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Background Cardiac implantable electronic device (CIED) generally excludes patients from undergoing fixed, high-field magnetic resonance imaging (MRI). Acute ischemic stroke patients undergo MRI as a standard part of an assessment of infarct burden. The use of a portable MRI scanner may be useful in patients who have contraindications to high-field MRI. We present the first case of a patient with a CIED who required an endovascular thrombectomy (EVT) for large vessel occlusion. She underwent a low-field MRI in the operating room with the Hyperfine portable system.Case The patient is an 80-year-old female status post-CIED, on Eliquis who presented with an acute ischemic stroke. Her National Institutes of Health Stroke Scale (NIHSS) of 8. Imaging demonstrated a left M2 occlusion on computed tomography angiogram (CTA) of the head and neck. No lytics were used due to concomitant gastrointestinal bleed. While, admitted, her NIHSS increased to 15. A subsequent CTA demonstrated a left internal carotid artery terminus and M1 occlusion. She underwent EVT with thrombolysis in cerebral infarction (TICI) 3 revascularization. An MRI was performed intraoperatively using a Hyperfine system, which is a low-field, portable MRI, to assess infarct volume.Conclusion Hyperfine Swoop brain MRI may be safe for use in patients with contraindications to high-field MRI scans. Continued technological refinement will improve the quality of diffusion-weighted imaging. Larger studies will be required to generalize Hyperfine MRI-based imaging for patients with devices that exclude them from high-field imaging.

Introduction Venous sinus stenting is a well established alternative to cerebrospinal fluid diversion for the treatment of idiopathic intracranial hypertension (IIH) with associated venous sinus stenosis. During this procedure, distal guide catheter placement within the venous sinuses may be desirable to facilitate stent delivery. We report our initial experience using the TracStar LDP™ (Imperative Care, Campbell, USA, 0.088-inch inner diameter) as the guide catheter for intracranial access during venous sinus stenting.Methods A multi-institutional retrospective chart review of a prospectively maintained IRB-approved database was performed. Consecutive patients who underwent venous sinus stenting from 1/1/2020-9/6/2021 for IIH were included. Patient characteristics, procedural details, TracStar distal reach, outcomes, and complications were collected and analyzed.Results Fifty-eight patients were included. The mean age was 33.8 years and 93.1% of patients were female. Visual changes prompted evaluation in 86.2% of patients. Stent placement was successful in all patients. The TracStar LDP catheter was advanced to the location of stent placement in 97.9% of cases in which it was attempted. The large 0.088-inch inner diameter lumen enabled compatibility with all desired stent sizes ranging from six to 10 millimeters. Gradient pressure across transverse sinus stenosis dropped from an average of 19.5 mmHg pre-procedure to 1.7 mmHg post-stent placement (p < 0.001). Clinical improvement was achieved in 87.9% (51/58) of patients. There were no catheter-related complications.Conclusion The TracStar LDP is a safe and effective access platform for reaching treatment locations in patients who present with idiopathic intracranial hypertension and who are candidates for venous sinus stent placement.

BackgroundMoyamoya is a chronic occlusive cerebrovascular disease of unknown etiology causing neovascularization of the lenticulostriate collaterals at the base of the brain. Although revascularization surgery is the most effective treatment for moyamoya, there is still no consensus on the best surgical treatment modality as different studies provide different outcomes.ObjectiveIn this large case series, we compare the outcomes of direct (DR) and indirect revascularisation (IR) and compare our results to the literature in order to reflect on the best revascularization modality for moyamoya.MethodsWe conducted a multicenter retrospective study in accordance with the Strengthening the Reporting of Observational studies in Epidemiology guidelines of moyamoya affected hemispheres treated with DR and IR surgeries across 13 academic institutions predominantly in North America. All patients who underwent surgical revascularization of their moyamoya-affected hemispheres were included in the study. The primary outcome of the study was the rate of symptomatic strokes.ResultsThe rates of symptomatic strokes across 515 disease-affected hemispheres were comparable between the two cohorts (11.6% in the DR cohort vs 9.6% in the IR cohort, OR 1.238 (95% CI 0.651 to 2.354), p=0.514). The rate of total perioperative strokes was slightly higher in the DR cohort (6.1% for DR vs 2.0% for IR, OR 3.129 (95% CI 0.991 to 9.875), p=0.052). The rate of total follow-up strokes was slightly higher in the IR cohort (8.1% vs 6.6%, OR 0.799 (95% CI 0.374 to 1.709) p=0.563).ConclusionSince both modalities showed comparable rates of overall total strokes, both modalities of revascularization can be performed depending on the patient’s risk assessment.

