Optimizing human apyrase to treat arterial thrombosis and limit reperfusion injury without increasing bleeding risk

Abstract

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.

Fig. 1. Mechanisms of action of endogenous apyrase

eATP and eADP are scavenged by apyrase, leading to the generation of cardioprotective adenosine (eADO). Thus, administration of exogenous apyrase (APT102) boosts the endogenous control mechanism that converts a proinflammatory and prothrombotic environment, characterized by dominant P2 purinoceptor signaling, into an eADO P1 receptor signaling that is largely characterized by prevention of inflammatory responses and thrombosis, and cardioprotective mechanisms. Apyrase attenuates platelet activation mediated by P2Y₁, P2Y₁₂, and P2X₁ receptors, whereas clopidogrel blocks only P2Y₁₂ receptors. Thus, apyrase may be more effective for inhibiting platelet-rich thrombosis than P2Y₁₂ receptor antagonists.

Fig. 2. In vitro inhibition of ADP-induced platelet aggregation and disaggregation by APT102 in platelet-rich plasma from a healthy human volunteer

(A) Dose response of ADP added to platelet-rich plasma (at arrow). (B) Inhibition of 5 µM ADP–induced platelet aggregation by APT101 (25 µg/ml) and its optimized derivative, APT102 (6.5 and 25 µg/ml), added before ADP. (C) APT102 (2.5 µg/ml), adenosine (20 µM), or AMP (20 µM) was added at the “inhibitor” arrow 6 min after maximal aggregation. Only APT102 completely reversed aggregation. (D) Adenosine dose dependently (1, 10, and 100 µM) but only partially reversed platelet aggregation. Blood was collected in tubes containing heparin to maintain physiologic calcium concentration. Results shown are representative of triplicate assays. Similar results were obtained in two other healthy volunteers.

Fig. 3. Coronary patency after reperfusion assessed by electromagnetic flow probe profiles in individual dogs

Open bars, patent arteries; black bars, occluded arteries; crosshatched bars, an interval of no recorded flow because of computer or power shutdown.

Fig. 4. Bleeding times after coronary occlusion in individual dogs

Data were averaged (n = 6) relative to baseline values before administration of either clopidogrel or APT102 followed by rt-PA and heparin (horizontal bars). (A to C) Each data point represents an average of duplicate bleeding time measurements for an individual animal. (D) Data are presented as the means ± SD. *P < 0.05 versus baseline; ^#P < 0.05 versus APT102 by one-way analysis of variance (ANOVA) and a two-tailed t test.

Fig. 5. Effect of clopidogrel and APT102 on myocardial ischemic damage in dogs

(A) Area at risk (AAR) and infarcted area (IA; nonviable) after coronary artery occlusion of left ventricular (LV) heart muscle of dogs (n = 6) expressed as a percent of the total LV and as a ratio (29). (B) Photographs of representative LV slices from two hearts at the same level showing (left) similar AAR after clopidogrel (a) or high-dose APT102 (c) defined by the area that was nonperfused (N) by Evans blue dye compared to the total surface area including the area perfused (P) with dye. IA, defined as unstained (U) tissue after incubation with triphenyltetrazolium chloride (TTC), was compared to the total area including still viable TTC-stained (S) myocardium for the dog given APT102 (d, arrows) compared to the area in the dog given clopidogrel (b). Infarction in clopidogrel-treated hearts was sometimes marked by more abundant areas of hemorrhage seen in the pre-TTC–stained tissue (*, panel a). Bars represent means ± SEM. *P < 0.02, **P < 0.002 by t test.

Fig. 6. Effect of APT102 and clopidogrel on myocardial tissue after ischemia-reperfusion injury in mice

C57BL/6J mice were given equivalent volumes of saline (intravenously as a control) or APT102 (1.0 mg/kg, intravenously) 5 min before or 50 min after the onset of 60 min of ischemia (via LAD coronary occlusion), clopidogrel (10.0 mg/kg, orally) pretreatment 24 hours before ischemia, or a combination of clopidogrel pretreatment and APT102 5 min before ischemia. Bars represent means ± SEM. Control, n = 12; APT102 before ischemia, n = 12; APT102 after ischemia, n = 30; clopidogrel, n = 15; clopidogrel plus APT102, n = 33. *P < 0.05, **P < 0.0005, ***P < 0.0001, ****P < 0.00001 by t tests.

Fig. 7. Representative transverse slices taken at a similar level of the left ventricle from mice undergoing 60 min of ischemia (via LAD coronary occlusion)

The slices were obtained after 24 hours and show a similar area at risk (outlined in red) for the four treatments defined by the area that was nonperfused by Evans blue dye compared to the total surface area including the area perfused with Evans blue dye (outlined in blue). Shown in black outline are the same heart slices after incubation with TTC to identify the area of unstained and therefore infarcted myocardium relative to the total area, including the TTC-stained myocardium that was still viable.