Heart, Lung and Circulation

A Novel Recombinant CD39 Targeting Activated Platelets via a Fused Single-chain Antibody: Achieving Efficient Anti-coagulation While Minimising Bleeding Side Effects by Clot Directed Enrichment

      Introduction: CD39, an NTPDase with strong antithrombotic properties, has previously been shown to be protective in models of stroke, transplantations, pulmonary embolism and myocardial infarctions by hydrolysing/removing the platelet agonist ADP. However CD39's high potency comes at the cost of an increased bleeding risk. We hypothesise that targeting CD39 to activated platelets allows localised enrichment at the growing thrombus despite a low and safe systemic concentration.
      Methods and results: CD39 was recombinantly fused to a single-chain antibody specific to activated platelets via selective binding to the active conformation of GPIIb/IIIa. The fusion construct was produced in Hek293 and purified using a His-tag chromatography step. Targeted-CD39 was significantly more effective at preventing platelet activation (flow cytometry) and platelet aggregation (aggregometry) with ADP and collagen as agonist than its non-targeted control (CD39 fused to a non-functional mutated single-chain antibody). Most importantly in a mouse model of ferric chloride-induced carotid artery thrombosis, targeted-CD39 was protective against vessel occlusion at a concentration at which the non-targeted-CD39 was ineffective (p < 0.005). At the same concentration no tail bleeding prolongation was observed for the targeted-CD39 while the ineffective non-targeted-CD39 showed a bleeding tendency (p < 0.01).
      Conclusion: 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 growing thrombi prevents the previously limiting bleeding side effects and advances CD39 towards potential clinical use.