BackgroundPlatelets and von Willebrand factor (vWF) are key components of acute ischemic stroke (AIS) emboli. We aimed to investigate the CD42b (platelets)/vWF expression, its association with stroke etiology and the impact these components may have on the clinical/procedural parameters.MethodsCD42b/vWF immunostaining was performed on 288 emboli collected as part of the multicenter STRIP Registry. CD42b/VWF expression and distribution were evaluated. Student’s t-test and χ2test were performed as appropriate.ResultsThe mean CD42b and VWF content in clots was 44.3% and 21.9%, respectively. There was a positive correlation between platelets and vWF (r=0.64, p<0.001**). We found a significantly higher vWF level in the other determined etiology (p=0.016*) and cryptogenic (p=0.049*) groups compared with cardioembolic etiology. No significant difference in CD42b content was found across the etiology subtypes. CD42b/vWF patterns were significantly associated with stroke etiology (p=0.006*). The peripheral pattern was predominant in atherosclerotic clots (36.4%) while the clustering (patchy) pattern was significantly associated with cardioembolic and cryptogenic origin (66.7% and 49.8%, respectively). The clots corresponding to other determined etiology showed mainly a diffuse pattern (28.1%). Two types of platelets were distinguished within the CD42b-positive clusters in all emboli: vWF-positive platelets were observed at the center, surrounded by vWF-negative platelets. Thrombolysis correlated with a high platelet content (p=0.03*). vWF-poor and peripheral CD42b/vWF pattern correlated with first pass effect (p=0.03* and p=0.04*, respectively).ConclusionsThe vWF level and CD42b/vWF distribution pattern in emboli were correlated with AIS etiology and revascularization outcome. Platelet content was associated with response to thrombolysis.

BackgroundWide-necked bifurcation aneurysms (WNBAs) present unique challenges for endovascular treatment. The Woven EndoBridge (WEB) device is an intrasaccular braided device, recently approved by the FDA for treatment of WNBAs. While treatment of intracranial aneurysms with the WEB device has been shown to yield an adequate occlusion rate of 85% at 1 year, few data have been published for patients with ruptured aneurysms.ObjectiveTo present a multi-institutional series depicting the safety and efficacy of using the WEB device as the primary treatment modality in ruptured intracranial aneurysms.MethodsA multi-institutional retrospective analysis was conducted, assessing patients presenting with aneurysmal subarachnoid hemorrhage treated with the WEB between January 2014 and April 2020. Baseline demographics, aneurysm characteristics, adverse events, and long-term outcomes (occlusion, re-treatment, functional status) were collected. A descriptive analysis was performed, and variables potentially associated with aneurysm recurrence or re-treatment were assessed.ResultsForty-eight patients were included. Anterior communicating artery aneurysms were the most common (35.4%) location for treatment, followed by middle cerebral artery (20.8%) and basilar apex (16.7%). Procedural success was noted in 95.8% of patients, and clinically significant periprocedural adverse events occurred in 12.5%. After a median follow-up of 5.5 months, 54.2% of patients had follow-up angiographic imaging. Complete occlusion was seen in 61.5% of cases with adequate occlusion in 92.3%. Re-treatment was required in only 4.2% of patients during the study period. Tobacco use was significantly higher in patients with aneurysm recurrence (88.9% vs 35.7%; p=0.012). No other characteristics were associated with recurrence/re-treatment. At 30 days, 81.1% were functionally independent (modified Rankin Scale score ≤2).ConclusionTreatment of acutely ruptured aneurysms with the WEB device demonstrates both safety and efficacy on par with rates of conventional treatment strategies.

