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. Author manuscript; available in PMC: 2015 Sep 5.
Published in final edited form as: Eur J Pharmacol. 2014 Jun 13;738:368–373. doi: 10.1016/j.ejphar.2014.05.052

Combination treatment of r- tPA and an optimized human apyrase reduces mortality rate and hemorrhagic transformation 6h after ischemic stroke in aged female rats

Zhenjun Tan 1, Xinlan Li 1, Ryan C Turner 1, Aric F Logsdon 2, Brandon Lucke-Wold 1, Kenneth DiPasquale 2, Soon Soeg Jeong 3, Ridong Chen 3, Jason D Huber 2, Charles L Rosen 1
PMCID: PMC4126582  NIHMSID: NIHMS611274  PMID: 24933645

Abstract

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.

Keywords: Acute ischemic stroke, APT102/ apyrase, hemorrhagic transformation, infarction size, recombinant tissue plasminogen activator

1. Introduction

Recombinant tissue plasminogen activator (r-tPA) is the only FDA approved drug for treatment of focal ischemic stroke. Unfortunately, less than 5% of people suffering an ischemic stroke receive r-tPA due to increased risk of secondary cerebral hemorrhage and edema formation. Thus, an unmet clinical need exists to develop novel therapeutics that work in combination with r-tPA to improve stroke outcome, reduce secondary complications, and extend the time window for administering tPA.

Reasons for the failed translation of therapeutics from bench-to-bedside are unclear but notable contrasts exist between preclinical models of ischemic stroke and the clinical population having ischemic strokes. Specifically, stroke is a disease of the elderly with approximately 72% of stroke patients being over the age of 65 (Goldstein et al., 2006). In 2009, the Stroke Therapy Academic Industry Roundtable (STAIR) committee recommended that following evaluation of proposed therapeutics in young, healthy male animals, further studies should be performed in aged female animals and animals with comorbid conditions (Fisher et al., 2009; Sacco et al., 2006).

Apyrase is a calcium-activated enzyme that catalyzes the hydrolysis of ATP and ADP to yield AMP and inorganic phosphate. Apyrase has found clinical application as a novel pathway for inhibition of platelet aggregation through the increased hydrolysis of extracellular ADP. ATP 102 is an experimental ayrase formulation that has shown both antithrombotic and anti-inflammatory activity with minimal risk of increased bleeding. Previous studies have demonstrated that APT102 alone or combination with r-tPA prevents ischemia-reperfusion injury following lung infarction (Eckle et al., 2007; Sugimoto et al., 2009), myocardial infarction (Kohler et al., 2007; Moeckel Douglas, 2011) and acute ischemic stroke in young male rats (Belayev et al., 2003; Sun et al., 2011; Wang et al., 2010). Thus, in this study, we used a combination therapy of r-tPA and APT102 in a clinically relevant embolic stroke model in aged female rats to evaluate whether the combination could safely extend the therapeutic time window for r-tPA out to 6 h following ischemic stroke. By using aged female rodents, tissue plasminogen activator mediated reperfusion, and functional/neurological assessment; we successfully address numerous STAIR recommendations for improving translation of therapeutics from bench-to-bedside. Furthermore, we attempt to increase the number of patients potentially eligible for rtPA-mediated reperfusion by extending the therapeutic window to 6 hours post-ischemia, a significant finding with regards to reducing ischemic stroke morbidity and mortality.

2. Materials and methods

2.1 Animals

Seventy-two female Sprague-Dawley rats (18-20 months old) were purchased from Hilltop Laboratories (Scottdale, PA) and housed under 12-h:12-h light-dark conditions with food and water ad libitum. All studies involving rats were approved by the West Virginia University Animal Care and Use Committee.

2.2 Drug Treatment

Rats were randomly divided into three treatment groups: r-tPA group (N=30), saline group (N=23), and r-tPA + APT102 group (N=19). The r-tPA group received r-tPA (5mg/kg, Genentech; South San Francisco, CA, USA) as an intravenous infusion (30% bolus and 70% infused over 30 min) at 6 h after MCAO. The saline group received an intravenous infusion of saline at 6 h after MCAO. The r-tPA+APT102 group received r-tPA (5 mg/kg) plus APT102 (2.25 mg/kg, APT Therapeutics Inc, St Louis, Missouri, 63108, USA) as an intravenous infusion (30% bolus and 70% infused over 30 min) at 6 h after MCAO. Standard physiologic parameters were monitored before, during, and after MCAO in a subset of rats (n=3 per group) to ensure uniformity and consistency with prior studies. All parameters were consistent with the ranges we have previously published (DiNapoli et al., 2006), indicating that APT102 did not induce obvious physiologic changes in blood pressure, blood pH, and blood gas composition. Rats from all three groups were evaluated at 24 h following MCAO.

