Introduction: Venous thromboembolism (VTE ) manifesting as deep vein thrombosis (DVT) and pulmonary embolus (PE) and arterial thromboembolism (ATE) manifesting as acute ischemic stroke (AIS) result in ~1 million US deaths annually. Increased levels of circulating inflammatory markers, particularly von Willebrand factor (VWF), may indicate poor outcomes in ATE, but previous studies limit this response to high shear stress milieu. We compared VTE and AIS thrombi inflammatory markers at time of intervention. Methods: Clots were harvested from 20 PE, 9 DVT, and 74 AIS patients and immunofluorescent staining completed in duplicate with VWF, plasminogen activator inhibitor 1 (PAI-1), glycophorin A (RBCs), CD42b (platelets), fibrinogen, and neutrophil endothelial trap constituents (NETs). NETs were defined as citrullinated histones (CitH3), neutrophil elastase (NE) and myeloperoxidase (MPO). Clot sections were analyzed with Image J. Results: VWF levels were lower in AIS clots (21.02 +/- 12.02%) compared to DVT (24.87 +/- 12.98%, p=0.0212) and higher in PE (12.21 +/- 5.96%, p=0.0001). PAI-1 levels were higher in AIS clots (42.80 +/- 16.28%) compared to DVT (27.43 +/- 15.61%, p=0.0001) and lower in PE (51.23 +/- 10.89%, p=0.0016). Although RBCs were not significantly different in AIS (35.34 +/- 15.32%) compared to DVT, they were more prevalent in PE (52.93 +/- 8.31%, p=0.0001). Surprisingly, although platelets were lower in DVT thrombi (15.30 +/- 12.33%, p=0.0105) compared to AIS (23.06 +/- 13.71%), they were increased in PE (32.70 +/- 8.46%, p=0.0001. Lastly, although there was no difference in DVT thrombi compared to AIS clots, fibrinogen (21.307 +/- 8.75%) was lower in PE clots (14.85 +/- 7.56%, p=0.0001), as was CitH3 (8.42 +/- 10.42% vs 7.03 +/- 4.18%, p=0.0001, and NE (31.10 +/- 18.31% vs 37.18 +/- 14.31% in PE, p=0.0025). MPO was unremarkable. Conclusion: Inflammatory marker levels in AIS vs VTE have a complexity beyond shear stress and offer insights into targeting thrombolytics.

INTRODUCTION: Acute ischemic stroke (AIS) is the leading cause of long term disability. rtPA treatment of AIS results in intracranial hemorrhage (ICH) in 7% of patients with 40% mortality. Increased matrix metalloprotease-9 (MMP-9) correlates with ICH, while reduced levels maintain integrity of the blood brain barrier (BBB), preventing ICH. Von Willebrand Factor (VWF) induces both thrombosis and inflammation in large vessel occlusion (LVO) stroke. BB-031 targets VWF, and lyses thrombotic occlusion. BB-025 reverses BB-031. METHODS: After 6 hours of autologous canine MCAO, animals were treated with vehicle, BB-031, and BB-031+BB-025. VWF inhibition, TICI scores, and plasma inflammatory markers [VWF, interleukin-1 (IL-1b), interleukin-6 (IL-6), tumor necrosis factor-a (TNF- a), and matrix metalloproteinase-9 (MMP-9)] were analyzed over the course of the study. RESULTS: Within 15 minutes of vehicle treatment, VWF increased by 3.102 ± .382 ng/ml (n = 6) whereas BB-031 reduced VWF by 0.223 ± .154 ng/ml (p < 0.05) (n = 8). BB-031-treated animals had TICI > 2B in 62.5% of the animals. Although IL-1b and IL-6 were unchanged, and TNF-a was not detectable, MMP-9 levels were lower in BB-031-treated animals at sacrifice compared to both vehicle and BB-031+BB-025 (p < 0.0001). CONCLUSIONS: BB-031 treatment inhibited VWF, increased reperfusion, and reduced MMP-9 after canine MCAO, suggesting that in addition to recanalization, inhibiting VWF mitigates inflammation and iatrogenic ICH.

