ca2+ ions and platelet aggregation with an increase in their intracellular concentration. The inhibition of P-vWF binding to GP lb, obtained with anti GP lb monoclonal antibody (LJ-Ib1), inhibits the increase of intracellular Ca2+ concentration ([Ca2+]i) and platelet aggregation. This effect is not observed with LJ-Ib10, an anti GP lb monoclonal antibody which does not inhibit the vWF binding to GP lb. An anti GP Ilb-IIIa monoclonal antibody (LJ-CP8) shown to inhibit the binding of both vWF and fibrinogen to the GP IIb-IIIa complex, had only a slight effect on the [Ca2+]i rise elicited by the addition of P-vWF. No inhibition was also observed with a different anti GP IIb-IIIa monoclonal antibody (LJ-P5), shown to block the binding of vWF and not that of fibrinogen to the GP IIb-IIIa complex. PGE1, apyrase and indomethacin show a minimal effect on [Ca2+]i rise, while EGTA completely blocks it. The GP lb occupancy by recombinant vWF fragment rvWF445-733 completely inhibits the increase of [Ca2+]i and large aggregates formation. Our results suggest that, in analogy to what is seen with human vWF under high shear stress, the binding of P-vWF to platelet GP lb, at low shear stress and through the formation of aggregates of an appropriate size, induces a transmembrane flux of Ca2+, independently from platelet cyclooxy-genase metabolism, perhaps through a receptor dependent calcium channel. The increase in [Ca2+]i may act as an intracellular message and cause the activation of the GP IIb-IIIa complex.

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