In patients with acute myocardial infarction undergoing reperfusion therapy to restore blood flow through blocked arteries, simultaneous inhibition of platelet P2Y12 receptors with the current standard of care neither completely prevents recurrent thrombosis nor provides satisfactory protection against reperfusion injury. Additionally, these antiplatelet drugs increase the risk of bleeding. To devise a different strategy, we engineered and optimized the apyrase activity of human nucleoside triphosphate diphosphohydrolase-3 (CD39L3) to enhance scavenging of extracellular adenosine diphosphate, a predominant ligand of P2Y12 receptors. The resulting recombinant protein, APT102, exhibited greater than four times higher adenosine diphosphatase activity and a 50 times longer plasma half-life than did native apyrase. Treatment with APT102 before coronary fibrinolysis with intravenous recombinant human tissue-type plasminogen activator in conscious dogs completely prevented thrombotic reocclusion and significantly decreased infarction size by 81% without increasing bleeding time. In contrast, clopidogrel did not prevent coronary reocclusion and increased bleeding time. In a murine model of myocardial reperfusion injury caused by transient coronary artery occlusion, APT102 also decreased infarct size by 51%, whereas clopidogrel was not effective. These preclinical data suggest that APT102 should be tested for its ability to safely and effectively maximize the benefits of myocardial reperfusion therapy in patients with arterial thrombosis.

Recombinant tissue plasminogen activator (r-tPA) is the only FDA-approved drug treatment for ischemic stroke and must be used within 4.5 hours. Thrombolytic treatment with r-tPA has deleterious effects on the neurovascular unit that substantially increases the risk of intracerebral hemorrhage if administered too late. These therapeutic shortcomings necessitate additional investigation into agents that can extend the therapeutic window for safe use of thrombolytics. In this study, combination of r-tPA and APT102, a novel form of human apyrase/ADPase, was investigated in a clinically-relevant aged-female rat embolic ischemic stroke model. We propose that successfully extending the therapeutic window of r-tPA administration would represent a significant advance in the treatment of ischemic stroke due to a significant increase in the number of patients eligible for treatment. Results of our study showed significantly reduced mortality from 47% with r-tPA alone to 16% with co-administration of APT102 and r-tPA. Co-administration decreased cortical (47±5% vs 29±5%), striatal (50±2%, vs 40±3%) and total (48±3%vs 33±4%) hemispheric infarct volume compared to r-tPA alone. APT102 improved neurological outcome (8.9±0.6, vs 6.8±0.8) and decreased hemoglobin extravasation in cortical tissue (1.9±0.1 mg/dlvs 1.4±0.1 mg/dl) striatal tissue (2.1±0.3 mg/dl vs 1.4±0.1 mg/dl) and whole brain tissue (2.0±0.2 mg/dl vs 1.4±0.1 mg/dl). These data suggest that APT102 can safely extend the therapeutic window for r-tPA mediated reperfusion to 6 h following experimental stroke without increased hemorrhagic transformation. APT102 offers to be a viable adjunct therapeutic option to increase the number of clinical patients eligible for thrombolytic treatment after ischemic stroke.

Summary Background Occlusion of vein grafts (VGs) after bypass surgery due to thrombosis and intimal hyperplasia (IH) is a major clinical problem. Apyrases are enzymes that scavenge extracellular ATP and ADP and promote adenosine formation at sites of vascular injury and hence have potential to inhibit vein graft pathology. Objectives We examined the effects of recombinant soluble human apyrase, APT102, on platelets, smooth muscle cells (SMCs), and endothelial cells (ECs) in vitro and thrombosis and IH in murine VGs. Methods SMC and EC proliferation and migration were studied in vitro. Inferior vena cava segments from donor mice were grafted into carotid arteries of recipient mice. Results While potently inhibiting ADP-induced platelet aggregation and VG thrombosis, APT102 did not impair surgical hemostasis. APT102 did not directly inhibit SMC or EC proliferation, but significantly attenuated the effects of ATP on SMC and EC proliferation. APT102 significantly inhibited SMC migration, but did not inhibit EC migration, which may be mediated, at least in part, by inhibition of SMC, but not EC, migration by adenosine. At 4 weeks after surgery, IH was significantly less in VGs of APT102-treated mice than in control VGs. APT102 significantly inhibited cell proliferation in VGs, but did not inhibit re-endothelialization. Conclusions Systemic administration of a recombinant human apyrase inhibits thrombosis and IH in VGs without increasing bleeding or compromising re-endothelialization. These results suggest that APT102 has the potential to become a novel, single-drug treatment strategy to prevent multiple pathological processes that drive early adverse remodeling and occlusion of VGs.

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.

Early coronary reperfusion attenuates myocardial infarction, but flow‐limiting rethrombosis and increased bleeding with use of P2Y12 antagonists limits the benefits of therapy. We tested whether an optimized human recombinant apyrase (APT102), which degrades circulating ATP and ADP causing inhibition of platelet activation and generation of cardioprotective adenosine, improves reperfusion blood flow and functional recovery over 7 d without increasing bleeding when given in combination with clopidogrel. Conscious dogs sedated with morphine had coronary thrombotic occlusion induced by electrical current followed by fibrinolysis with IV rt‐PA (Activase, 1 mg/kg) an average of 91min after ischemia. Animals received either clopidogrel (4 mg/kg, PO) or IV APT102 (1.0 mg/kg) 10 min before rt‐PA plus heparin and aspirin (4 mg/kg), and then daily clopidogrel (1 mg/kg) for 7 d. Bleeding time was measured over 24 h and then hemorrhage around surgical incisions was assessed daily. Myocardial function and regional microvascular perfusion were quantified with magnetic resonance imaging at baseline, 24 h and 1 wk. Coronary flow (implanted probe) was at or above baseline with APT102 followed by clopidogrel (n=4), but remained below baseline in dogs given clopidogrel alone (n=5). Ejection fraction and microvascular perfusion both increased back to or above baseline by 1 wk with the combination, but not clopidogrel alone. Bleeding times returned to baseline before clopidogrel in the combination group. Interestingly, APT102 + clopidogrel had less hemorrhage from incisions requiring pressure bandages (1/4) than clopidogrel alone (3/5). We conclude that APT102 improves reperfusion blood flow and myocardial function compared with standard P2Y12 inhibition without increasing bleeding. Moreover, APT102 may actually decrease hemorrhage when used before P2Y12 inhibition. APT102 warrants further study as a conjunctive agent to optimize reperfusion therapy.