Introduction: Venous thromboembolism (VTE) manifesting as deep vein thrombosis and pulmonary embolus results in ~300,000 US deaths annually. Risk factors include obesity and current therapy limitations leave ~ 50% of patients with no effective treatment. Circulating von Willebrand Factor (VWF) levels portend poor clinical outcomes. BB-031, an aptamer-based VWF inhibitor with prophylactic and arterial thrombolytic efficacy may translate to VTE. Methods: Jugular vein occlusion (JVO) was induced by FeCl 3 either before or after intravenous treatment in 14 week old male and female C57/BL6 wild type (WT) or male diet-induced obese (DIO) mice. 5.0 mg/kg BB-031 was administered as a bolus, 10.0 mg/kg rtPA with 10% bolus and remaining 90% over 60 minutes, 400 U heparin as a bolus, or vehicle. Doppler jugular vein flow velocity was recorded from baseline to determine patency 30 minutes after injury. Thrombolytic clots were stabilized for 20 minutes. Results: Prophylactic mice occluded ~20 minutes after FeCl 3 application with rtPA=18.32 +/- 6.54 min, heparin 17.14 +/- 8.53 min, and vehicle 17.29 +/- 7.26 min. Thrombosis did not occur with BB-031 treatment (n=4-12/group). Treatment after JVO resulted in no difference compared to vehicle in male WT mice, but BB-031-treated WT females resulted in 76.09% recanalization 35 minutes after occlusion compared to rtPA (9.64%, p<0.0001), heparin (30.95%, p=0.7306), or vehicle (22.07%, p<0.0001), (n=5/group). Male DIO mice significantly reperfused with BB-031 (100%) vs rtPA (10.52%), heparin (4.54%), and vehicle (22.50%), p<0.0001, (n=4-6/group). Conclusion: VWF inhibition by BB-031 maintains patency in a VTE murine model offering future therapeutic alternatives.

Preclinical models of large-vessel occlusion (LVO) stroke play an important role in testing novel thrombolytic and neuroprotective agents, as well as assessing endovascular devices for endovascular thrombectomy. Depending on the modality used to model LVO stroke, different aspects of human stroke pathophysiology can be recapitulated. Thus, variables including size and region of the occlusion, composition of the clot, and degree of reperfusion can be controlled. We conducted a thorough literature review of all the preclinical models of stroke used currently. We searched for studies using the PubMed database and included original studies, reviews, systematic reviews, and meta-analyses. In this review, we describe the considerations in choosing a preclinical model of LVO stroke. We provide an overview of available small- and large-animal LVO stroke models that can be used in stroke research as well as the benefits and limitations of each. We then discuss the current outcome measures that can be evaluated through these models, in terms of both tissue damage and behavioral outcome. We also discuss the advantages and disadvantages of each animal model. Finally, based on this summary, we propose that mice represent the most versatile small-animal LVO model and dogs are the most appropriate large-animal LVO stroke model to translate candidate drugs and devices into clinical trials.

Background and purposePosterior circulation strokes, accounting for 20% of acute ischemic strokes, significantly contribute to morbidity and mortality. Fibrinolysis by rtPA improves outcomes in stroke but the risk of intracranial hemorrhage limits benefit. Arterial recanalization of basilar artery occlusion by thrombolysis or endovascular thrombectomy improves outcomes in posterior circulation strokes. This study investigates a VWF-targeting RNA aptamer as a safer and more effective alternative to rtPA in a canine model.Materials and methodsAutologous clots were placed into the basilar artery to induce stroke in 24 beagles. To compare reperfusion, 0.9 mg/kg rtPA, 0.5 mg/kg BB-031, or vehicle were administered 60 min after the initiation of occlusion. Digital subtraction angiography, laser speckle imaging and magnetic resonance imaging were used to assess recanalization, reperfusion and infarct volume, respectively.ResultsTreatment with BB-031 resulted in recanalization of the posterior circulation on digital subtraction angiography with no evidence of microembolism assessed at sacrifice. 66.5% of animals treated with BB-031 resulted in reperfusion with a TICI score of ≥1 whereas vehicle remained at TICI score 0 as did all but one rtPA animal at sacrifice. Improved perfusion was seen in the basilar artery and surrounding blood vessels visualized through the cranial window with laser speckle imaging to ~47% of its original baseline in BB-031 group compared to rtPA at 37% and vehicle at 22%. Finally, BB-031-treatment resulted in an approximate 32% mean infarct volume, significantly smaller on magnetic resonance imaging compared to 56% in vehicle treated and 48% with rtPA treatment.ConclusionTargeted inhibition of VWF by BB-031 increased recanalization and reperfusion, and reduced infarct volume in a canine model of BAO stroke. It represents a promising target based on preliminary results for treating acute ischemic stroke.