Coronavirus disease 2019 (COVID-19) results from infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It was first reported in Wuhan, China in patients suffering from severe pneumonia and acute respiratory distress syndrome and has now grown into the first pandemic in over 100 years. Patients infected with SARS-CoV-2 develop arterial thrombosis including stroke, myocardial infarction and peripheral arterial thrombosis, all of which result in poor outcomes despite maximal medical, endovascular, and microsurgical treatment compared with non-COVID-19-infected patients. In this review we provide a brief overview of SARS-CoV-2, the infectious agent responsible for the COVID-19 pandemic, and describe the mechanisms responsible for COVID-19-associated coagulopathy. Finally, we discuss the impact of COVID-19 on ischemic stroke, focusing on large vessel occlusion.

Background: The discovery that short oligonucleotides, termed aptamers, can fold into three-dimensional structures that allow them to selectively bind and inhibit the activity of pathogenic proteins is now over 25 years old. The invention of the SELEX methodology heralded in an era in which such nucleic acid-based ligands could be generated against a wide variety of therapeutic targets. Results: A large number of aptamers have now been identified by combinatorial chemistry methods in the laboratory and moreover, an increasing number have been discovered in nature. The affinities and activities of such aptamers have often been compared to that of antibodies, yet only a few of these agents have made it into clinical studies compared to a large and increasing number of therapeutic antibodies. One therapeutic aptamer targeting VEGF has made it to market, while 3 others have advanced as far as phase III clinical trials. Conclusion: In this manuscript, we hope the reader appreciates that the success of aptamers becoming a class of drugs is less about nucleic acid biochemistry and more about target validation and overall drug chemistry.

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.

Background The modified Rankin Scale (mRS) is a clinician-reported scale that measures the degree of disability in patients who suffered a stroke. Patients’ perception of a meaningful recovery from severe stroke, expected value of a stroke intervention, and the effect of disparities are largely unknown.Methods We conducted a survey of patients, their family members, and accompanying visitors to understand their personal preferences and expectations for acute strokes potentially eligible for acute endovascular intervention using a hypothetical scenario of a severe stroke in a standardized questionnaire.Results Of 164 survey respondents, 65 (39.6%) were the patient involved, 93 (56.7%) were a family member, and six (3.7%) were accompanied visitors (friends, other). Minimally acceptable disability after a stroke intervention was considered as mRS 2 by 42 respondents (25.6%), as mRS 3 by 79 (48.2%), and as mRS 4 by 43 (26.2%) respondents. Race was associated with different views on this question (p< 0.001; Hispanic and Black patients being more likely to accept disability than Caucasian and Asian patients), while sex (p= 0.333) and age (p= 0.560) were not. Sixty-three respondents (38.4%) viewed minimally acceptable probability of improvement with an intervention as over 50%, 57 (34.8%) as 10–50%, and 44 (26.8%) as less than 10%.Conclusions A wide range of acceptable outcomes were reported regardless of gender or age. However, race was associated with different acceptable outcome. This is an important finding to demonstrate because of the persistent racial and ethnic disparities in the utilization of endovascular therapy for acute stroke in the United States.

