Abstract 14752: Clot-targeting Of CD39 Through Genetic Fusion To An Activated-platelet Specific Single-chain Antibody Provides Efficient Inhibition Of ADP Receptor-mediated Platelet Activation Without Systemic Platelet Inhibition
CD39, a NTPDase with strong antithrombotic properties, has previously been shown to be protective in models of stroke, transplantations, pulmonary embolism and myocardial infarction by hydrolyzing/removing the platelet agonist ADP and thus inhibiting ADP receptor-mediated platelet activation. However, CD39's high potency comes with an increased bleeding risk, as also noted with ADP receptor inhibitors in clinical use. We hypothesize that targeting CD39 to activated platelets allows localized enrichment to effective concentrations at thrombi despite a low systemic and thus safe concentration with a low risk of bleeding side effects. CD39 was recombinantly fused to a single-chain antibody (scFv) specific to the active conformation of GPIIb/IIIa on activated platelets. Targeting was shown by binding to microthrombi (flow chamber). Targeted CD39 (Targ-CD39) was significantly more effective at preventing ADP-induced platelet activation (flow cytometry) and aggregation (light transmission aggregometry) than its non-targeted control (NT-CD39, CD39 fused to a non-functional scFv). In a mouse model of ferric chloride-induced carotid artery thrombosis NT-CD39 (150U/g), while being protective against vessel occlusion (n=7, p<0.05 vs. control), showed a significant bleeding risk as demonstrated by mouse tail transsection (n=5, p<0.01). Targ-CD39 concentrates at the thrombus site, allowing a lower dose to be administrated. A dose ∼10x lower (15U/g) is able to prevent vessel occlusion (n=7, p<0.01 vs. control) to a similar extend as the high dose NT-CD39. An equimolar dose of NT-CD39 at this low concentration is ineffective at preventing vessel occlusion. The improved efficiency in preventing thrombosis as a result of the targeting also means that the systemic dose of CD39 administered as part of the Targ-CD39 construct is below that causing a bleeding time prolongation in mice (n=5). Targeting CD39 to its desired site of action enables administration of such a low concentration as to avoid the previously observed bleeding tendencies while still being a highly effective antithrombotic drug. Thus, enriching CD39 to activated platelets at thrombi prevents the previously limiting bleeding side effects and advances CD39 towards potential clinical use.
- © 2011 by American Heart Association, Inc.