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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2007;27:1206.)
© 2007 American Heart Association, Inc.

Thrombosis

Targeting Ligand-Induced Binding Sites on GPIIb/IIIa via Single-Chain Antibody Allows Effective Anticoagulation Without Bleeding Time Prolongation

Patrick Stoll; Nicole Bassler; Christoph E. Hagemeyer; Steffen U. Eisenhardt; Yung Chih Chen; Rene Schmidt; Meike Schwarz; Ingo Ahrens; Yasuhiro Katagiri; Benedikt Pannen; Christoph Bode; Karlheinz Peter

From the Centre for Thrombosis & Myocardial Infarction (P.S., N.B., C.E.H., S.U.E., Y.C.C., K.P.), Baker Heart Research Institute, Melbourne, Australia; the Departments of Cardiology (P.S., M.S., I.A., C.B.) and Anaesthesiology (R.S.), University of Freiburg, Germany; the National Institutes of Health (Y.K.), Bethesda, Md; and the Department of Anaesthesia (B.P.), University Hospital Duesseldorf, Germany.

Correspondence to Karlheinz Peter, MD, Christoph E. Hagemeyer, PhD, Baker Heart Research Institute, PO Box 6492 St Kilda Road Central, Melbourne, Victoria 8008, Australia. E-mail christoph.hagemeyer{at}baker.edu.au

Objective— Therapeutic anticoagulation is widely used, but limitations in efficacy and bleeding complications cause an ongoing search for new agents. However, with new agents developed it seems to be an inherent problem that increased efficiency is accompanied by an increase in bleeding complications. We investigate whether targeting of anticoagulants to activated platelets provides a means to overcome this association of potency and bleeding.

Methods and Results— Ligand-induced binding sites (LIBS) on fibrinogen/fibrin-binding GPIIb/IIIa represent an abundant clot-specific target. We cloned an anti-LIBS single-chain antibody (scFv_anti-LIBS) and genetically fused it with a potent, direct factor Xa (fXa) inhibitor, tick anticoagulant peptide (TAP). Specific antibody binding of fusion molecule scFv_anti-LIBS-TAP was proven in flow cytometry; anti-fXa activity was demonstrated in chromogenic assays. In vivo anticoagulative efficiency was determined by Doppler-flow in a ferric chloride–induced carotid artery thrombosis model in mice. ScFv_anti-LIBS-TAP prolonged occlusion time comparable to enoxaparine, recombinant TAP, and nontargeted mutant-scFv-TAP. ScFv_anti-LIBS-TAP revealed antithrombotic effects at low doses at which the nontargeted mutant-scFv-TAP failed. In contrast to the other anticoagulants tested, bleeding times were not prolonged by scFv_anti-LIBS-TAP.

Conclusions— The novel clot-targeting approach of anticoagulants via single-chain antibody directed against a LIBS-epitope on GPIIb/IIIa promises effective anticoagulation with reduced bleeding risk.

A new strategy of targeting anticoagulants to activated platelets is evaluated. A newly cloned single-chain antibody directed against a LIBS-epitope on GPIIb/IIIa and the potent, direct factor-Xa inhibitor TAP were genetically fused. Anticoagulative efficiency and safety was proven in a mouse model with carotid artery thrombosis and bleeding time measurements.

Key Words: GPIIb/IIIa • anticoagulation • single-chain • fXa • thrombosis

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