2.3 Surgical procedure for MCAO

Rats were given a reversible embolic MCAO as previously described (Dinapoli et al., 2006; DiNapoli et al., 2008; Tan et al., 2009). Briefly, rats were anesthetized with 2% isoflurane in a mixture of 30% oxygen and 70% nitrous oxide. A servo-controlled homeothermic heating blanket, equipped with a rectal thermometer, was used to maintain body temperature at 37°C. Cerebral blood flow was monitored with a laser Doppler probe precisely positioned over the area supplied by the middle cerebral artery (MCA). A modified PE 50 microcatheter was inserted into the external carotid artery stump and advanced into the MCA. Placement in the proximal MCA was verified by a sudden decrease in cerebral blood flow as measured by laser Doppler. The microcatheter was withdrawn ~1 mm, allowing cerebral blood flow to return to baseline, and then a 25 mm fibrin-enriched clot was injected. Successful MCA occlusion was confirmed by a drop in perfusion greater than 80% of baseline flow. Recanalization was achieved when cerebral blood flow increased to at least 75% of initial baseline. Rats not achieving these standards were excluded from the study.

2.4 Modified Neurological Severity Score (mNSS)

Neurological function was assessed at 24 h post-MCAO by an investigator blinded to the treatment group using the modified Neurological Severity Score (mNSS). The modified Neurological Severity Score (mNSS) evaluated motor, sensory, balance and reflex measures. Scores range from 0 to 17 with higher scores indicating greater neurologic injury (Seyfried et al., 2004).

2.5 TTC staining for determination of infarction volume

At 24 h following MCAO, rats were sacrificed and infarct size and volume were measured by 2,3,5-triphenyltetrazolium chloride (TTC; 2%) staining. 2 mm coronal brain slices were stained for 15 min at 37°C. Hemispheres were scanned on a flatbed scanner and analyzed using Image J software. On each slice, the non-stained area (ischemic brain) was outlined, and the infarct volume was calculated. The corrected infarction volume (CIV) was calculated using the following equation: CIV=(CHV-[IHV-IHI])x d, where CHV was the area of the contralateral cortex or striatum in mm2, IHV was the area of the ipsilateral cortex or striatum in mm2, IHI was the infarct area (cortical or striatal) in mm2, and d was slice thickness (2 mm). Edema index was calculated as follows: Edema index = ([volume of ipsilateral hemisphere-volume of contralateral hemisphere]/volume of contralateral hemisphere) x100% (Yang et al., 1998).

2.6 Spectrophotometric assay of hemoglobin

Hemorrhagic transformation was quantified using a spectrophotometric assay of brain hemoglobin content (QuantiChromTM Hemoglobin Assay Kit). At 24 h after MCAO, the rats were anesthetized with ketamine (90 mg/kg; i.p.; Webster Veterinary; Sterling, MA) and xylazine (5 mg/kg; Webster Veterinary), and then transcardially perfused with 0.1 mol/L phosphate buffered saline until the outflow fluid from the right atrium was clear. MCA areas in left and right hemisphere were dissected out, homogenized, and centrifuged at 13,000 × g for 30 min. Supernatant (50 μl) from each sample was aliquoted into a 96 well plate and 200 μl of assay reagent was added. After 15 min incubation, optical density was measured at 400 nm with a spectrophotometer. Hemoglobin content was expressed as mg/dl tissue (Christoforidis et al., 2007).

2.7 Statistical analysis

Data were presented as mean±S.E.M. Differences in infarct volume, mNSS, edema index and hemoglobin content between treatment groups were compared using Students t-test. Differences in mortality rate between treatment groups were compared using Fisher’s exact test. Level of significance was set at P<0.05.

3. Results

3.1. APT102 and r-tPA in combination significantly decreased mortality rate at 24 h following middle cerebral artery occlusion (MCAO)

No rats were excluded from the study for not meeting the ischemic brain injury criteria and no differences in reperfusion were seen amongst groups receiving r-tPA. Mortality rate was evaluated at 24 h following MCAO. Combination therapy of APT102 and r-tPA significantly reduced the mortality rate from 47% in the r-tPA group to 16% in the r-tPA+APT102 group (P=0.027). No significant difference (P=0.163) in mortality rate was found between saline and rtPA+APT102 group rats. There was no significant difference (P=0.384) in mortality rate between saline and r-tPA rats (Fig.1). Some rats dying prior to 24 h were autopsied with typical findings including larger hemispheric infarctions, extensive brain edema, and macroscopic hemorrhage. Rats dying prior to 24 h were not included in the histological, hemoglobin assay, and performance studies. Many of the rats that died prior to 24 h, particularly within the r-tPA group, exhibited extensive hemorrhagic transformation and enlarged infarcts. The higher mortality rate in the r-tPA group compared to the r-tPA+APT102 group, introduces a survival bias in that the most severely infracted and impaired animals died and were excluded from analysis. Therefore, the difference in parameters between r-tPA and r-tPA+APT102 is likely even larger than reported herein due to this bias.