Background A number of large bore guide catheters are currently available for use in neuroendovascular surgery. This study represents a multi-institutional retrospective series of patients undergoing mechanical thrombectomy with the use of a TracStar Large Distal Platform (LDP) guide catheter and assessed its performance in vivo in 107 patients.Objective To review a multi-institutional initial experience with the TracStar LDP guide catheter during mechanical thrombectomy for emergent large vessel occlusion (ELVO).Methods A retrospective review was performed at two level one stroke centres to include all patients who underwent mechanical thrombectomy and had the TracStar LDP guide catheter used during the intervention.Results The TracStar LDP guide catheter was successfully used in 107 mechanical thrombectomies. In anterior circulation ELVO, the guide catheter advanced into the cavernous segment of the internal carotid artery in 62.6% (62/99) of cases. In posterior circulation cases, the guide catheter advanced to the basilar artery in 87.5% (7/8) of cases. A thrombolysis in cerebral infarction 2b or greater reperfusion was obtained in 90.7% (97/107). No complications occurred related to the TracStar LDP guide catheter. Three complications occurred with aspiration catheters including a small dissection that did not require further intervention and fracturing of the AXS Catalyst 6 catheter tip in two cases. No thromboembolic events occurred.Conclusions The TracStar LDP large bore guide catheter is safe and effective at navigating the tortuous vascular anatomy often encountered during mechanical thrombectomy for stroke. The flexible distal and stiffer proximal components provide a good combination of navigability and support for use in neuroendovascular interventions.

Although antiplatelet medications and anticoagulants are necessary for numerous cardiac comorbidities, prevention of stroke, and treatment and prevention of venous thromboembolic events, they pose a significant treatment dilemma in neurosurgical patients, particularly in the setting of intracranial hemorrhage or prior to emergent neurosurgical procedures. For the majority of current anticoagulation and antiplatelet therapies, no direct reversal agents exist; however, there are numerous strategies that can be used to reverse or mitigate their antithrombotic properties. This review provides a comprehensive summary of the latest antiplatelet and anticoagulant therapies and the role of emergency reversal prior to emergent neurosurgical procedures.

Antiplatelet agents used to treat neurovascular disease include aspirin; P2Y12 receptor antagonists clopidogrel, prasugrel, and ticagrelor; ADP antagonist ticlopidine; phosphodiesterase inhibitor dipyridamole; and glycoprotein IIb/IIIa inhibitors abciximab, eptifibatide, and tirofiban. Numerous studies have been performed evaluating their efficacy in stroke, extracranial carotid artery disease and dissection, intracranial atherosclerotic disease, and moyamoya disease. The rapid technological advancements in endovascular neurosurgical devices have also made antiplatelet therapy a necessary part of treating intracranial aneurysms. This article presents the relevant data supporting the use of antiplatelet agents in vascular neurosurgery and recommendations based on the described studies.

Thromboembolic complications remain a major risk of endovascular neurosurgery during the treatment of intracranial aneurysms, despite the use of therapeutic heparinization and oral antiplatelet therapy when indicated. Glycoprotein (GP) IIb/IIIa inhibitors target a nonredundant pathway of platelet aggregation following adhesion and activation. Initially established and implemented in the cardiovascular arena, this drug class has provided a new tool in the neurovascular armamentarium as well. Numerous case reports, case series, and retrospective reviews have evaluated the safety and efficacy of abciximab, eptifibatide, and tirofiban in the treatment of acute thromboembolic complications during the endovascular treatment of intracranial aneurysms. The use of this drug class has also been found to be beneficial as a prophylactic agent, providing ischemia protection during the placement of intracranial stents, flow diverters, and thrombogenic coils in the setting of subarachnoid hemorrhage and during elective aneurysmal embolization. While the current published literature clearly establishes efficacy and safety of GP IIb/IIIa inhibitors in the prevention of thromboembolic complications, there does not yet exist an established protocol for their administration in endovascular neurosurgery. This review provides a comprehensive evaluation of the current published literature pertaining to the use of all available GP IIb/IIIa inhibitors for thromboembolic complications, providing recommendations for dosing and administration of abciximab, eptifibatide, and tirofiban based on previously published rates of efficacy and intracranial hemorrhage.

