Increased surface expression of the membrane glycoprotein IIb/IIIa complex induced by platelet activation. Relationship to the binding of fibrinogen and platelet aggregation

Platelet activation altered the binding of three monoclonal antibodies (monovalent Fab' fragment) directed against the glycoprotein (GP) IIb/IIIa complex. An increased binding of two- to threefold occurred after stimulation with thrombin or phorbol myristate acetate (PMA), with slight but significant increase in the dissociation constants (Kd) of two antibodies (LJ-CP8 and LJ-P9). 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 30% to 40% in the presence of EDTA at 22 to 25 degrees C. Platelets stimulated by thrombin or PMA bound more fibrinogen than did those stimulated by ADP, and significant differences in the extent but not in the affinity of fibrinogen binding were observed with various platelet agonists. When the pool of GP IIb/IIIa molecules exposed on the surface of unstimulated platelets was reacted with the monoclonal antibody LJ-CP3 to block ADP-induced fibrinogen binding and platelet aggregation, stimulation with thrombin or PMA still induced substantial binding of antibody and fibrinogen, and aggregation ensued. Therefore, platelets exposed to “strong” agonists exhibit an increased number of surface-oriented epitopes associated with GP IIb/IIIa. The GP IIb/IIIa molecules bearing these newly exposed epitopes are functional in that they can bind fibrinogen and mediate platelet aggregation.

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Increased surface expression of the membrane glycoprotein IIb/IIIa complex induced by platelet activation. Relationship to the binding of fibrinogen and platelet aggregation

Blood ◽

10.1182/blood.v70.2.475.475 ◽

1987 ◽

Vol 70 (2) ◽

pp. 475-483 ◽

Cited By ~ 176

Author(s):

K Niiya ◽

E Hodson ◽

R Bader ◽

V Byers-Ward ◽

JA Koziol ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Dissociation Constants ◽

Surface Expression ◽

The Other ◽

Membrane Glycoprotein ◽

Myristate Acetate ◽

Fab Fragment ◽

Activated Platelets ◽

Fibrinogen Binding

Abstract Platelet activation altered the binding of three monoclonal antibodies (monovalent Fab' fragment) directed against the glycoprotein (GP) IIb/IIIa complex. An increased binding of two- to threefold occurred after stimulation with thrombin or phorbol myristate acetate (PMA), with slight but significant increase in the dissociation constants (Kd) of two antibodies (LJ-CP8 and LJ-P9). 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 30% to 40% in the presence of EDTA at 22 to 25 degrees C. Platelets stimulated by thrombin or PMA bound more fibrinogen than did those stimulated by ADP, and significant differences in the extent but not in the affinity of fibrinogen binding were observed with various platelet agonists. When the pool of GP IIb/IIIa molecules exposed on the surface of unstimulated platelets was reacted with the monoclonal antibody LJ-CP3 to block ADP-induced fibrinogen binding and platelet aggregation, stimulation with thrombin or PMA still induced substantial binding of antibody and fibrinogen, and aggregation ensued. Therefore, platelets exposed to “strong” agonists exhibit an increased number of surface-oriented epitopes associated with GP IIb/IIIa. The GP IIb/IIIa molecules bearing these newly exposed epitopes are functional in that they can bind fibrinogen and mediate platelet aggregation.

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

10.1055/s-0038-1643957 ◽

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.

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Heparin-induced thrombocytopenia: new evidence for the dynamic binding of purified anti-PF4–heparin antibodies to platelets and the resultant platelet activation

Blood ◽

10.1182/blood.v96.1.182.013k18_182_187 ◽

2000 ◽

Vol 96 (1) ◽

pp. 182-187 ◽

Cited By ~ 3

Author(s):

Peter M. Newman ◽

Beng H. Chong

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Surface Expression ◽

Dynamic Binding ◽

Platelet Factor ◽

Heparin Induced Thrombocytopenia ◽

Activated Platelets ◽

Igg Binding ◽

Iodine 125 ◽

First Time

Immune heparin-induced thrombocytopenia (HIT) is associated with antibodies directed against a complex of platelet factor 4 (PF4) and heparin. We were able to affinity purify anti-PF4–heparin IgG (HIT IgG) from the plasma of 2 patients with HIT. Under conditions that were more physiological and sensitive than those in previous studies, we observed that this HIT IgG caused platelet aggregation on the addition of heparin. Platelets activated with HIT IgG increased their release and surface expression of PF4. We quantitated, for the first time, the binding of affinity-purified HIT iodine 125–IgG to platelets as they activated in a plasma milieu. Binding of the HIT IgG was dependent on heparin and required some degree of platelet activation. Blocking the platelet FcγRII with the monoclonal antibody IV.3 did not prevent HIT IgG binding to activated platelets. We concluded that anti-PF4–heparin IgG is the component in these HIT plasmas that induces platelet aggregation. The Fab region of HIT IgG binds to PF4–heparin on the surface of activated platelets. We propose that only then does the Fc portion of the bound IgG further activate the same or adjacent platelets through the Fc receptor. Our data support a dynamic model of platelet activation in which released PF4 enhances further antibody binding and more release.

