Recent emphasis has been on ADP as a mediator of thrombotic events characterized by excessive platelet reactivity. In contrast, neither ATP nor AMP induces platelet activation. Thus, (enzymatic) removal of ADP from the milieu of activated platelets constitutes a novel antithrombotic strategy. Apyrases constitute a group of enzymes catalyzing metabolism of ATP to ADP, and ADP to AMP. Therefore, we cloned a human apyrase isozyme with 35% sequence identity to CD39 (NTPDase1) from a human genome cDNA library and enhanced its ADPase activity by protein engineering. The novel engineered apyrase, named APT102 was transfected into and secreted from HEK293 cell lines. It was purified from conditioned medium using anion exchange chromatography, size exclusion chromatography and affinity chromatography, and enzymatically characterized. APT102 was highly soluble and stable. It exhibited a 2.5-fold increase in ADPase activity relative to its ATPase activity, when compared to the parent compound, with catalytic efficiencies (Vmax/Km) of 0.135 and 0.164 respectively. APT102 strongly inhibited platelet reactivity in vitro when added to citrated and heparinized human platelet-rich plasma upon stimulation with ADP or collagen. ATP is released upon tissue damage and could potentially lead to formation of prothrombotic levels of ADP following its metabolism by apyrases. We demonstrated that with APT102, aggregation was efficiently inhibited and reversed by the enzyme. This occurred even when human platelets were initially stimulated with 10 μM ADP simultaneously with up to 100 μM ATP, followed within 5 min with another dose of 300 μM ATP. Following tail vein infusion of APT102 to male Sprague-Dawley rats (~300 g) at 0.75 mg/kg body weight, platelet aggregation ex vivo in PRP in response to ADP and collagen was strongly inhibited. The pharmacokinetics of APT102 were determined by enzyme activity assays with either ADP or ATP as substrate, as analyzed by our radio-TLC procedure. The data indicate that APT102 has a fast onset of action and a prolonged duration of action. This will be advantageous clinically since a bolus injection will suffice without a requirement for continuous infusion. The pharmacodynamics of APT102 were determined using ex vivo platelet aggregation to ADP, and showed identical properties. In keeping with the demonstrated lack in mice of a direct anti-platelet effect of soluble apyrase, the rat tail bleeding time was minimally prolonged in parallel experiments. Thus, APT102 strongly inhibits the recruitment of platelets into an evolving occlusive thrombus, but it is anticipated to be safer than currently employed antithrombotics since they all have a narrow therapeutic window between therapeutic effectiveness and undesirable side effects. In separate experiments involving experimental stroke, male Wistar rats were randomly infused via the tail vein with either saline or 0.75 mg APT102 per kg body weight prior to being subjected to temporary middle cerebral artery occlusion. Brain infarct volumes were determined 24 h following stroke induction. Treatment with APT102 resulted in a 40% reduction in brain infarct volume. We conclude that APT102 will represent a new therapeutic modality for platelet-driven thrombotic disorders, which appears to be safe and effective.