Introduction: Acute ischemic strokes (AIS) are a leading cause of death and long-term disability worldwide. Von Willebrand Factor (VWF) is an attractive target for anti-thrombotic therapeutics as it plays a pivotal role in platelet adhesion, activation, and aggregation with large vessel occlusion (LVO) stroke. VWF is stored in an ultra large form in platelet α-granules and Weibel-Palade bodies of endothelial cells from where it is released during injury or inflammation. BB-031 is an RNA aptamer that binds and inhibits VWF, and both prevents and lyses an occlusive thrombus. Hypothesis: We hypothesize BB-031 treatment will inhibit VWF thereby reducing platelet reactivity and attenuating the increase in VWF levels in a canine model of LVO stroke. Methods: A canine embolic MCAO model was used to assess BB-031 efficacy of VWF inhibition and platelet activity compared to placebo. After 6 hours of LVO with an autologous clot, intravenous treatment was initiated with placebo (n=7), or 0.5 (n=8), 1.0 (n=4), and 5.0 (n=4) mg/kg BB-031. Whole blood was collected at baseline, 6 hours after occlusion then at 10, 15, 20, 25, 30, 60 min after treatment and lastly, at time of sacrifice 9 hours after MCAO induction. Enzyme-linked immunosorbent assay (ELISA) was used to measure circulating VWF and platelet function analysis (PFA-100 Siemens) was utilized to determine platelet reactivity. Results: VWF levels immediately after LVO in vehicle control were significantly attenuated within 10 minutes of administration with all BB-031 concentrations (p<0.05). Within 15 minutes after vehicle treatment, VWF levels increased by 3.102 ± .382 ng/ml whereas BB-031 administration of 0.5, 1.0, and 5.0 mg/kg reduced VWF levels by 0.223 ± .154 ng/ml, 0.269 ± .304 ng/ml and 1.053 ± .267 ng/ml respectively. Platelet reactivity to ADP/Collagen (PFA-100) revealed significantly increased platelet closing time in all BB-031 treated canines from immediately after administration through 60 minutes. (p<0.05). Conclusion: BB-031 treatment demonstrated significant VWF inhibition and reduced platelet reactivity in a canine AIS LVO model compared to placebo. These findings indicate that BB-031 may offer a superior therapeutic treatment paradigm for ischemic stroke.

Introduction: Von Willebrand Factor (VWF) is a ubiquitous component of thrombi extracted by endovascular thrombectomy from patients with large vessel occlusion (LVO) stroke. BB-031 is a modified RNA aptamer that targets von Willebrand Factor (VWF) that lyses occlusive thrombi and improves outcomes in pre-clinical models of acute ischemic stroke (AIS). Hypothesis: BB-031 is safe, tolerable, and inhibits both VWF binding and activity in a dose-dependent manner. Methods: Healthy participants were randomized to receive BB-031 or placebo (6:2) by intravenous bolus injection at single ascending doses (SAD) of 0.1, 0.3, 1.0, 2.0 and 4.0 mg/kg (total n=40). Serial Clinical assessment, and blood sample collection for pharmacokinetic (PK) and pharmacodynamic (PD) analysis were performed. Non-compartmental PK analysis was conducted using Phoenix WinNonlin. PD evaluation included measurement of VWF inhibition and whole blood thrombosis (Platelet Function Analyzer [PFA-200]). Results: BB-031 was safe and well-tolerated for 28 days following single IV doses up to 4.0 mg/kg. There were no significant adverse events (SAEs), or treatment-emergent adverse events (TEAEs) leading to dose discontinuation. Minor events included bleeding at an IV site in 1 participant, and minor bleeding of an ulcer on the tongue in 1 participant. None of the minor TEAEs were dose-dependent. BB-031 demonstrated nonlinear, dose-dependent plasma PK across the dose range tested, with an apparent mean terminal half-life (t 1/2 ) of 18 min at 0.1 mg/kg to 67 min at 4.0 mg/kg. Dose-dependent changes in VWF binding were observed; >95% maximal mean change from baseline was reached following a single IV dose of 4.0 mg/kg. Finally, PFA-200 results showed complete inhibition of clot formation (closing time ≥300 seconds) at all doses tested with a dose-dependent duration of inhibition and return to normal range. Conclusion: This first-in-human SAD study of BB-031 demonstrated safety following a single IV dose of 0.1 to 4 mg/kg, and dose-dependent patterns of VWF binding and platelet function changes. These results lay the foundation for future studies in patients suffering from thrombotic conditions including AIS.