BackgroundWe sought to determine the rate of early neurologic decline (END) in patients with acute ischemic stroke (AIS) with large vessel occlusion (LVO) who presented with mild deficits and received intravenous tissue plasminogen activator (IVtPA).MethodsAmong 1022 patients with AIS who received IVtPA from 2014 to 2019, we identified 313 (30.6%) with LVO, of which 94 (30%) presented with National Institute of Health Stroke Scale (NIHSS) score ≤7. Thirteen patients were excluded, leaving 81 for analysis. END was defined as NIHSS worsening of ≥4 points within 24 hours.ResultsAmong 81 patients with LVO and low NIHSS score, the mean age was 65.8 years (range 25–93) and 41% were female. The mean time to IVtPA from last known well was 2.5 hours (range 0.8–7). LVO sites were as follows: 5 (6%) carotid, 23 (28%) M1, and 53 (65%) M2 occlusions. Among the 81 patients, 28 (34.6%) had END, and these patients were older (70.8 vs 63.2 years, p=0.036). The mean change in NIHSS score at 24 hours in those with END was 10.4 (range 4–22). Patients with END were less likely to be discharged home (25% vs 66%, p=0.004).ConclusionsAmong patients with LVO AIS who received IVtPA, 30% presented with initial mild deficits. END occurred in one-third of LVO patients with initial mild deficits despite receiving IVtPA. Clinicians should be aware that the natural history of LVO with initial mild deficits is not benign and these patients are eligible for rescue thrombectomy in the 24-hour window if they deteriorate.

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 (t1/2) 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.

To our knowledge, an ex vivomodel of thrombotic occlusion that (1) mimics arterial setting, (2) allows true occlusion, and (3) drug delivery without disturbing the thrombus does not exist. A platform with these capabilities would allow for high-throughput mechanistic study of thrombosis and thrombolysis. Here we present a microfluidic system that achieves these goals, providing a biofidelic ex vivoplatform to study ischemic stroke.A mold with an upstream Y and a region of stenosis was fabricated and used to manufacture microfluidic chambers. Channels were coated with collagen at the stenosis. Heparinized blood was collected from healthy human donors (N=3) and platelets were labelled with fluorescent anti-CD41. Samples were perfused at 10000/s and outflow mass collected on a scale. Occlusion was indicated by mass plateau, and thrombi were left to retract in situfor 0 or 6 hrs. Kinetic immunofluorescent images were captured and used to derive surface area and the image profile by averaging MFI in each pixel column. Sample from the same donor dosed with 0.7 or 7nM alteplase (ALT) was perfused using the Y inlet using a constant pressure head (1000/s; mimicking middle cerebral artery) for 120 min.After 6 hours of post-occlusion retraction, thrombi substantially remodeled. The MFI curves (Fig 1) demonstrate even distribution of MFI post-occlusion that shifts to a central peak after 6 hr. Dosed sample from the same donor was able to be delivered upstream of the thrombus without disruption. Immediate reperfusion resulted in a reduction in thrombus surface area of (mean (SD)) 2% (10%) with 0.7nM ALT, 23% (7%) with 7nM, and 15% (1%) with vehicle. Reperfusion after 6 hrs resulted in a reduction of 14% (14%) with 0.7nM, 45% (12%) with 7nM, and 17% (8%) with vehicle.We have established an ex vivohigh throughput system capable of recapitulating arterial occlusion, thrombus retraction in situ, drug delivery, and thrombolysis. This system can be used for mechanistic inquiry and drug discovery.

