Targeting ligand-induced binding sites on GPIIb/IIIa via single-chain antibody allows effective anticoagulation without bleeding time prolongation.
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.


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.
Stoll
Patrick
P
Centre for Thrombosis & Myocardial Infarction, Baker Heart Research Institute, PO Box 6492 St Kilda Road Central, Melbourne, Victoria 8008, Australia.
Bassler
Nicole
N
Hagemeyer
Christoph E
CE
Eisenhardt
Steffen U
SU
Chen
Yung Chih
YC
Schmidt
Rene
R
Schwarz
Meike
M
Ahrens
Ingo
I
Katagiri
Yasuhiro
Y
Pannen
Benedikt
B
Bode
Christoph
C
Peter
Karlheinz
K
D000818:Animals; D000911:Antibodies, Monoclonal / Q000627:therapeutic use*; D001666:Binding Sites, Antibody; D001792:Blood Platelets / Q000187:drug effects; D015153:Blotting, Western; D000939:Epitopes; D005434:Flow Cytometry; D015870:Gene Expression; D006470:Hemorrhage / Q000453:epidemiology / Q000209:etiology; D006801:Humans; D006825:Hybridomas / Q000473:pathology; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology / Q000627:therapeutic use*; D007155:Immunologic Factors / Q000627:therapeutic use*; D008024:Ligands; D051379:Mice; D008810:Mice, Inbred C57BL; D010974:Platelet Aggregation / Q000187:drug effects; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000187:drug effects* / Q000276:immunology; D011485:Protein Binding; D012307:Risk Factors; D013927:Thrombosis / Q000097:blood / Q000150:complications / Q000188:drug therapy*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007128:Immunoglobulin Fragments; D007155:Immunologic Factors; D008024:Ligands; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; C055966:immunoglobulin Fv