Fig. 1.

Fig. 1

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Mortality rate was evaluated at 24 h following MCAO. Results showed a 47% mortality rate at 24 h when r-tPA was administered alone at 6 h following MCAO (N=30). Combination of APT102 and r-tPA administration (N=19) at 6 h after MCAO reduced mortality at 24 h from 47% to 16% (P<0.05). No significant difference (P>0.05) in mortality rate was observed between saline group (35%, n=23) and r-tPA+TPA102 (16%, n=19). There was no significant difference (P>0.05) in mortality rate between saline group (35%, n=23) and r-tPA group (47%, n=30). * = P<0.05.

3.2. APT102 and r-tPA in combination reduced cortical, striatal, and total hemispheric infarction size at 24 h following MCAO

Stroke volume of the cortex, striatum, and total cerebral hemisphere was measured at 24 h following MCAO (Fig. 2). Cortical (P=0.011), striatal (P=0.013), and total hemispheric (P=0.004) infarction volume were significantly reduced by r-tPA + APT102 combination treatment compared with r-tPA alone. Cortical (P=0.033), striatal (P=0.010), and total hemispheric (P=0.011) infarction volume were significantly reduced by r-tPA + APT102 combination treatment compared with saline treated rats. No significant difference in cortical (P=0.698), striatal (P=0.409), and total hemispheric (P=0.844) infarction volume was observed between saline and r-tPA treated rats.

Fig. 2.

Fig. 2

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Measurement of stroke volume of cortex, striatum, and total cerebral hemisphere at 24 h following MCAO. Results showed a significant decrease (P<0.05 in cortex and striatum, P<0.01 in total hemisphere) for stroke volume in cortex (29±4% vs 47±5%), striatum (40±3% vs 50±2%), and total hemisphere (33±4% vs 48±3%) in r-tPA+APT102 group (n=14) compared with r-tPA group (n=14). Results also showed a significant decrease (P<0.05) in stroke volume in cortex (29±4% vs 44±5%), striatum (40±3% vs 54±4%), and total hemisphere (33±4% vs 47±4%) in rtPA+APT102 group (n=14) compared with saline group (n=13). No significant difference (P>0.05) in stroke volume in cortex (44±5% vs 47±5%), striatum (54±4% vs 50±2%), and total hemisphere (47±4% vs 48±3%) was observed in saline group (n=13) compared with r-tPA group (n=14). Bars represent mean ± S.E.M. * = P<0.05. ), * * = P<0.01.

3.3. APT102 and r-tPA in combination improved neurological function at 24 h post-MCAO

Before MCAO, neurological score was normal (score=0) in all animals. Neurological function using the modified neurological severity score (mNSS) was assessed at 24 h following MCAO. Higher numbers indicate more severe deficits. APT102 and r-tPA in combination significantly (P=0.045) improved the neurological score compared with r-tPA alone. No significant (P=0.331) difference in mNSS was noted between saline and r-tPA+APT102 treated rats. No significant (P=0.138) difference in neurological scores was observed between saline and r-tPA treated rats (Fig. 3).

Fig. 3.

Fig. 3

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Assessment of neurological function using a 17 point mNSS following MCAO. Results showed that combination of r-tPA with APT102 had significantly improved mNSS (P<0.05) from r-tPA group (8.9±0.6, n=14) to r-tPA+ APT102 group (6.8±0.8, n=16). No significant difference (P>0.05) in mNSS scores was demonstrated between saline group (7.3±0.5, n=15) and r-tPA+ APT102 group (6.8±0.8, n=16). No significant difference (P>0.05) was observed between saline group (7.3±0.5, n=15) and r-tPA group (8.9±0.6, n=14). Bars represent mean ± S.E.M. * = P<0.05.

3.4 APT102 and r-tPA in combination significantly decreased hemorrhage transformation in cortex, striatum and total hemisphere at 24 following MCAO

Hemoglobin levels in cortex, striatum, and total cortical hemisphere were measured by hemoglobin assay at 24 h following MCAO. r-tPA and APT102 combination significantly decreased hemoglobin levels in cortex (P=0.009), striatum (P=0.045), and total cerebral hemisphere (P=0.018) compared to r-tPA treatment alone. Results also showed a significant decrease of hemoglobin levels in the cortex (P=0.017) and total hemisphere (P=0.008) in r-tPA+ APT102 group compared with saline group. No significant difference (P=0.056) in hemoglobin levels in striatum was found in r-tPA+ APT102 group compared with saline group. No significant difference in hemoglobin levels in cortex (P=0.893), striatum (P=0.234), and total cerebral hemisphere (P=0.420) was observed comparing saline group with r-tPA group rats (Fig. 4 A). Representative coronal sections showed there were different levels of hemorrhage with saline (moderate), r-tPA (severe) and r-tPA+APT102 treated rats (mild) following MCAO at 24 h (Fig. 4 B).