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Heparin-induced thrombocytopenia: new evidence for the dynamic binding of purified anti-PF4–heparin antibodies to platelets and the resultant platelet activation

Blood ◽

10.1182/blood.v96.1.182 ◽

2000 ◽

Vol 96 (1) ◽

pp. 182-187 ◽

Cited By ~ 130

Author(s):

Peter M. Newman ◽

Beng H. Chong

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Surface Expression ◽

Dynamic Binding ◽

Platelet Factor ◽

Heparin Induced Thrombocytopenia ◽

Activated Platelets ◽

Igg Binding ◽

Iodine 125 ◽

First Time

Abstract Immune heparin-induced thrombocytopenia (HIT) is associated with antibodies directed against a complex of platelet factor 4 (PF4) and heparin. We were able to affinity purify anti-PF4–heparin IgG (HIT IgG) from the plasma of 2 patients with HIT. Under conditions that were more physiological and sensitive than those in previous studies, we observed that this HIT IgG caused platelet aggregation on the addition of heparin. Platelets activated with HIT IgG increased their release and surface expression of PF4. We quantitated, for the first time, the binding of affinity-purified HIT iodine 125–IgG to platelets as they activated in a plasma milieu. Binding of the HIT IgG was dependent on heparin and required some degree of platelet activation. Blocking the platelet FcγRII with the monoclonal antibody IV.3 did not prevent HIT IgG binding to activated platelets. We concluded that anti-PF4–heparin IgG is the component in these HIT plasmas that induces platelet aggregation. The Fab region of HIT IgG binds to PF4–heparin on the surface of activated platelets. We propose that only then does the Fc portion of the bound IgG further activate the same or adjacent platelets through the Fc receptor. Our data support a dynamic model of platelet activation in which released PF4 enhances further antibody binding and more release.

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

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650238 ◽

1996 ◽

Vol 75 (01) ◽

pp. 168-174 ◽

Cited By ~ 13

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.

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Extracellular fibrinogen binding protein, Efb, from Staphylococcus aureus binds to platelets and inhibits platelet aggregation

Thrombosis and Haemostasis ◽

10.1160/th03-05-0287 ◽

2004 ◽

Vol 91 (04) ◽

pp. 779-789 ◽

Cited By ~ 24

Author(s):

Oonagh Shannon ◽

Jan-Ingmar Flock

Keyword(s):

Platelet Aggregation ◽

Potent Inhibitor ◽

Specific Binding ◽

Binding Protein ◽

Dependent Manner ◽

Platelet Surface ◽

Putative Receptor ◽

Activated Platelets ◽

Fibrinogen Binding ◽

Dose Dependent

Summary S. aureus produces and secretes a protein, extracellular fibrinogen binding protein (Efb), which contributes to virulence in wound infection. We have shown here that Efb is a potent inhibitor of platelet aggregation. Efb can bind specifically to platelets by two mechanisms; 1) to fibrinogen naturally bound to the surface of activated platelets and 2) also directly to a surface localized component on the platelets. This latter binding of Efb is independent of fibrinogen. The specific binding of Efb to the putative receptor on the platelet surface results in a stimulated, non-functional binding of fibrinogen in a dose dependent manner, distinct from natural binding of fibrinogen to platelets. The natural binding of fibrinogen to GPIIb/IIIa on activated platelets could be blocked by a monoclonal antibody against this integrin, whereas the Efb-mediated fibrinogen binding could not be blocked. The enhanced Efb-dependent fibrinogen binding to platelets is of a nature that does not promote aggregation of the platelets; instead it inhibits aggregation. The anti-thrombotic action of Efb may explain the effect of Efb on wound healing, which is delayed in the presence of Efb.

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Abstract 40: Platelet Fragmentation During the Hemostatic Response and its Prevention by a P2Y12 Antagonist

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.37.suppl_1.40 ◽

2017 ◽

Vol 37 (suppl_1) ◽

Author(s):

Maurizio Tomaiuolo ◽

Chelsea N Matzko ◽

Izmarie Poventud-Fuentes ◽

Leonard Nettey ◽

Brad A Herbig ◽

...

