In vitro test methods for evaluating high molecular weight polyethylene oxide polymer induced hemolytic and thrombotic potential.

To combat opioid abuse, the U.S. Food and Drug Administration (FDA) released a comprehensive action plan to address opioid addiction, abuse, and overdose that included increasing the prevalence of abuse-deterrent formulations (ADFs) in opioid tablets. Polyethylene oxide (PEO) has been widely used as an excipient to deter abuse via nasal insufflation. However, changes in abuse patterns have led to unexpected shifts in abuse from the nasal route to intravenous injection. Case reports identify adverse effects similar to thrombotic thrombocytopenic purpura (TTP) syndrome following the intravenous (IV) abuse of opioids containing PEO excipient. Increased risk of IV opioid ADF abuse compared to clinical benefit of the drug led to the removal of one opioid product from the market in 2017. Because many generic drugs containing PEO are still in development, there is interest in assessing safety consistent with generic drug regulation and unintended uses. Currently, there are no guidelines or in vitro assessment tools to characterize the safety of PEO excipients taken via intravenous injection. To create a more robust excipient safety evaluation tool and to study the mechanistic basis of HMW PEO-induced TMA, a dynamic in vitro test system involving blood flow through a needle model has been developed.

Structure of shear-induced platelet aggregated clot formed in an in vitro arterial thrombosis model.

The structure of occlusive arterial thrombi is described herein. Macroscopic thrombi were made from whole blood in a collagen-coated, large-scale stenosis model with high shear flow similar to an atherosclerotic artery. The millimeter-sized thrombi were harvested for histology and scanning electron microscopy. Histological images showed 3 distinctive structures of the thrombus. (1) The upstream region showed string-like platelet aggregates growing out from the wall that protrude into the central lumen, with red blood cells trapped between the strings. The strings were >10 times as long as they were wide and reached out to join the strings from the opposite wall. (2) Near the apex, the platelet strings coalesced into a dense mass with microchannels that effectively occluded the lumen. (3) In the expansion region, the thrombus ended abruptly with an annulus of free blood in the flow-separation zone. Scanning electron microscopy showed dense clusters of spherical platelets upstream and downstream, with amorphous platelets in the occluded throat consistent with prior activation. The total clot is estimated to contain 1.23 billion platelets with pores 10 to 100 µm in diameter. The results revealed a complex structure of arterial thrombi that grow from their tips under high shear stress to bridge the 2.5-mm lumen quickly with von Willebrand factor platelet strings. The occlusion leaves many microchannels that allow for some flow through the bulk of the thrombus. This architecture can create occlusion or hemostasis rapidly with minimal material, yet can remain porous for potential delivery of lytic agents to the core of the thrombus.

Development of a humidity pretreatment method for the measurement of ozone in ambient air.

A frost filter (FRF) was developed as a humidity pretreatment device (HPD) to improve the measurement of ambient ozone (O3). The FRF was produced in a tube, which was supercooled by a thermoelectric cooling device based on the Peltier effect. The relative humidity (RH) of the air samples varied from 30% to 80% at 25 °C, and the O3 concentration was set as 100 ppbv. Besides O3, SO2 at 150 ppbv was used for comparison. The density of the FRF was evaluated. Comparison studies on the humidity removal efficiencies and loss ratios of analytes among a FRF HPD, a short Nafion™ tube (NS), and a long Nafion™ tube (NL) HPDs were conducted. As results, the density of the FRF was dependent on the temperature at a fixed sampling flow rate. The outlet humidity of both the FRF and the NL HPDs were less than 8% RH at 25 °C. The mean concentrations of O3 and SO2 after the FRF HPD were similar to the initial concentrations at all humidity levels, whereas they were significantly different for both the NS and NL HPDs at higher humidity. This suggests that the FRF HPD is a reliable humidity pretreatment for O3 measurements.

Lysis of arterial thrombi by perfusion of N,N'-Diacetyl-L-cystine (DiNAC).