Fig. 4.

Fig. 4

Fig. 4

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(A) Hemoglobin levels were measured in cortex, striatum and total hemisphere by hemoglobin assay at 24 h following MCAO. Results showed a significant decrease (P<0.05 in striatum and total hemisphere, P<0.01 in cortex) in hemoglobin levels in cortex (1.39±0.08 vs 1.88±0.14mg/dl), striatum (1.37±0.10 vs 2.04±0.29mg/dl), and total hemisphere (1.37±0.08 vs 1.95±0.20 mg/dl) in r-tPA+ APT102 group (n=13) compared with r-tPA group (n=12). Results also showed a significant decrease (P<0.05 in cortex, P<0.01 in total hemisphere) in hemoglobin levels in cortex (1.39±0.08 vs 1.85±0.14mg/dl) and total hemisphere (1.37±0.08 vs 1.76±0.10 mg/dl) in r-tPA+ APT102 group (n=13) compared with saline group (n=15). No significant difference (P>0.05) in hemoglobin levels was observed in striatum (1.37±0.10 vs 1.66±0.10) in r-tPA+ APT102 group (n=13) compared with saline group (n=15). No significant difference (P>0.05) in hemoglobin levels in cortex (1.85±0.14 vs 1.88±0.14mg/dl), striatum (1.66±0.10 vs 2.04±0.29mg/dl), and total hemisphere (1.76±0.10 vs 1.95±0.20 mg/dl) was demonstrated in saline group (n=15) compared with r-tPA group (n=12).Bars represent mean ± S.E.M. * = P<0.05,* * = P<0.01. (B) Representative coronal sections through the infarct regions of brains in saline (moderate hemorrhage), r-tPA (severe hemorrhage) and r-tPA+APT102 (mild hemorrhage) rats following MCAO at 24 h.

3.5. APT102 and r-tPA in combination improved edema formation in saline group rats but not in r-tPA group rats at 24 h following MCAO

Edema formation (brain swelling) was measured at 24 h following MCAO (Fig. 5). Results showed a significant decrease (P=0.038) in edema formation between r-tPA+APT102 and saline treated rats. No significant difference (P=0.366) in edema formation was observed between r-tPA and r-tPA +APT102 treated rats. No significant difference (P=0.408) in edema formation was found between saline and r-tPA treated rats

Fig. 5.

Fig. 5

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Measurement of edema formation in the infarct hemisphere following MCAO. Results showed a significant decrease (P<0.05) in edema formation between r-tPA+APT102 group (22.4±2.3%, n=14) and saline group (28.4±1.5%, n=13).No significant (P>0.05) difference in edema formation was observed between r-tPA+APT102 group (22.4±2.3%, n=14) and r-tPA group (25.7±2.8%, n=14). No significant (P>0.05) difference in edema formation was shown between saline group (28.4±1.5%, n=13) and r-tPA group(25.7±2.8%, n=14). Bars represent mean ± S.E.M. * =P<0.05.

4. Discussion

This is the first report of the evaluation of a soluble apyrase in a reproducible and clinically relevant embolic stroke model of aged female rats. The protective effect of r-tPA over placebo (saline) was lost at 6 h following ischemic stroke. The main finding of this study, taking into account prior findings demonstrating a protective effect of r-tPA when administered at 2 hours after ischemic stroke (DiNapoli et al., 2008; Tan et al., 2009), was that r-tPA in combination with the recombinant human apyrase (APT102) safely extended the time window for r-tPA out to 6 h after ischemic stroke in aged female rats. This study showed that co-administration of APT102 with r-tPA significantly reduced mortality rate, decreased infarct volume and cerebral hemorrhage, and improved neurological functional outcome at 24 h following MCAO as compared to subjects treated with r-tPA alone. These findings have significant relevance in that this work can potentially translate to the clinical environment. The management of ischemic stroke would thereby change radically. Specifically, r-tPA could potentially be administered safely and efficaciously until 6 hours post-ischemia, significantly reducing morbidity and mortality.