Von Willebrand factor (VWF) is critical in arterial thrombosis. Previous studies have shown that aptamer BB-031, a VWF inhibitor, induces thrombolysis in vivoin ischemic stroke models, making it a promising stroke therapeutic. We hypothesized that BB-031 will induce thrombolysis in an ex vivoischemic stroke model and that advanced imaging techniques will provide mechanistic information. A microfluidic model was utilized to simulate ischemic stroke and drug delivery without disturbing the occluding thrombus. Heparinized whole blood was obtained from healthy volunteers (N=10). A gravity-based constant pressure microfluidic system mimicking arterial perfusion induced occlusive thrombosis at a collagen-coated region of stenosis. After resting for 0, 3, or 6 hours to allow retraction in situthrombi were reperfused with blood containing 1692nM (~1mg/kg) BB-031, 0.7nM alteplase (ALT), 0.7nM tenecteplase (TEN), or vehicle 500um upstream of the thrombus using a separate inlet. Platelets were labelled prior to thrombus formation and reperfusion using fluorescent anti-CD41. Thrombolysis and patency were then monitored for 2hrs via outlet mass recording and z-stacked immunofluorescent imaging. Upon immediate reperfusion, 1692nM (~1mg/kg) BB-031 increased mean mass output by 0.250±0.629g compared to 0.031±0.065g with vehicle, 0.074±0.061g with ALT, and 0.101±0.150g with 0.7nM TEN. After a 3-hour retraction period, BB-031 reperfusion mean mass output was 0.951±1.279g compared to 0.301±0.380g with vehicle, 0.412±0.681g with ALT, and 0.569±0.714g with TEN. After 6-hours resting, reperfusion mass output increased by 1.354±1.433g with BB-031, 0.368±0.538g with vehicle, 0.158±0.195g with ALT, and 0.520±0.539g with TEN. Representative z-stacked renderings of the thrombus pre and post reperfusion from a 0-hour resting period show a volumetric decrease in thrombus size of 39% with BB-031 compared to 25% and 18% reduction with vehicle and ALT respectively in a donor-matched dataset. BB-031 improves patency in a microfluidic model of biofidelic thrombolysis compared to current standards of care, which is enhanced at 3 and 6 hours post-occlusion. This may be due to VWF-dependent lysis resulting in reduced thrombus volume.

Systematic review.

Retrospective review of prospective collected data on 22 patients.

Patients with sickle cell disease (SCD) often suffer painful vaso-occlusive episodes caused in part by the adhesion of sickle erythrocytes (SS-RBC) to the vascular endothelium. To investigate inhibition of SS-RBC adhesion as a possible treatment for vaso-occlusion, 2 adhesion molecules, avß3and P-selectin, were targeted by high-affinity RNA aptamers. An in vitroflow chamber assay was used to test the antiadhesion activity of avß3aptamer clone 17.16. Human SS-RBC were passed across a confluent monolayer of thrombin-stimulated human umbilical vein endothelial cells (HUVEC) at a constant rate. avß3aptamer reduced SS-RBC adhesion to activated endothelial cells to the level seen with untreated HUVEC. An aptamer reactive with complement component 8 was used as a negative control and exerted no inhibition, confirming the specificity of avß3aptamer (P=0.04). At 2?dyn/cm2shear stress, 30?nM avß3aptamer showed maximal effect in decreasing SS-RBC adhesion to HUVEC. The antiadhesive activity of the P-selectin aptamer clone PF377 was also tested using HUVEC pretreated with IL-13 to upregulate expression of P-selectin as seen in activated endothelial cells. At 1?dyn/cm2shear stress, 60?nM of P-selectin aptamer had antiadhesion activity similar to heparin, a known inhibitor of SS-RBC adhesion to P-selectin. A negative control did not prevent adhesion (P=0.05). These data show the potential utility of aptamers to block endothelial adhesion molecules to prevent or treat vaso-occlusion in SCD.

Thrombin is a multifunctional protease that plays a key role in hemostasis, thrombosis, and inflammation. Most thrombin inhibitors currently used as antithrombotic agents target thrombin's active site and inhibit all of its myriad of activities. Exosites 1 and 2 are distinct regions on the surface of thrombin that provide specificity to its proteolytic activity by mediating binding to substrates, receptors, and cofactors. Exosite 1 mediates binding and cleavage of fibrinogen, proteolytically activated receptors, and some coagulation factors, while exosite 2 mediates binding to heparin and to platelet receptor GPIb-IX-V. The crystal structures of two nucleic acid ligands bound to thrombin have been solved. Previously Padmanabhan and colleagues solved the structure of a DNA aptamer bound to exosite 1 and we reported the structure of an RNA aptamer bound to exosite 2 on thrombin. Based upon these structural studies we speculated that the two aptamers would not compete for binding to thrombin. We observe that simultaneously blocking both exosites with the aptamers leads to synergistic inhibition of thrombin-dependent platelet activation and procoagulant activity. This combination of exosite 1 and exosite 2 inhibitors may provide a particularly effective antithrombotic approach.

Abstract