Stimulation of platelet glycoprotein IIb‐IIIa (α<sub>IIb</sub>β<sub>3</sub>‐integrin) functional activity by a monoclonal antibody to the N‐terminal region of glycoprotein IIIa

Мазуров, А. В.; Хаспекова, С. Г.; Byzova, Tatiana V.; Tikhomirov, Oleg; Berndt, Michael C.; Steiner, Beat; Kouns, W C

doi:10.1016/0014-5793(96)00709-0

Cited by 27 publications

(28 citation statements)

References 21 publications

(31 reference statements)

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“…Fibrinogen binding to GP IIb/IIIa induces conformational changes, collectively termed ligand-induced binding sites. [15][16][17][18][19] Besides fibrinogen, small molecule GP IIb/IIIa antagonists, including RGD peptides, induce similar conformational changes. The observation that the DDABs analyzed here detect GP IIb/IIIa-antagonist complexes over a wide agonist concentration range suggests that the drug may not be part of the epitope.…”

Section: Discussionmentioning

confidence: 99%

“…In vitro, exposure of platelets to arginine-glycine-aspartic acid (RGD) peptides or small-molecular GP IIb/IIIa antagonists induces alterations in the conformation of GP IIb/IIIa. These neoepitopes, also referred to as ligand-induced binding sites, have been monitored by using conformationally sensitive monoclonal antibodies [15][16][17][18][19] or gel shift analysis. 20 It is currently unknown whether similar conformationally sensitive antibodies can be elicited in response to GP IIb/IIIa antagonist therapy.…”

Section: Introductionmentioning

confidence: 99%

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Evidence that thrombocytopenia observed in humans treated with orally bioavailable glycoprotein IIb/IIIa antagonists is immune mediated

Billheimer¹,

Dicker²,

Wynn³

et al. 2002

Blood

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Evidence that thrombocytopenia observed in humans treated with orally bioavailable glycoprotein IIb/IIIa antagonists is immune mediated

Billheimer¹,

Dicker²,

Wynn³

et al. 2002

Blood

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“…Activation index ϭ LIBS mAb MFI/AP2 MFI for the patient and for the control separately. Note that the epitope of CRC54 is located within the first 100 N-terminal residues of GPIIIa, 28 and to date no information on the epitope of 7C7 is available. Flow cytometric analysis of GPIIb-Ala5IIIa and GPIIb-Ala435IIIa expressed on the cell surface.…”

mentioning

confidence: 99%

Disruption of the long-range GPIIIa Cys5-Cys435 disulfide bond results in the production of constitutively active GPIIb-IIIa (αIIbβ3) integrin complexes

Sun

Liu

Wang

et al. 2002

Blood

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The major platelet integrin ␣ IIb ␤ 3 , also known as the platelet glycoprotein (GP) IIb-IIIa complex, mediates platelet aggregation by serving as the receptor for fibrinogen and von Willebrand factor. In addition to its physiologic role, GPIIb-IIIa also bears a number of clinically important alloantigenic determinants. Previous studies have shown that disruption of the long-range Cys 5 -Cys 435 disulfide bond of the ␤ 3 subunit results in the production of isoforms that bind some, but not all, anti-Pl A1 alloantibodies, suggesting that mutations in this so-called long-range disulfide bond can alter the conformation of GPIIIa. The purpose of this study was to examine the effects of either the Cys5Ala or Cys435Ala substitution of GPIIIa on the adhesive properties of the GPIIb-IIIa complex. We found that both Ala5GPIIIa and Ala435GPIIIa were capable of associating with GPIIb and were expressed normally on the cell surface when cotransfected into Chinese hamster ovary (CHO) cells. CHO cells expressing GPIIb-Ala5GPIIIa or GPIIb-Ala435IIIa bound well-characterized, conformationally sensitive ligand-induced binding site (LIBS) antibodies, and were capable of constitutively binding the fibrinogenmimetic monoclonal antibodies Pl-55 and PAC-1, as well as soluble fibrinogen. Both GPIIb-Ala5IIIa-and GPIIb-Ala435IIIa-transfected CHO cells also bound more avidly to immobilized fibrinogen and were capable of mediating the tyrosine phosphorylation of pp125 FAK on cell adhesion. These data are consistent with the notion that these regions of GPIIIa participate in the conformational change associated with receptor activation. Additionally, these studies may provide a molecular explanation for the previously reported ability of mild reducing agents to activate the GPIIb-IIIa complex and promote platelet aggregation. IntroductionIntegrins, one of several gene families that encode cell surface adhesive receptors capable of mediating cell-cell and cellextracellular matrix (ECM) interactions, 1 are heterodimers consisting of a 120-to 180-kDa ␣ subunits noncovalently associated with a 90-to 110-kDa ␤ subunits. To date, 19 ␣ and 8 ␤ subunits have been characterized, and 24 members of this heterodimer family have been identified. 2,3 Integrins mediate both adhesion and bidirectional transmembrane signaling. The binding of these integrins to soluble macromolecules is tightly regulated by integrin activation or inside-out signaling, also known as affinity modulation, which involves structural changes intrinsic to the heterodimer, and avidity modulation due to lateral diffusion and clustering of heterodimers into oligomers. 4,5 In addition, ligand-induced binding site (LIBS) antibodies, which preferentially bind the ligandoccupied form of receptor, can lock the integrin into a higheraffinity state without the need for signals from inside the cell. 6,7 Following receptor occupancy, information is transduced across the plasma membrane in a process termed outside-in signaling. These signals include elevation of intracellular pH and calcium, ...