Consistent with our previous studies, higher mortality and larger infarct volumes were observed in aged female rats at 24 h following MCAO and r-tPA reperfusion (Dinapoli et al., 2006; DiNapoli et al., 2008; Tan et al., 2009). Prior studies in our lab also show that experimental therapeutics (EEMMID and apocynin) combined with r-tPA were neuroprotective follwing ischemic stroke in young animals but not in aged animals (Tan et al., 2009; Kelly et al., 2009). As a thrombolytic agent, r-tPA has direct and deleterious effects on the neurovascular unit that substantially increases death rate and infarct volume and causes severe hemorrhage when administered intravenously following an MCAO in a delayed fashion. Recent studies indicate that aged rats were more severely impaired following ischemic stroke than younger rats with diminished functional recovery (Popa-Wagner et al., 2006a; Popa-Wagner et al., 2006b; Popa-Wagner et al., 2007). Clinically, increased age negatively affects spontaneous recovery of stroke patients and is a primary risk factor for fragility of the blood brain barrier and hemorrhagic transformation following thrombolytic therapy (Fisher et al., 2009). In the largest clinical trial of rtPA use in stroke, the International Stroke Trial 3 (IST-3), r-tPA treatment increased symptomatic intracranial hemorrhage within 7 d from 1% to 7%. This increase was then associated with a mortality of 11% in the r-tPA group versus 7% in the control group at 7 d following ischemic stroke (Davalos et al, 2012). Much of the mortality following ischemic stroke is due to increased intracranial pressure produced by edema formation.

Vascular endothelium and platelets maintain vascular integrity and promote primary and secondary hemostasis following interruption of vessel continuity. Cerebral artery occlusion and reperfusion is associated with endothelial damage and activation of platelets (Marcus et al., 2001; Marcus et al., 2005; Sugimoto et al., 2009). ATP and ADP are released in high concentrations by activated endothelial cells and platelets following aggregation on endothelium. In turn, ATP and ADP further increase additional platelet aggregation, leading to microthrombus formation and the eventual worsening of endothelial injury. Platelet and fibrin deposition downstream of an occlusive lesion contribute significantly to the postischemic hypoperfusion and tissue injury; thus, complicating stroke injury (Belayev et al., 2003; Bours et al., 2006; Dwyer et al., 2004; Imai et al., 2000; Kohler et al., 2007; Wang and Guidotti, 1996).

The beneficial effects of co-administration of APT102 with r-tPA at 6h following MCAO may likely be explained by the effect of apyrase. APT102, a novel form of the naturally occurring human apyrase/ADPase (CD39, recombinant soluble human CD39), promotes the breakdown of extracellular ATP and ADP by catalyzing the removal of the gamma phosphate from ATP and the beta phosphate from ADP (Imai et al., 2000; Marcus et al., 1991; Pinsky et al., 2002; Robson, 2006; Robson et al., 2005). APT102 does not act on platelets per se but rather on prothrombolic mediators (ATP and ADP). Increased MMP9 activity along the basement membrane of the blood brain barrier is believed to cause hemorrhagic transformation due to increased release and extravasation of neutrophils. Moreover, APT102 most likely prevents hemorrhagic transformation by inhibiting ATP-induced endothelial cell activation and neutrophil infiltration and re-sensitizing platelets (Belayev et al., 2003; Guckelberger et al., 2004; Kohler et al., 2007; Marcus et al., 2005; Moeckel Douglas, 2011; Robson, 2006). As such, the protective effect of APT102 likely requires reperfusion to be observed, a finding consistent with prior work in a thrombo-embolic model of ischemic stroke with r-tPA mediated reperfusion (Sun et al., 2011). For this reason, APT102 was not studied in isolation in this study and consequently, we cannot comment on any protective effect of APT102 in isolation within our clinically-relevant model of ischemic stroke in aged female rats.

The effects of exogenously administered apyrase are consistent with a prior study, in which CD39 knockout mice demonstrated increased cerebral infarction volumes compared with control CD39 wild type mice following experimental stroke. Additionally, the study showed that endogenous CD39 was protective after stroke, and administration of pharmacological doses of soluble CD39 was effective in inhibiting thrombosis and tissue injury following ischemic stroke (Pinsky et al., 2002). Since soluble CD39 does not interfere with primary GPIb-mediated platelet adhesion at the site of vessel damage, soluble CD39 administration should not prevent a layer of platelets from forming at the site of injury or interfere with hemostatic mechanisms that prevent intracerebral hemorrhage (Belayev et al., 2003; Marcus et al., 2005). Compared with aspirin, soluble CD39 may induce more potent inhibition of platlet aggregation by blocking ADP-induced platelet recruitment. Soluble CD39 was shown to be more efficient in inhibiting platelet recruitment than aspirin via blockade of the arachidonate/thromboxane pathway. Soluble CD39 disaggregates platelets under recruitment, but it does not have a deleterious effect on primary hemostasis. These actions may explain how soluble CD39 can inhibit microvascular thrombosis and confer cerebroprotection in ischemic stroke without worsening intracerebral hemorrhage (Kohler et al., 2007; Marcus et al., 1991; Marcus et al., 2005; Moeckel Douglas, 2011).

5. Conclusion

In conclusion, combination of r-tPA and APT102 reduces mortality rate, decreases cerebral infarct volume, improves neurological deficit scores, and prevents r-tPA-induced hemorrhage transformation in our clinical relevant ischemic stroke model in aged female rats. APT102 can extend the therapeutic window out to at least 6 h by reducing r-tPA-induced intracerebral hemorrhage following MCAO.

