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.

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

NORMAL EXPRESSION OF THROMBOSPONDIN RECEPTOR SITES ON PLATELETS OF THREE GLANZMANN THROMBASTHENIC PATIENTS

1987 ◽  
Author(s):  
J L McGregor ◽  
H Boukerche
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Aggregation ◽  
Whole Blood ◽  
Binding Sites ◽  
Competitive Binding ◽  
Fibrinogen Binding ◽  
Receptor Sites ◽  
Low Concentrations ◽  
Normal Expression ◽  
Stimulation Of

A well characterised anti-thrombospondin (TSP) monoclonal antibody (Mab) LYP8 was used to investigate the presence of TSP receptors on Glanzmann thrombasthenic (G.T.) platelets. LYP8 inhibited platelet aggregation induced by low concentrations of thrombin (0.05 U/ml) or collagen (0.5 ug/ml). The presence of LYP8 did not affect the number of sites and Kd of 125I-fibrinogen binding to thrombin-stimulated normal platelets. Binding of LYP8 to normal platelets was minimal in whole blood (300 IgG molecules/ olatelet), increased in citrated PRP (1187 ± 209 IgG molecules/ platelet) and washed platelets (2967 ± 1278 IgG molecules/platelet) . Thrombin stimulation of platelets, washed in the presence of 2 mM calcium, increased the number of LYP8 binding sites (14917 ± 42n IgG molecules/platelet). Addition of EDTA (5mM) to thrombin-stimulated platelets did not reduce the number of LYP8 binding sites. The number of LYP8 binding sites on thrombin-stimulated platelets of three Glanzmann thrombasthenic patients (showing an absence of the glycoprotein (GP) lib and IIIa)was similar to normals in the presence of 2 mM calcium or 5 mM EDTA. In competitive binding, Mab LYP18 directed against the GPIIb-IIIa complex did not inhibit the binding of labelled monoclonal antibody LYP8. These results strongly suggest that TSP binds to a membrane receptor different from the GPIIb-IIIa complex in the presence of calcium or EDTA. This unidentified receptor may be GPIV also known as GPIIIb (Asch, A. et al. Clin. Res. 1986, 34:450A).

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

1998 ◽  
Vol 79 (01) ◽  
pp. 177-185 ◽  
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.

Specific Cross-reaction of IgG Anti-phospholipid Antibody with Platelet Glycoprotein IIIa

1996 ◽  
Vol 75 (01) ◽  
pp. 168-174 ◽  
Author(s):  
Shigeru Tokita ◽  
Morio Arai ◽  
Naomasa Yamamoto ◽  
Yasuhiro Katagiri ◽  
Kenjiro Tanoue ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Heparan Sulfate ◽  
Disulfide Bonds ◽  
Dextran Sulfate ◽  
Human Platelets ◽  
Platelet Glycoprotein ◽  
Activated Platelets ◽  
Fibrinogen Binding ◽  
Dose Dependent Fashion ◽  
Glycoprotein Iiia

SummaryTo study the pathological functions of anti-phospholipid (anti-PL) antibodies, we have analyzed their effect on platelet function. We identified an IgG anti-PL mAb, designated PSG3, which cross-reacted specifically with glycoprotein (GP) IIIa in human platelets and inhibited platelet aggregation. PSG3 bound also to certain polyanionic substances, such as double-stranded DNA, heparan sulfate, dextran sulfate and acetylated-LDL, but not to other polyanionic substances. The binding of PSG3 to GPIIIa was completely inhibited by heparan sulfate and dextran sulfate, indicating that PSG3 recognizes a particular array of negative charges expressed on both GPIIIa and the specified polyanionic substances. Since neither neuraminidase- nor endoglycopeptidase F-treatment of GPIIIa had any significant effect on the binding of PSG3, this array must be located within the amino acid sequence of GPIIIa but not in the carbohydrate moiety. Reduction of the disulfide bonds in GPIIIa greatly reduced its reactivity, suggesting that the negative charges in the epitope are arranged in a particular conformation. PSG3 inhibited platelet aggregation induced by either ADP or collagen, it also inhibited fibrinogen binding to activated platelets in a dose-dependent fashion. PSG3, however, did not inhibit the binding of GRGDSP peptide to activated platelets. These results suggest that the PSG3 epitope on GPIIIa contains a particular array of negative charges, and possibly affects the fibrinogen binding to GPIIb/IIIa complex necessary for platelet aggregation.

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.

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

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.

