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

Abstract

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...

Immunoglobulin M antiplatelet autoantibodies from mice immunized with rat platelets induce thrombocytopenia and platelet function impairment

“…Although antiplatelet monoclonal antibodies (mAbs) that are of great interest for analyzing platelet surface molecule functions have already been reported, 2,3 these mAbs were derived after xenogenic immunization and are therefore not fully reflective of antiplatelet autoantibodies. Mouse monoclonal antiplatelet autoantibodies have also been derived from (NZBxBXSB)F1 animals.…”

LRRFIP1 expression triggers platelet agglutination by enhancing αIIbβ3 expression

“…Aggregation processes are initiated by the activation of αIIbβ3, which binds to fibrinogen and adjacent platelets (30,31). Chen et al (32) previously reported that anti-αIIbβ3 complex antibodies inhibited the aggregation of platelets. The present study examined the levels of αIIbβ3 to identify the effects of the LRRFIP1 on platelet agglutination.…”

Immunodiagnosis of platelet activation in immune thrombocytopenia through scFv antibodies cognate to activated IIb3 integrins

“…The monoclonal antibody 3C7, which is directed against the complex aIIbb3, is also known to inhibit platelet aggregation. 16 However, this antibody was observed to bind to both non-activated and activated platelets, and as such would not be of use simultaneously in the diagnosis of the activation status of platelets in patients.…”