Whilst therapeutic advances using anti-platelet and anti-coagulation drugs have been made to address cardiovascular diseases, they have significant drawbacks, including bleeding complications. Our aim was to develop a targeted anti-thrombotic therapy to deliver a therapeutic payload directly to the site of the thrombi, thereby preventing bleeding side effects.

Methods:
We have genetically designed and engineered an activated platelet targeted recombinant fusion protein with the tick anticoagulant peptide (Targ-TAP). A non-binding single-chain antibody fused with an active TAP (Mut-TAP) was used as control.

Results
Flow cytometry assays (both human and mouse) demonstrated the binding affinity of Targ-TAP specifically to activated platelets (ADP, CRP and TRAP), while no binding was observed with Mut-TAP (p<0.05). Competitive assay using an anti-fibrinogen antibody demonstrated the binding of Targ-TAP to the activated GPIIb/IIIa integrin, thereby inhibiting fibrinogen binding(p<0.05). No inhibition was observed using Mut-TAP. Light transmission aggregometry demonstrated strong platelet inhibition using Targ-Tap as compared to Mut-TAP (p<0.05). Flow chamber assays using bright-field microscopy demonstrated the potent anticoagulant capacity of Targ-TAP, as well as its specificity toward aggregated platelets. Using a FeCl3-induced mouse model of arterial thrombosis, Targ-TAP significantly delayed arterial occlusion (p<0.05). More importantly, Targ-TAP does not increase bleeding time nor blood volume loss.

Conclusions:
Targ-TAP allows for the enrichment of the anticoagulant effect directly at the site of the developing thrombus, whilst ensuring low systemic concentrations. Targ-TAP represents a novel, effective and safe
anticoagulant drug candidate. The recombinant character of this drug allows tailoring of the pharmacokinetic (e.g. different MW) ready either for intravenous or subcutaneous injections.

01 - TARGETED DRUG THERAPY TO PROVIDE EFFECTIVE AND LOCALISED ANTICOAGULATION WITHOUT IMPAIRING HAEMOSTASIS