Acknowledgments

This work was supported by NIH grant NS060175 (to R.C.,C.R) and NIH Training Grant 5T32GM08174 (to R.T.).

Footnotes

Competing interests: R.C. is the founder of APT Therapeutics, Inc. and the shareholder of the company.

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References

  1. Belayev L, Khoutorova L, Deisher TA, Belayev A, Busto R, Zhang Y, Zhao W, Ginsberg MD. Neuroprotective effect of SolCD39, a novel platelet aggregation inhibitor, on transient middle cerebral artery occlusion in rats. Stroke. 2003;34:758–63. doi: 10.1161/01.STR.0000056169.45365.15. [DOI] [PubMed] [Google Scholar]
  2. Benchenane K, Berezowski V, Ali C, Fernandez-Monreal M, Lopez-Atalaya JP, Brillault J, Chuquet J, Nouvelot A, MacKenzie ET, Bu G, Cecchelli R, Touzani O, Vivien D. Tissue-type plasminogen activator crosses the intact blood-brain barrier by low-density lipoprotein receptor-related protein-mediated transcytosis. Circulation. 2005;111:2241–9. doi: 10.1161/01.CIR.0000163542.48611.A2. [DOI] [PubMed] [Google Scholar]
  3. Bours MJ, Swennen EL, Di Virgilio F, Cronstein BN, Dagnelie PC. Adenosine 5′- triphosphate and adenosine as endogenous signaling molecules in immunity and inflammation. Pharmacol Ther. 2006;112:358–404. doi: 10.1016/j.pharmthera.2005.04.013. [DOI] [PubMed] [Google Scholar]
  4. Christoforidis GA, Slivka A, Mohammad Y, Karakasis C, Avutu B, Yang M. Size matters: hemorrhage volume as an objective measure to define significant intracranial hemorrhage associated with thrombolysis. Stroke. 2007;38:1799–804. doi: 10.1161/STROKEAHA.106.472282. [DOI] [PubMed] [Google Scholar]
  5. Dawson TM, Dawson VL. Taming the clot-buster tPA. Nat Med. 2006;12:993–4. doi: 10.1038/nm0906-993. [DOI] [PubMed] [Google Scholar]
  6. Dinapoli VA, Rosen CL, Nagamine T, Crocco T. Selective MCA occlusion: a precise embolic stroke model. J Neurosci Methods. 2006;154:233–8. doi: 10.1016/j.jneumeth.2005.12.026. [DOI] [PubMed] [Google Scholar]
  7. DiNapoli VA, Huber JD, Houser K, Li X, Rosen CL. Early disruptions of the blood-brain barrier may contribute to exacerbated neuronal damage and prolonged functional recovery following stroke in aged rats. Neurobiol Aging. 2008;29:753–64. doi: 10.1016/j.neurobiolaging.2006.12.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Eckle T, Fullbier L, Wehrmann M, Khoury J, Mittelbronn M, Ibla J, Rosenberger P, Eltzschig HK. Identification of ectonucleotidases CD39 and CD73 in innate protection during acute lung injury. J Immunol. 2007;178:8127–37. doi: 10.4049/jimmunol.178.12.8127. [DOI] [PubMed] [Google Scholar]
  9. Fisher M, Feuerstein G, Howells DW, Hurn PD, Kent TA, Savitz SI, Lo EH. Update of the stroke therapy academic industry roundtable preclinical recommendations. Stroke. 2009;40:2244–50. doi: 10.1161/STROKEAHA.108.541128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Goldstein LB, Adams R, Alberts MJ, Appel LJ, Brass LM, Bushnell CD, Culebras A, DeGraba TJ, Gorelick PB, Guyton JR, Hart RG, Howard G, Kelly-Hayes M, Nixon JV, Sacco RL. Primary prevention of ischemic stroke: a guideline from the American Heart Association/American Stroke Association Stroke Council: cosponsored by the Atherosclerotic Peripheral Vascular Disease Interdisciplinary Working Group; Cardiovascular Nursing Council; Clinical Cardiology Council; Nutrition, Physical Activity, and Metabolism Council; and the Quality of Care and Outcomes Research Interdisciplinary Working Group. Circulation. 2006;113:e873–923. doi: 10.1161/01.STR.0000223048.70103.F1. [DOI] [PubMed] [Google Scholar]
  11. Guckelberger O, Sun XF, Sevigny J, Imai M, Kaczmarek E, Enjyoji K, Kruskal JB, Robson SC. Beneficial effects of CD39/ecto-nucleoside triphosphate diphosphohydrolase-1 in murine intestinal ischemia-reperfusion injury. Thromb Haemost. 2004;91:576–86. doi: 10.1160/TH03-06-0373. [DOI] [PubMed] [Google Scholar]