Purpose: To develop a large animal preclinical model of thromboembolic stroke with stable, protracted large vessel occlusion (LVO) utilizing an autologous clot. Materials and methods: A reproducible canine model of large vessel occlusion stroke was established by endovascular placement of an autologous clot into the middle cerebral artery (MCA) of six adult hounds and confirmed using digital subtraction angiography (DSA). Infarct volume and evidence of hemorrhage were determined by magnetic resonance imaging (MRI) 7 h after occlusion and Thrombolysis in Cerebral Infarction scale (TICI) was assessed before and after clot placement and at 1, 6, 7, and 9 h after middle cerebral artery occlusion (MCAO). Heart rate (HR) and blood pressure (BP) were monitored continuously and invasively through an arterial sheath throughout the procedures and complete blood count and blood gas analysis completed at time of sacrifice. Histopathological findings at time of sacrifice were used to confirm stroke volume and hemorrhage. Results: MCAO with resulting TICI 0 flow was observed in all six animals, verified by serial DSA, and lack of collateral flow persisted for 9 h after clot placement until time of sacrifice. The mean infarct volume was 47.0 ± 6.7% of the ipsilateral hemisphere and no events of spontaneous recanalization or clot autolysis were observed. Conclusion: We demonstrate a thromboembolic canine model of MCAO that is both feasible and results in consistent infarct volumes to generate a clinically relevant LVO. This model is important to evaluate treatment of LVO in acute ischemic stroke (AIS) outside the established 4.5 h recombinant tissue plasminogen activator (rTPA) therapeutic window utilizing a prolonged occlusive thrombus.

Extracellular purine nucleotides and nucleosides released from activated or injured cells influence multiple aspects of cardiac physiology and pathophysiology. Ectonucleoside triphosphate diphosphohydrolase-1 (ENTPD1; CD39) hydrolyzes released nucleotides and thereby regulates the magnitude and duration of purinergic signaling. However, the impact of CD39 activity on post-myocardial infarction (MI) remodeling is incompletely understood. We measured the levels and activity of ectonucleotidases in human left ventricular samples from control and ischemic cardiomyopathy (ICM) hearts and examined the impact of ablation of Cd39 expression on post-myocardial infarction remodeling in mice. We found that human CD39 levels and activity are significantly decreased in ICM hearts (n = 5) compared with control hearts (n = 5). In mice null for Cd39, cardiac function and remodeling are significantly compromised in Cd39-/- mice following myocardial infarction. Fibrotic markers including plasminogen activator inhibitor-1 (PAI-1) expression, fibrin deposition, α-smooth muscle actin (αSMA), and collagen expression are increased in Cd39-/- hearts. Importantly, we found that transforming growth factor β1 (TGF-β1) stimulates ATP release and induces Cd39 expression and activity on cardiac fibroblasts, constituting an autocrine regulatory pathway not previously appreciated. Absence of CD39 activity on cardiac fibroblasts exacerbates TGF-β1 profibrotic responses. Treatment with exogenous ectonucleotidase rescues this profibrotic response in Cd39-/- fibroblasts. Together, these data demonstrate that CD39 has important interactions with TGF-β1-stimulated autocrine purinergic signaling in cardiac fibroblasts and dictates outcomes of cardiac remodeling following myocardial infarction. Our results reveal that ENTPD1 (CD39) regulates TGF-β1-mediated fibroblast activation and limits adverse cardiac remodeling following myocardial infarction.NEW & NOTEWORTHY We show that CD39 is a critical modulator of TGF-β1-mediated fibroblast activation and cardiac remodeling following myocardial infarction via modulation of nucleotide signaling. TGF-β1-induced CD39 expression generates a negative feedback loop that attenuates cardiac fibroblast activation. In the absence of CD39 activity, collagen deposition is increased, elastin expression is decreased, and diastolic dysfunction is worsened. Treatment with ecto-apyrase attenuates the TGF-β1-induced profibrotic cardiac fibroblast phenotype, revealing a novel approach to combat post-myocardial infarction cardiac fibrosis.