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“…These data indicated that 3C7 recognized a LIBS epitope. It was reported that certain anti-LIBS antibodies activated ␣ IIb ␤ 3 and promoted fibrinogen binding to platelets (22,23). However, 3C7 was not an activating antibody (Fig.…”

Section: Generation and Characterization Of Monoclonal Antibody-mentioning

confidence: 88%

A Novel Anti-platelet Monoclonal Antibody (3C7) Specific for the Complexof Integrin αIIbβ3 Inhibits PlateletAggregation and Adhesion

Chen¹,

Sun²,

Liu

2005

Journal of Biological Chemistry

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Activation or ligand binding induces conformational changes in ␣ IIb ␤ 3 , resulting in exposure of neoepitopes named ligand-induced binding sites. We reported here a novel monoclonal antibody developed by using Chinese hamster ovary (CHO) cells expressing an activated ␣ IIb ␤ 3 mutant (CHO ␣ IIb ␤ 3 ⌬717) as the immunogen. This IgG 2b named 3C7 was specific for the complex of ␣ IIb ␤ 3 as demonstrated by flow cytometry, immunoprecipitation, and EDTA chelating. The binding of 3C7 to platelets increased significantly when platelets were activated by ADP/thrombin or occupied by RGDS peptides, fibrinogen, or PAC-1, suggesting that 3C7 was an antiligand-induced binding site antibody. The antibody failed to bind to the CHO cells expressing another ␣ IIb ␤ 3 mutant (␤ 3 Y178A) suggesting that the Cys 177 -Cys 184 loop of ␤ 3 was likely the epitope for 3C7. 3C7 inhibited platelet aggregation, which was initiated by ADP or thrombin in a dose-dependent manner (IC 50 s of 5.6 and 0.05 g/ml, respectively). The antibody also inhibited platelet adhesion to immobilized fibrinogen but not to fibronectin or collagen. These findings suggested that 3C7 was a potent antagonist of integrin ␣ IIb ␤ 3 and a potential anti-thrombotic agent.Platelet aggregation and adhesion are the central events in thrombosis and homeostasis (1). Integrin ␣ IIb ␤ 3 (platelet glycoprotein GPIIb/IIIa) is the fibrinogen receptor of platelets. It is in a resting state in the circulation but is activated during platelet aggregation and adhesion. Although the mechanism is not yet fully elucidated, the activation is believed to be induced by conformational changes of ␣ IIb ␤ 3 , resulting in a higher affinity for fibrinogen and other adhesive molecules (2). When stimulated by platelet agonists such as ADP or thrombin, integrin ␣ IIb ␤ 3 undergoes rapid conformational changes, which exposes fibrinogen binding sites and enables the rapid formation of platelet clots (3). Ligand binding to activated integrin ␣ IIb ␤ 3 further induces expression of neoepitopes or ligand-induced binding sites (LIBS) 1 (4, 5).Platelet activation, aggregation, and adhesion are importantly involved in acute coronary syndromes and following certain intravascular therapeutic interventions (6 -9). The central role of integrin ␣ IIb ␤ 3 in thrombosis has led to the development of pharmaceutical agents that block interactions between integrin ␣ IIb ␤ 3 and fibrinogen. The ␣ IIb ␤ 3 antagonists are capable of inhibiting platelet adhesion and aggregation and formation of platelet thrombi at the site of plaque rupture or plaque fissure (10). Because platelet-rich rather than fibrinrich thrombosis was found to be responsible for many acute complications of angioplasty, the blockade of platelet glycoprotein IIb/IIIa receptor was appreciated as valuable in interventional cardiology. Currently there are three integrin ␣ IIb ␤ 3 antagonists used clinically, abciximab (11), tirofiban (12), and eptifibatide (13). Abciximab is a chimeric Fab created based on a murine monoclonal antibody 7E3. Its mec...

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Stimulation of platelet glycoprotein IIb‐IIIa (α_IIbβ₃‐integrin) functional activity by a monoclonal antibody to the N‐terminal region of glycoprotein IIIa

Cited by 27 publications

References 21 publications

Evidence that thrombocytopenia observed in humans treated with orally bioavailable glycoprotein IIb/IIIa antagonists is immune mediated

Evidence that thrombocytopenia observed in humans treated with orally bioavailable glycoprotein IIb/IIIa antagonists is immune mediated

Disruption of the long-range GPIIIa Cys5-Cys435 disulfide bond results in the production of constitutively active GPIIb-IIIa (αIIbβ3) integrin complexes

A Novel Anti-platelet Monoclonal Antibody (3C7) Specific for the Complexof Integrin αIIbβ3 Inhibits PlateletAggregation and Adhesion

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Stimulation of platelet glycoprotein IIb‐IIIa (αIIbβ3‐integrin) functional activity by a monoclonal antibody to the N‐terminal region of glycoprotein IIIa

Cited by 27 publications

References 21 publications

Evidence that thrombocytopenia observed in humans treated with orally bioavailable glycoprotein IIb/IIIa antagonists is immune mediated

Evidence that thrombocytopenia observed in humans treated with orally bioavailable glycoprotein IIb/IIIa antagonists is immune mediated

Disruption of the long-range GPIIIa Cys5-Cys435 disulfide bond results in the production of constitutively active GPIIb-IIIa (αIIbβ3) integrin complexes

A Novel Anti-platelet Monoclonal Antibody (3C7) Specific for the Complexof Integrin αIIbβ3 Inhibits PlateletAggregation and Adhesion

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About

Stimulation of platelet glycoprotein IIb‐IIIa (α_IIbβ₃‐integrin) functional activity by a monoclonal antibody to the N‐terminal region of glycoprotein IIIa