The search persists for a safe and effective agent to lyse arterial thrombi in the event of acute heart attacks or strokes due to thrombotic occlusion. The culpable thrombi are composed either primarily of platelets and von Willebrand Factor (VWF), or polymerized fibrin, depending on the mechanism of formation. Current thrombolytics were designed to target red fibrin-rich clots, but may be not be efficacious on white VWF-platelet-rich arterial thrombi. We have developed an in vitro system to study the efficacy of known and proposed thrombolytic agents on white clots formed from whole blood in a stenosis with arterial conditions. The agents and adjuncts tested were tPA, ADAMTS-13, abciximab, N-acetyl cysteine, and N,N'-Diacetyl-L-cystine (DiNAC). Most of the agents, including tPA, had little thrombolytic effect on the white clots. In contrast, perfusion of DiNAC lysed thrombi as quickly as 1.5 min, which ranged up to 30 min at lower concentrations, and resulted in an average reduction in surface area of 71 ± 20%. The clot burden was significantly reduced compared to both tPA and a saline control (p<0.0001). We also tested the efficacy of all agents on red fibrinous clots formed in stagnant conditions. DiNAC did not lyse red clots, whereas tPA significantly lysed red clot over 48 h (p<0.01). These results lead to a novel use for DiNAC as a possible thrombolytic agent against acute arterial occlusions that could mitigate the risk of hyper-fibrinolytic bleeding.

Clot Permeability, Agonist Transport, and Platelet Binding Kinetics in Arterial Thrombosis.

The formation of wall-adherent platelet aggregates is a critical process in arterial thrombosis. A growing aggregate experiences frictional drag forces exerted on it by fluid moving over or through the aggregate. The magnitude of these forces is strongly influenced by the permeability of the developing aggregate; the permeability depends on the aggregate's porosity. Aggregation is mediated by formation of ensembles of molecular bonds; each bond involves a plasma protein bridging the gap between specific receptors on the surfaces of two different platelets. The ability of the bonds existing at any time to sustain the drag forces on the aggregate determines whether it remains intact or sheds individual platelets or larger fragments (emboli). We investigate platelet aggregation in coronary-sized arteries using both computational simulations and in vitro experiments. The computational model tracks the formation and breaking of bonds between platelets and treats the thrombus as an evolving porous, viscoelastic material, which moves differently from the background fluid. This relative motion generates drag forces which the fluid and thrombus exert on one another. These forces are computed from a permeability-porosity relation parameterized from experimental measurements. Basing this relation on measurements from occlusive thrombi formed in our flow chamber experiments, along with other physiological parameter values, the model produced stable dense thrombi on a similar timescale to the experiments. When we parameterized the permeability-porosity relation using lower permeabilities reported by others, bond formation was insufficient to balance drag forces on an early thrombus and keep it intact. Under high shear flow, soluble agonist released by platelets was limited to the thrombus and a boundary layer downstream, thus restricting thrombus growth into the vessel lumen. Adding to the model binding and activation of unactivated platelets through von Willebrand-factor-mediated processes allowed greater growth and made agonist-induced activation more effective.

A Potential Approach to Compensate the Gas Interference for the Analysis of NO by a Non-dispersive Infrared Technique.

Interference is a pivotal issue of a non-dispersive infrared (NDIR) sensor and analyzer. Therefore, the main contribution of this study is to introduce a potential method to compensate for the interference of the NDIR analysis. A potential method to compensate for the interference of a nitric oxide (NO) NDIR analyzer was developed. Double bandpass filters (BPFs) with HITRAN (high-resolution transmission molecular absorption database)-based wavelengths were used to create an ultranarrow bandwidth, where there were least-interfering effects with respect to the coal-fired power plant emission gas compositions. Key emission gases from a coal-fired power plant, comprising carbon monoxide (CO), NO, sulfur dioxide (SO2), nitrogen dioxide (NO2), carbon dioxide (CO2), and water (H2O) (in the form of vapor), were used to investigate the gas interference. The mixtures of those gases were also used to investigate the performance of the double BPFs. We found that CO, CO2, SO2, and H2O significantly affected the detection of NO when a commercial, single narrow BPF was used. In contrast, the double BPFs could remove the interference of CO, NO2, SO2, and CO2 in terms of their concentrations. In the case of H2O, the filter performed well until a level of 50% relative humidity at 25 °C. Moreover, the signal-to-noise ratio of the analyzer was approximately 10 when the double BPFs were applied. In addition, the limit of detection of the analyzer with the double BPFs was approximately 4 ppm, whereas that with the commercial one was 1.3 ppm. Therefore, double BPFs could be used for an NO NDIR analyzer instead of a gas filter correlation to improve the selectivity of the analyzer under the condition of a known gas composition, such as a coal-fired power plant. However, the sensitivity of the analyzer would be decreased.