Introduction: We demonstrated that VWF inhibition thrombolyses stabilized clots in and decreases infarct size after middle cerebral artery occlusion (MCAO) in pre-clinical models of stroke. Objective: To compare the thrombolytic efficacy of von Willebrand Factor (VWF) inhibition by DTRI-031 versus rTPA and aspirin utilizing an ex vivo Halo assay. Hypothesis: We hypothesized that DTRI-031 treatment, would result in greater combinatorial efficacy with aspirin or rTPA than any one treatment alone. Methods: Ex vivo Halo assay was performed with whole blood from male and female, 15-month-old wild-type C57BL/6J adult mice, 1-year-old hounds, and healthy control patients 40-60 years of age). Results: Healthy patients The mean CLR max of the negative control group (n=6) was 0.024 ± 0.17 min -1 . Addition of 250 nM DTRI-031 (n=8) or 0.7 nM rtPA (n=8) increased the CLR max to 1.67 ± 1.56 min -1 and 1.80 ± 1.41 min -1 respectively. Addition of 500 nM DTRI-031 and 0.7 rtPA (n=4) resulted in a synergistic effect of both compounds with a CLR max of 10.15 ± 2.4 min -1 (p=.0003). Mice The CLR max of the negative control (n=6) was 0.013 ± 0.011 min -1 . Addition of 250 nM DTRI-031 (n=6) increased the CLR max to 1.19 ± 1.27 min -1 . Addition of 7 nM rtPA (n=6) increased the CLR max to 0.13 ± 0.13 min -1 , which was considerably muted compared to the assay with human blood. The synergistic effect of DTRI-031+rtPA was seen at doses of 250 nM DTRI + 7 nM rtPA (n=4) with a CLR max of 2.08 ± 1.45 min -1 and 500 nM + 7 nM rtPA (n=6) with a CLR max of 4.3 ± 2.5 min -1 compared to monotherapy (p<0.0001). Canine The CLR max of the negative control (n=33) was 2.611 ± 2.659 min -1 . The addition of 250 nM DTRI-031 or 500 nM DTRI-031 and 0.7 rtPA (n=18) resulted in a synergistic effect of both compounds which were significantly greater than all monotherapies with a CLR max of 13.52 ± 8.985 min -1 and 9.152± 6.943 min -1 , respectively. Conclusion: VWF inhibition by DTRI-031 aptamer + rTPA significantly improves clot lysis rate compared to monotherapy with rTPA, DTRI-031, and aspirin in mouse, canine, and human blood.

Introduction: Acute ischemic stroke (AIS) is the leading cause of combined morbidity and mortality worldwide. Recombinant tissue plasminogen activator (rtPA) is the only FDA-approved drug to treat AIS, however, it is limited to treating patients within 4.5 hours of stroke onset because of the risk of intracranial hemorrhage. This limits rtPA administration to ~6% of patients. Moreover, it poorly lyses large vessel occlusion (LVO) stroke. Endovascular mechanical thrombectomy (MT) effectively recanalizes LVO but is limited to highly specialized hospitals, leaving the majority without acute therapy. Hypothesis: We hypothesize that targeted von Willebrand Factor (VWF) inhibition by DTRI-031 will recanalize arterial thrombosis in a canine model of LVO stroke, and DTRI-031 activity will be rapidly reversed by DTRI-025, which is specifically designed to inhibit DTRI-031. Methods: Utilizing a canine embolic middle cerebral artery occlusion (eMCAO) model of LVO stroke, we assessed DTRI-031 on vessel recanalization after 6 hours of LVO stroke by digital subtraction angiography (DSA). We also measured the effect of recanalization on infarct volume (efficacy) and hemorrhage (safety) using magnetic resonance imaging (MRI). Finally, we assessed the ability of DTRI-025, an oligonucleotide that binds DTRI-031 to reverse DTRI-031 activity. Results: DTRI-031 administration after 6 hours of LVO stroke resulted in ≥TICI 2A in 62.5% and ≥TICI 2B in 50% of canines (n=8). The negative control group demonstrated no revascularization (n=7). Recanalization resulted in reduced infarct volume compared to the negative control (p<0.05, unpaired t-test). None of the animals that received DTRI-031 had an intracranial hemorrhage on MRI. Finally, DTRI-025 completely reversed the activity of DTRI-031 within 5 minutes of administration in both whole blood impedance aggregometry (WBA) and PFA-100. There was no difference in heart rate, blood pressure, or temperature among the 3 groups studied. Conclusion: DTRI-031 effectively recanalizes LVO after 6 hours of stroke onset with reduced infarct volume and no incidence of hemorrhage. DTRI-025 rapidly reverses DTRI-031. This drug/reversal agent combination represents a robust yet safe approach to treat AIS.