  12. Handa M, Guidotti G. Purification and cloning of a soluble ATP-diphosphohydrolase (apyrase) from potato tubers (Solanum tuberosum) Biochem Biophys Res Commun. 1996;218:916–23. doi: 10.1006/bbrc.1996.0162. [DOI] [PubMed] [Google Scholar]
  13. Imai M, Takigami K, Guckelberger O, Lin Y, Sevigny J, Kaczmarek E, Goepfert C, Enjyoji K, Bach FH, Rosenberg RD, Robson SC. CD39/vascular ATP diphosphohydrolase modulates xenograft survival. Transplant Proc. 2000;32:969. doi: 10.1016/s0041-1345(00)01065-4. [DOI] [PubMed] [Google Scholar]
  14. Kohler D, Eckle T, Faigle M, Grenz A, Mittelbronn M, Laucher S, Hart ML, Robson SC, Muller CE, Eltzschig HK. CD39/ectonucleoside triphosphate diphosphohydrolase 1 provides myocardial protection during cardiac ischemia/reperfusion injury. Circulation. 2007;116:1784–94. doi: 10.1161/CIRCULATIONAHA.107.690180. [DOI] [PubMed] [Google Scholar]
  15. Lees KR, Bluhmki E, von Kummer R, Brott TG, Toni D, Grotta JC, Albers GW, Kaste M, Marler JR, Hamilton SA, Tilley BC, Davis SM, Donnan GA, Hacke W, Allen K, Mau J, Meier D, del Zoppo G, De Silva DA, Butcher KS, Parsons MW, Barber PA, Levi C, Bladin C, Byrnes G. Time to treatment with intravenous alteplase and outcome in stroke: an updated pooled analysis of ECASS, ATLANTIS, NINDS, and EPITHET trials. Lancet. 375:1695–703. doi: 10.1016/S0140-6736(10)60491-6. [DOI] [PubMed] [Google Scholar]
  16. Lloyd-Jones D, Adams RJ, Brown TM, Carnethon M, Dai S, De Simone G, Ferguson TB, Ford E, Furie K, Gillespie C, Go A, Greenlund K, Haase N, Hailpern S, Ho PM, Howard V, Kissela B, Kittner S, Lackland D, Lisabeth L, Marelli A, McDermott MM, Meigs J, Mozaffarian D, Mussolino M, Nichol G, Roger VL, Rosamond W, Sacco R, Sorlie P, Thom T, Wasserthiel-Smoller S, Wong ND, Wylie-Rosett J. Heart disease and stroke statistics--2010 update: a report from the American Heart Association. Circulation. 121:e46–e215. doi: 10.1161/CIRCULATIONAHA.109.192667. [DOI] [PubMed] [Google Scholar]
  17. Marcus AJ, Safier LB, Hajjar KA, Ullman HL, Islam N, Broekman MJ, Eiroa AM. Inhibition of platelet function by an aspirin-insensitive endothelial cell ADPase. Thromboregulation by endothelial cells. J Clin Invest. 1991;88:1690–6. doi: 10.1172/JCI115485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Marcus AJ, Broekman MJ, Drosopoulos JH, Islam N, Alyonycheva TN, Safier LB, Hajjar KA, Posnett DN, Schoenborn MA, Schooley KA, Gayle RB, Maliszewski CR. The endothelial cell ecto-ADPase responsible for inhibition of platelet function is CD39. J Clin Invest. 1997;99:1351–60. doi: 10.1172/JCI119294. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Marcus AJ, Broekman MJ, Drosopoulos JH, Pinsky DJ, Islam N, Maliszewsk CR. Inhibition of platelet recruitment by endothelial cell CD39/ecto-ADPase: significance for occlusive vascular diseases. Ital Heart J. 2001;2:824–30. [PubMed] [Google Scholar]
  20. Marcus AJ, Broekman MJ, Drosopoulos JH, Olson KE, Islam N, Pinsky DJ, Levi R. Role of CD39 (NTPDase-1) in thromboregulation, cerebroprotection, and cardioprotection. Semin Thromb Hemost. 2005;31:234–46. doi: 10.1055/s-2005-869528. [DOI] [PubMed] [Google Scholar]
  21. Moeckel Douglas BP, Nguyen Annie JS, Chen R, Abendschein A novel human apyrase prevents reocclusion and substantially decreases infarct size without bleeding prolongation after coronary fibrinolysis in dogs. Arteriosclerosis, thrombosis and vascular biology 2011 scientific sessions of American Heart Association oral presentation. 2011 Vol., ed.^eds. [Google Scholar]