Platelet α-granules are required for occlusive high-shear-rate thrombosis.

von Willebrand factor (VWF) is essential for the induction of arterial thrombosis. In this study, we investigated the critical role of platelet VWF in occlusive thrombosis formation at high shear in mice that do not express platelet VWF (Nbeal2-/-). Using in silico modeling, in vitro high-shear microfluidics, and an in vivo Folts model of arterial thrombosis we reproduced the platelet dynamics that occur under pathological flow in a stenosed vessel. Computational fluid dynamics (CFDs) simulated local hemodynamics in a stenosis based on arterial geometries. The model predicted shear rates, time course of platelet adhesion, and time to occlusion. These predictions were validated in vitro and in vivo. Occlusive thrombosis developed in wild-type control mice that had normal levels of plasma VWF and platelet VWF in vitro and in vivo. Occlusive thrombosis did not form in the Nbeal2-/- mice that had normal plasma VWF and an absence of platelet VWF. Occlusive thrombosis was corrected in Nbeal2-/- microfluidic assays by the addition of exogenous normal platelets with VWF. Combining model and experimental data, we demonstrated the necessary requirement of platelet VWF in α-granules in forming an occlusive thrombus under high shear. These results could inspire new pharmacological targets specific to pathological conditions and prevent arterial thrombosis.

Occlusive thrombosis in arteries.

Thrombus formation in major arteries is life threatening. In this review article, we discuss how an arterial thrombus can form under pathologically high shear stresses, with bonding rates estimated to be the fastest K o n values in biochemistry. During occlusive thrombosis in arteries, the growth rate of the thrombus explodes to capture a billion platelets in about 10 min. Close to 100% of all platelets passing the thrombus are captured by long von Willebrand factor (vWF) strands that quickly form tethered nets. The nets grow in patches where shear stress is high, and the local concentration of vWF is elevated due to α -granule release by previously captured platelets. This rapidly formed thrombus has few red blood cells and so has a white appearance and is much stronger and more porous than clots formed through coagulation. Understanding and modeling the biophysics of this event can predict totally new approaches to prevent and treat heart attacks and strokes.

Shear-induced platelet aggregation: 3D-grayscale microfluidics for repeatable and localized occlusive thrombosis.

Atherothrombosis leads to complications of myocardial infarction and stroke as a result of shear-induced platelet aggregation (SIPA). Clinicians and researchers may benefit from diagnostic and benchtop microfluidic assays that assess the thrombotic activity of an individual. Currently, there are several different proposed point-of-care diagnostics and microfluidic thrombosis assays with different design parameters and end points. The microfluidic geometry, surface coatings, and anticoagulation may strongly influence the precision of these assays. Variability in selected end points also persists, leading to ambiguous results. This study aims to assess the effects of three physiologically relevant extrinsic design factors on the variability of a single end point to provide a quantified rationale for design parameter and end-point standardization. Using a design of experiments approach, we show that the methods of channel fabrication and collagen surface coating significantly impact the variability of occlusion time from porcine whole blood, while anticoagulant selection between heparin and citrate did not significantly impact the variability. No factor was determined to significantly impact the mean occlusion time within the assay. Occlusive thrombus was found to consistently form in the first third (333 µm) of the high shear zone and not in the shear gradient regions. The selection of these factors in the design of point-of-care diagnostics and experimental SIPA assays may lead to increased precision and specificity in high shear thrombosis studies.

Effects of Water Removal Devices on Ambient Inorganic Air Pollutant Measurements.

Water vapor is a pivotal obstacle when measuring ambient air pollutants. The effects of water vapor removal devices which are called KPASS (Key-compound PASSer) and Cooler. On the measurement of O3, SO2, and CO at ambient levels were investigated. Concentrations of O3, SO2, and CO were 100 ppb, 150 ppb, and 25 ppm, respectively. The amount of water vapor varied at different relative humidity levels of 30%, 50%, and 80% when the temperature was 25 °C and the pressure was 1 atm. Water vapor removal efficiencies and recovery rates of target gases were also determined. The KPASS showed a better performance than the Cooler device, removing 93.6% of water vapor and the Cooler removing 59.2%. In terms of recovery, the KPASS showed a better recovery of target gases than the Cooler. Consequently, it is suggested that the KPASS should be an alternative way to remove water vapor when measuring O3, SO2, and CO.