BACKGROUND Basilar artery occlusion (BAO) is a subset of posterior circulation stroke that carries a mortality as high as 90%.  The current clinical standard to diagnose ischemic stroke include computerized tomography (CT), CT angiography and perfusion and magnetic resonance imaging (MRI). Large animal pre-clinical models to accurately reflect the clinical disease as well as methods to assess stroke burden and evaluate treatments are lacking. METHODS We describe a canine model of large vessel occlusion (LVO) stroke in the posterior circulation, and developed a laser speckle imaging (LSI) protocol to monitor perfusion changes in real time.  We then utilized high b-value DWI (b=1800s/mm2) MRI to increase detection sensitivity. We also evaluated the ability of magnetic resonance angiography (MRA) to assess arterial occlusion and correlate with DSA. Finally, we verified infarct size from apparent diffusion coefficient (ADC) mapping with histology.  Results:  Administration of thromboembolism occluded the basilar artery as tracked by DSA (n=7).   LSI correlated with DSA, demonstrating a reduction in perfusion after stroke onset that persisted throughout the experiment, allowing us to monitor perfusion in real time.  DWI with an optimized b-value for dogs illustrated the stroke volume and allowed us to derive ADC and magnetic resonance angiography (MRA) images. The MRA performed at the end of the experiment correlated with DSA performed after occlusion. Finally, stroke burden on MRI correlated with histology. CONCLUSIONS Our studies demonstrate real time perfusion imaging using LSI of a canine thromboembolic LVO model of posterior circulation stroke, which utilizes multimodal imaging important in the diagnosis and treatment of ischemic stroke.

Introduction: Von Willebrand Factor (VWF) is a glycoprotein critical to initiate, propagate and stabilize an occlusive thrombus seen in a significant segment of large vessel occlusion stroke as well as during neuroendovascular procedures, which approach 15% despite using heparin. Recombinant tissue plasminogen activator (rtPA) is the only drug approved to treat ischemic stroke despite poor recanalization rates (~10%), significant risk of intracranial hemorrhage and lack of reversibility. Hypothesis: An antidote-controlled RNA aptamer targeting VWF will significantly reduce stroke volume in a canine model of large vessel occlusion (LVO) stroke. Methods: We developed an RNA aptamer DTRI-031 and a matched reversal agent. A canine model of basilar artery occlusion (BAO) stroke was used to assess DTRI-031 efficiency in thromboembolic stroke compared to rtPA and vehicle control in vivo. Laser speckle imaging (LSI) and digital subtraction angiography (DSI) was used to study the microvasculature after BAO and subsequent treatment with DTRI-031, rtPA or negative control. Magnetic resonance imaging (MRI) was used to assess stroke volume and platelet aggregometry was used to assess platelet function during the experiment. Results: Botrocetin-induced aggregometry demonstrated >95% inhibition compared to rtPA and negative control (p<0.0001) (n=6 per group). Laser speckle imaging demonstrated improved revascularization and MRI demonstrated the lowest stroke volume in DTRI-031-treated dogs compared to rtPA and negative control (p<0.05). Conclusions: DTRI-031 demonstrated superior recanalization, reduced stroke burden and robust platelet inhibition in the setting of LVO stroke compared to rtPA. These findings suggest that this drug-antidote combination targeting VWF may represent a superior treatment paradigm in stroke ischemic stroke and as well as in acute thrombosis in neurointerventional procedures.

Developing endothelial-protective, nonthrombogenic antirestenotic treatments has been a challenge. A major hurdle to this has been the identification of a common molecular target in both smooth muscle cells and endothelial cells, inhibition of which blocks dysfunction of both cell types. The authors' findings suggest that the PERK kinase could be such a target. Importantly, PERK inhibition mitigated both restenosis and thrombosis in preclinical models, implicating a low-thrombogenic antirestenotic paradigm.