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

1996 ◽  
Vol 76 (06) ◽  
pp. 1038-1046 ◽  
Author(s):  
Michihide Tokuhira ◽  
Makoto Handa ◽  
Tetsuji Kamata ◽  
Atsushi Oda ◽  
Masahiko Katayama ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Aggregation ◽  
Human Platelets ◽  
Antibody Binding ◽  
Murine Monoclonal Antibody ◽  
Conformational Structure ◽  
Dependent Manner ◽  
Binding Parameters ◽  
Fibrinogen Binding ◽  
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.

STIMULUS-DEPENDENT CHANGES IN THE SURFACE EXPRESSION OF GPIIb/IIIa AND FIBRINOGEN RECEPTORS. RELATIONSHIP TO PLATELET AGGREGATION

1987 ◽  
Author(s):  
K Niiya ◽  
E Hodson ◽  
R Bader ◽  
V Byers-Ward ◽  
E F Plow ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Platelet Aggregation ◽  
Divalent Cation ◽  
Surface Expression ◽  
The Other ◽  
Fab Fragment ◽  
Activated Platelets ◽  
Fibrinogen Binding ◽  
Dissociation Constant Kd

Platelet stimulation altered the binding of three monoclonal antibodies (monovalent Fab’ fragment) directed against the glycoprotein (GP)IIb/IIIa complex. We found that 47,600-60,300 molecules of antibody bound per platelet before stimulation, as compared to 89,200-146,500 molecules per platelet after thrombin stimulation. These changes were observed in parallel with a small but significant increase in the dissociation constant (Kd) of two antibodies. In contrast, no statistically significant changes were observed with ADP-stimulated platelets. The increased binding of LJ-CP3, but not of the other two antibodies, to activated platelets decreased by 3040% in the presence of EDTA at 22-25°C, suggesting the occurrence of divalent-cation mediated, activation-dependent changes in the corresponding GPIIb/IIIa epitope. Platelets stimulated by thrombin bound more fibrinogen than those stimulated by ADP, and significant differences in the extent but not in the affinity of fibrinogen binding were observed with different platelet agonists. When the pool of GPIIb/IIIa molecules exposed on the surface of unstimulated platelets was reacted with monoclonal antibody LJ-CP3 to block ADP-induced fibrinogen binding and platelet aggregation, thrombin stimulation still induced substantial binding and aggregation. This effect of thrombin required exposure of platelets to the active agonist and was not mediated by molecules released by thrombin into the medium. Therefore, platelets activated with “strong” agonists exhibit increased number of surface-oriented epitopes associated with GPIIb/IIIa. The GPIIb/IIIa molecules bearing these newly exposed epitopes are functional in that they bind fibrinogen and mediate platelet aggregation.

Relationship Between Aggregation and Binding of 125I-Fibrinogen and 45Calcium to Human Platelets

1979 ◽  
Author(s):  
E.I. Peerschke ◽  
R.A. Grant ◽  
M.B. Zucker
Keyword(s):  
Binding Sites ◽  
Calcium Binding ◽  
Silicone Oil ◽  
Human Platelets ◽  
Mechanisms Of Action ◽  
Ionized Calcium ◽  
Clot Retraction ◽  
Fibrinogen Binding ◽  
Induced Aggregation ◽  
Stimulation Of

Since calcium and fibrinogen are essential cofactors for ADP-induced aggregation, their mechanisms of action were investigated. Aspirin-treated platelets were filtered through Sepharose 2B equilibrated with cation-poor Tyrode’s solution. After adding the radioactive compounds at 22, platelets were centrifuged through silicone oil. Trapping was assessed in separate samples with 14C-sorbitoi. Calcium binding was maximal at 1 hr and with 200 uH CaCl2. Two binding sites could be demonstrated on normal and thrombasthenic platelets and on platelets which had lost their ability to aggregate (but not to change shape or promote clot retraction) after treatment with EDTA (8 min, 37°, pH 7.8). ADP did not alter calcium binding in the presence or absence of fibrinogen. Fibrinogen, however, bound to normal gel filtered platelets in the presence of ADP and ionized calcium “ but not to thrombasthenic or EDTA-treated platelets or to normal platelets in the presence of EDTA or at pH 6.5. Binding of fibrinogen increased with concentration but saturation was not observed even at physiologic levels. Fibrinogen binding was similar in gel filtered platelets and citrated piatelet-rich plasma. These studies indicate that stimulation of platelets with ADP under conditions favorable to aggregation is associated with binding of fibrinogen but not of additional calcium.

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