Keyword(s):

Platelet Activation ◽

Surface Expression ◽

Large Mass ◽

Major Step ◽

Jugular Veins ◽

Injury Site ◽

Activated Platelets ◽

Platelet Signaling ◽

P2y12 Antagonist

Previous studies using intravital microscopy have shown that hemostatic plugs formed in the mouse microvasculature have a characteristic architecture in which the extent of platelet activation reflects gradients in the distribution of platelet agonists radiating outwards from the injury site. In that setting, we found minimal overlap of thrombin and ADP signaling, with thrombin primarily responsible for robust platelet activation close to the injury site and P2Y 12 -mediated ADP signaling resulting in accumulation of minimally activated platelets. Here we have taken these studies a major step forward by integrating fluorescence with scanning electron microscopy. Hemostatic plugs produced by needle injury in mouse jugular veins were imaged in situ 1 to 20 min after injury. The results show with unprecedented detail what could only be inferred previously, showing that platelet size, morphology and packing density vary remarkably depending on spatial localization within the hemostatic plug. The intraluminal and extravascular portions of the hemostatic mass presented distinct architectures. A large mass comprised almost exclusively of platelets was observed on the interior surface of the vein. Platelets closest to the injury edge had a highly activated morphology, including P-selectin surface expression, dense packing and platelet fragmentation, while those farther from the injury edge often remained discoid. In contrast, the extravascular portion of the hemostatic mass was rich in densely-packed, platelet-derived fragments intertwined with fibrin. Hemostatic plugs from mice treated with a P2Y 12 inhibitor were significantly smaller. The platelet activation gradient described above was less apparent and, notably, fragmentation of the platelets close to the injury edge was not observed with the inhibitor present. In conclusion, our findings indicate that 1) the development of a platelet activation gradient is a conserved feature of the hemostatic response across different vessels, 2) fragmentation of platelets closest to the injury site occurs very rapidly following injury, and 3) clinically relevant platelet signaling pathways play a role in regulating its formation.

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

Blood ◽

10.1182/blood.v83.4.1006.1006 ◽

1994 ◽

Vol 83 (4) ◽

pp. 1006-1016 ◽

Cited By ~ 29

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.

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Prostaglandin E2 potentiates platelet aggregation by priming protein kinase C

Blood ◽

10.1182/blood.v82.9.2704.2704 ◽

1993 ◽

Vol 82 (9) ◽

pp. 2704-2713 ◽

Cited By ~ 53

Author(s):

R Vezza ◽

R Roberti ◽

GG Nenci ◽

P Gresele

Keyword(s):

Protein Kinase C ◽

Platelet Aggregation ◽

Protein Kinase ◽

Prostaglandin E2 ◽

Platelet Activation ◽

Human Platelets ◽

Platelet Surface ◽

Kinase C ◽

Activated Platelets ◽

Induced Aggregation

Abstract Prostaglandin E2 (PGE2) is produced by activated platelets and by several other cells, including capillary endothelial cells. PGE2 exerts a dual effect on platelet aggregation: inhibitory, at high, supraphysiologic concentrations, and potentiating, at low concentrations. No information exists on the biochemical mechanisms through which PGE2 exerts its proaggregatory effect on human platelets. We have evaluated the activity of PGE2 on human platelets and have analyzed the second messenger pathways involved. PGE2 (5 to 500 nmol/L) significantly enhanced aggregation induced by subthreshold concentrations of U46619, thrombin, adenosine diphosphate (ADP), and phorbol 12-myristate 13-acetate (PMA) without simultaneously increasing calcium transients. At a high concentration (50 mumol/L), PGE2 inhibited both aggregation and calcium movements. PGE2 (5 to 500 nmol/L) significantly enhanced secretion of beta-thromboglobulin (beta TG) and adenosine triphosphate from U46619- and ADP-stimulated platelets, but it did not affect platelet shape change. PGE2 also increased the binding of radiolabeled fibrinogen to the platelet surface and increased the phosphorylation of the 47-kD protein in 32P- labeled platelets stimulated with subthreshold doses of U46619. Finally, the amplification of U46619-induced aggregation by PGE2 (500 nmol/L) was abolished by four different protein kinase C (PKC) inhibitors (calphostin C, staurosporine, H7, and TMB8). Our results suggest that PGE2 exerts its facilitating activity on agonist-induced platelet activation by priming PKC to activation by other agonists. PGE2 potentiates platelet activation at concentrations produced by activated platelets and may thus be of pathophysiologic relevance.

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