Endothelial surface and circulating glycoprotein von Willebrand factor (vWF) regulates platelet adhesion and is associated with thrombotic diseases, including ischemic stroke, myocardial infarction, and peripheral vascular disease. Thrombosis, as manifested in these diseases, is the leading cause of disability and death in the western world. Current parenteral antithrombotic and thrombolytic agents used to treat these conditions are limited by a short therapeutic window, irreversibility, and major risk of hemorrhage. To overcome these limitations, we developed a novel anti-vWF aptamer, called DTRI-031, that selectively binds and inhibits vWF-mediated platelet adhesion and arterial thrombosis while enabling rapid reversal of this antiplatelet activity by an antidote oligonucleotide (AO). Aptamer DTRI-031 exerts dose-dependent inhibition of platelet aggregation and thrombosis in whole blood and mice, respectively. Moreover, DTRI-031 can achieve potent vascular recanalization of platelet-rich thrombotic occlusions in murine and canine carotid arteries. Finally, DTRI-031 activity is rapidly (<5 min) and completely reversed by AO administration in a murine saphenous vein hemorrhage model, and murine toxicology studies indicate the aptamer is well tolerated. These findings suggest that targeting vWF with an antidote-controllable aptamer potentially represents an effective and safer treatment for thrombosis patients having platelet-rich arterial occlusions in the brain, heart, or periphery.

Introduction: While recombinant tissue plasminogen activator (rTPA) is the mainstay of ischemic stroke treatment, recanalization is only achieved in 25-50% of patients. With a significant risk of intracranial hemorrhage, its use has been limited to within 4.5 hours of symptom onset. Previous work has demonstrated that aptamer inhibition of Von Willebrand Factor (VWF) effectively restores reperfusion following murine carotid artery occlusion. Hypothesis: We tested the hypothesis that VWF aptamer would promote recanalization following thrombotic middle cerebral artery (MCA) occlusion, ameliorating stroke burden with greater efficacy than rTPA. Methods: Adult wild-type (C57BL/6J) mice were anesthetized, and the right carotid artery was exposed. A 32-gauge intracranial catheter was advanced within the carotid artery. Murine autologous blood was then mixed with 10 μL 0.9% normal saline and 1 μL murine thrombin and was allowed to stabilize at 37 °C for 15 minutes, after which it was injected through the catheter into the MCA. Laser-doppler flowmetry monitoring measured decreased flow following injection of the embolus. Treatment (vehicle, platelet binding buffer, n=5; VWF aptamer, n=6; rTPA, n=7) was initiated 20 minutes after thrombus injection. An MRI was obtained at 24 hours to assess ischemic stroke volumes. Results: None of the mice receiving rTPA survived to 24 hours, while all mice treated with VWF aptamer and vehicle survived to 24 hours and received an MRI. Ischemic stroke volume was significantly decreased in mice treated with VWF aptamer (5.49 ± 5.01 mm 3 ) compared to vehicle (35.34 ± 9.57 mm 3 , p<0.05)(Figure 1). No evidence of intracranial hemorrhage was identified in either cohort. Conclusions: Treatment with VWF aptamer decreases stroke volume on MRI in a murine model of embolic stroke without the risk of hemorrhagic conversion seen in patients treated rTPA. VWF inhibition represents a promising therapy in stroke treatment.