  22. Pinsky DJ, Broekman MJ, Peschon JJ, Stocking KL, Fujita T, Ramasamy R, Connolly ES, Jr., Huang J, Kiss S, Zhang Y, Choudhri TF, McTaggart RA, Liao H, Drosopoulos JH, Price VL, Marcus AJ, Maliszewski CR. Elucidation of the thromboregulatory role of CD39/ectoapyrase in the ischemic brain. J Clin Invest. 2002;109:1031–40. doi: 10.1172/JCI10649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Popa-Wagner A, Badan I, Vintilescu R, Walker L, Kessler C. Premature cellular proliferation following cortical infarct in aged rats. Rom J Morphol Embryol. 2006a;47:215–28. [PubMed] [Google Scholar]
  24. Popa-Wagner A, Dinca I, Yalikun S, Walker L, Kroemer H, Kessler C. Accelerated delimitation of the infarct zone by capillary-derived nestin-positive cells in aged rats. Curr Neurovasc Res. 2006b;3:3–13. doi: 10.2174/156720206775541732. [DOI] [PubMed] [Google Scholar]
  25. Popa-Wagner A, Badan I, Walker L, Groppa S, Patrana N, Kessler C. Accelerated infarct development, cytogenesis and apoptosis following transient cerebral ischemia in aged rats. Acta Neuropathol. 2007;113:277–93. doi: 10.1007/s00401-006-0164-7. [DOI] [PubMed] [Google Scholar]
  26. Robson S. The therapeutic potential of CD39: interview with Dr Simon Robson by Emma Quigley. Expert Opin Ther Targets. 2006;10:649–52. doi: 10.1517/14728222.10.5.649. [DOI] [PubMed] [Google Scholar]
  27. Robson SC, Wu Y, Sun X, Knosalla C, Dwyer K, Enjyoji K. Ectonucleotidases of CD39 family modulate vascular inflammation and thrombosis in transplantation. Semin Thromb Hemost. 2005;31:217–33. doi: 10.1055/s-2005-869527. [DOI] [PubMed] [Google Scholar]
  28. Sacco RL, Adams R, Albers G, Alberts MJ, Benavente O, Furie K, Goldstein LB, Gorelick P, Halperin J, Harbaugh R, Johnston SC, Katzan I, Kelly-Hayes M, Kenton EJ, Marks M, Schwamm LH, Tomsick T. Guidelines for prevention of stroke in patients with ischemic stroke or transient ischemic attack: a statement for healthcare professionals from the American Heart Association/American Stroke Association Council on Stroke: co-sponsored by the Council on Cardiovascular Radiology and Intervention: the American Academy of Neurology affirms the value of this guideline. Stroke. 2006;37:577–617. doi: 10.1161/01.STR.0000199147.30016.74. [DOI] [PubMed] [Google Scholar]
  29. Seyfried D, Han Y, Lu D, Chen J, Bydon A, Chopp M. Improvement in neurological outcome after administration of atorvastatin following experimental intracerebral hemorrhage in rats. J Neurosurg. 2004;101:104–7. doi: 10.3171/jns.2004.101.1.0104. [DOI] [PubMed] [Google Scholar]
  30. Sugimoto S, Lin X, Lai J, Okazaki M, Das NA, Li W, Krupnick AS, Chen R, Jeong SS, Patterson GA, Kreisel D, Gelman AE. Apyrase treatment prevents ischemia-reperfusion injury in rat lung isografts. J Thorac Cardiovasc Surg. 2009;138:752–9. doi: 10.1016/j.jtcvs.2009.04.049. [DOI] [PubMed] [Google Scholar]
  31. Sun G, Zhao X, Grotta J, Savitz S, Chen C, Aronoslaw J. Apyrase, APT102, improves the r-tPA beneficial effect in thromboembolic stroke model. American Heart Associated International stroke. 2011 Feb 9; 2011. Poster presentation. [Google Scholar]
  32. Tan Z, Li X, Kelly KA, Rosen CL, Huber JD. Plasminogen activator inhibitor type 1 derived peptide, EEIIMD, diminishes cortical infarct but fails to improve neurological function in aged rats following middle cerebral artery occlusion. Brain Res. 2009;1281:84–90. doi: 10.1016/j.brainres.2009.05.042. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Wang C, Shabanzadeh AP, He C, Pegg CC. APT 102 protects the ischemically injured brain in rats. Oral presentation; Neuroscience 2010 meeting; Nov 14.2010. [Google Scholar]
  34. Wang TF, Guidotti G. CD39 is an ecto-(Ca2+, Mg2+)-apyrase. J Biol Chem. 1996;271:9898–901. [PubMed] [Google Scholar]
  35. Yang Y, Shuaib A, Li Q. Quantification of infarct size on focal cerebral ischemia model of rats using a simple and economical method. J Neurosci Methods. 1998;84:9–16. doi: 10.1016/s0165-0270(98)00067-3. [DOI] [PubMed] [Google Scholar]

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