Objective: To compare the prophylactic and thrombolytic effects of RNA aptamer (9.14T79VRT7) on ex vivo canine platelet function. Approach and Results: We previously demonstrated that inhibition of von Willebrand Factor (VWF) by a targeted RNA aptamer prevents thrombosis and thrombolyses stabilized clots in a FeCl 3 -induced murine vascular injury model suggesting a pivotal role for VWF in the pro-thrombotic and anti-thrombotic milieu. We hypothesize that 9.14T79VRT7, which demonstrated no hemorrhagic complications and greater re-perfusion compared to rTPA in murine and canine thrombotic models, may affect additional agonist pathways to mitigate platelet activation, aggregation, and adhesion. Platelet aggregation before and after 9.14T79VRT7 addition was analyzed utilizing WB aggregometry based on impedance in five adult beagles. Agonist concentrations were selected from previous publications which had optimized platelet reactivity in canine WB and included: collagen (3.2 ug/ul), ADP (20 uM), arachidonic acid (0.5 mM), and botrocetin (1 ug/ul). The 9.14T79VRT7 concentration used (25 nM) was selected as the most effective as assessed by Total Thrombus Analysis System (TTAS). Botrocetin elicited the greatest response before 9.14T79VRT7 addition, followed by Collagen, ADP, and Arachidonic acid. Impedance resulting from prophylactic addition of 9.14T79VRT7 before Collagen, ADP, and Botrocetin resulted in a 50.00%, 34.83%, and 38.26% change in amplitude, respectively and a 43.70%, 14.28%, and 35.14% change in slope, respectively (p<.0001). There was no significant difference in response after Arachidonic acid. Impedance resulting from thrombolytic addition of 9.14T79VRT7 after agonist response to Collagen, ADP, and Botrocetin resulted in a 78.66%, 94.08%, and 84.58% change in amplitude, respectively and a 67.28%, 61.28%, and 63.73% change in slope, respectively (p<.0001). Although amplitude and slope decreased with 9.14T79VRT7 addition after Arachidonic acid, the change was not significant. Both control groups (9.14T79VRT7 and platelet buffer diluent) resulted in no significant changes. Conclusion: 9.14T79VRT7 markedly changes platelet response both before and after agonist treatment in canine ex vivo impedance analysis.

Objective: To compare the functional effects of von Willebrand Factor (VWF) inhibition by RNA aptamer (T79) vs rTPA utilizing in vivo arterial thrombosis in mice. Approach and Results: We previously demonstrated that inhibition of VWF by a targeted RNA aptamer (T79) both prevents thrombosis and thrombolyses stabilized clots in a murine model of ferric chloride (FeCl 3 )-induced vascular injury suggesting a pivotal role for VWF in the pro-thrombotic and anti-thrombotic milieu. We hypothesized that T79 treatment, which demonstrated no hemorrhagic complications and greater re-perfusion compared to rTPA, would result in improved behavioral outcome with 7 day survival after vascular injury. Baseline locomotor testing in an open field was performed on both male and female, 8-16 week old, wild-type C57BL/6J mice. Occlusive arterial thrombus formation was induced by a 3 minute exposure to 10% FeCl 3 on the right common carotid monitored by Doppler flow and time to occlusion (blood flow of 0 ml/min) was measured. Twenty minutes after thrombus stabilization either T79 (0.1 mg/kg or 0.5 mg/kg bolus), rTPA (10 mg/kg 45 min infusion) or saline vehicle (45 min infusion) was introduced via a saphenous catheter. Animals were recovered and locomotor testing was repeated 48 hours and 7 days after injury with baseline set as 100%. All groups including vehicle control resulted in a statistically significant decrease in distance traveled (meters) and speed (meters/sec) at 48 hours after injury. At 7 days, 0.1 mg/kg T79 increased 29.89% from 48 hours, 0.5 mg/kg T79 increased 22.50% from 48 hours but rTPA treatment showed no significant improvement (2.79% increase) (n=4, p<0.001). In addition, 0.1 mg/kg and 0.5 mg/kg T79 resulted in a 28.46% and 22.44% increase in speed, respectively, at 7 days compared to 48 hours whereas rTPA did not (2.94% increase) (n=4, p<0.001). Interestingly, males scored lower on most behavioral parameters at 48 hours, but recovered to the same level as females by 7 days. No significant differences in time to occlusion or baseline locomotor testing were observed in any group. Conclusion: Inhibition of VWF by T79 aptamer markedly improves behavioral outcomes compared to rTPA after survival carotid artery occlusion following ferric chloride-induced injury in mice.

Occlusive arterial thrombosis leading to cerebral ischemic stroke and myocardial infarction contributes to ~13 million deaths every year globally. Here, we have translated a vascular injury model from a small animal into a large animal (canine), with slight modifications that can be used for pre-clinical screening of prophylactic and thrombolytic agents. In addition to the surgical methods, the modified protocol describes the step-by-step methods to assess carotid artery canalization by angiography, detailed instructions to process both the brain and carotid artery for histological analysis to verify carotid canalization and cerebral hemorrhage, and specific parameters to complete an assessment of downstream thromboembolic events by utilizing magnetic resonance imaging (MRI). In addition, specific procedural changes from the previously well-established small animal model necessary to translate into a large animal (canine) vascular injury are discussed.