Investigating thrombin-loaded fibrin in whole blood clot microfluidic assay via fluorogenic peptide.

During clotting under flow, thrombin rapidly generates fibrin, whereas fibrin potently sequesters thrombin. This co-regulation was studied using microfluidic whole blood clotting on collagen/tissue factor, followed by buffer wash, and a start/stop cycling flow assay using the thrombin fluorogenic substrate, Boc-Val-Pro-Arg-AMC. After 3 min of clotting (100 s-1) and 5 min of buffer wash, non-elutable thrombin activity was easily detected during cycles of flow cessation. Non-elutable thrombin was similarly detected in plasma clots or arterial whole blood clots (1000 s-1). This thrombin activity was ablated by Phe-Pro-Arg-chloromethylketone (PPACK), apixaban, or Gly-Pro-Arg-Pro to inhibit fibrin. Reaction-diffusion simulations predicted 108 nM thrombin within the clot. Heparin addition to the start/stop assay had little effect on fibrin-bound thrombin, whereas addition of heparin-antithrombin (AT) required over 6 min to inhibit the thrombin, indicating a substantial diffusion limitation. In contrast, heparin-AT rapidly inhibited thrombin within microfluidic plasma clots, indicating marked differences in fibrin structure and functionality between plasma clots and whole blood clots. Addition of GPVI-Fab to blood before venous or arterial clotting (200 or 1000 s-1) markedly reduced fibrin-bound thrombin, whereas GPVI-Fab addition after 90 s of clotting had no effect. Perfusion of AF647-fibrinogen over washed fluorescein isothiocyanate (FITC)-fibrin clots resulted in an intense red layer around, but not within, the original FITC-fibrin. Similarly, introduction of plasma/AF647-fibrinogen generated substantial red fibrin masses that did not penetrate the original green clots, demonstrating that fibrin cannot be re-clotted with fibrinogen. Overall, thrombin within fibrin is non-elutable, easily accessed by peptides, slowly accessed by average-sized proteins (heparin/AT), and not accessible to fresh fibrinogen.

Gabapentin as Add-on Therapy to Trihexyphenidyl in Children with Dyskinetic Cerebral Palsy: A Randomized, Controlled Trial.

OBJECTIVE: To compare the efficacy of gabapentin as add-on therapy to trihexyphenidyl in the treatment of children with dyskinetic cerebral palsy (CP). METHODS: An open-labelled, randomized, controlled trial was conducted among children aged 3-9 y with dyskinetic CP [Gross Motor Functional Classification System (GMFCS) 4-5]. Participants were assigned into two groups: gabapentin with trihexyphenidyl (n = 30) and trihexyphenidyl alone (n = 30). Dyskinesia Impairment Scale (DIS), Dystonia Severity Assessment Plan (DSAP), and International Classification of Functioning, Disability, and Health-Children and Youth Version (ICF-CY) were measured at baseline, 4 and 12 wk. RESULTS: There was significant reduction in baseline dystonia in both the groups (DIS: p < 0.001; DSAP: p = 0.007; ICF-CY: p < 0.001) but when data were compared between the groups, there was no significant difference in the severity of dystonia at 4 wk and at 12 wk (DIS: p = 0.09; DSAP: p = 0.49; ICF-CY: p = 0.25). Constipation was the commonest side effect observed in both the groups [3 (11.5%) vs. 4 (14.3%)]. CONCLUSION: Trihexyphenidyl alone is as effective as combination of gabapentin with trihexyphenidyl in decreasing the severity of dystonia at 12 wk. Hence, there is no added benefit of gabapentin as add-on therapy for dystonia among children with dyskinetic CP. TRIAL REGISTRATION: CTRI/2019/04/018603.

A three-dimensional multiscale model for the prediction of thrombus growth under flow with single-platelet resolution.

Modeling thrombus growth in pathological flows allows evaluation of risk under patient-specific pharmacological, hematological, and hemodynamical conditions. We have developed a 3D multiscale framework for the prediction of thrombus growth under flow on a spatially resolved surface presenting collagen and tissue factor (TF). The multiscale framework is composed of four coupled modules: a Neural Network (NN) that accounts for platelet signaling, a Lattice Kinetic Monte Carlo (LKMC) simulation for tracking platelet positions, a Finite Volume Method (FVM) simulator for solving convection-diffusion-reaction equations describing agonist release and transport, and a Lattice Boltzmann (LB) flow solver for computing the blood flow field over the growing thrombus. A reduced model of the coagulation cascade was embedded into the framework to account for TF-driven thrombin production. The 3D model was first tested against in vitro microfluidics experiments of whole blood perfusion with various antiplatelet agents targeting COX-1, P2Y1, or the IP receptor. The model was able to accurately capture the evolution and morphology of the growing thrombus. Certain problems of 2D models for thrombus growth (artifactual dendritic growth) were naturally avoided with realistic trajectories of platelets in 3D flow. The generalizability of the 3D multiscale solver enabled simulations of important clinical situations, such as cylindrical blood vessels and acute flow narrowing (stenosis). Enhanced platelet-platelet bonding at pathologically high shear rates (e.g., von Willebrand factor unfolding) was required for accurately describing thrombus growth in stenotic flows. Overall, the approach allows consideration of patient-specific platelet signaling and vascular geometry for the prediction of thrombotic episodes.

Thrombosis and Hemodynamics: external and intrathrombus gradients.

Distinct from dilute, isotropic, and homogeneous reaction systems typically used in laboratory kinetic assays, blood is concentrated, two-phase, flowing, and highly anisotropic when clotting on a surface. This review focuses on spatial gradients that are generated and can dictate thrombus structure and function. Novel experimental and computational tools have recently emerged to explore reaction-transport coupling during clotting. Multiscale simulations help bridge tissue length scales (the coronary arteries) to millimeter scales of a growing clot to the microscopic scale of single-cell signaling and adhesion. Microfluidic devices help create and control pathological velocity profiles, albeit at a low Reynolds number. Since rate processes and force loading are often coupled, this review highlights prevailing convective-diffusive transport physics that modulate cellular and molecular processes during thrombus formation.

Real-World Evidence and Glycemic Improvement Using Dexcom G6 Features.

Background: Optional features of continuous glucose monitoring (CGM) systems empower patients and caregivers to understand and manage diabetes in new ways. We examined associations between use of optional features, demographics, and glycemic outcomes. Methods: Retrospective cohort studies were performed with data from US-based users of the G6 CGM System (Dexcom, Inc.). For all cohorts, data included sensor glucose values (SGVs). In separate cohorts, use of alert features (for hyperglycemia, existing hypoglycemia, and impending hypoglycemia), remote data sharing feature (Share), software for retrospective pattern analysis (CLARITY), "virtual assistant" feature that announces the current SGV and trend in response to a spoken request were assessed. Descriptive statistics were used to summarize feature set utilization patterns and relate them to glycemic outcomes. Results: Most individual features were consistently adopted by high proportions of G6 users. Threshold SGVs chosen for activating hyperglycemia and hypoglycemia alerts varied with age and were higher among the youngest and oldest patients. Use of the Share feature was more common among young patients and those with type 1 diabetes. Individuals who used more of the alert and notification features had more favorable glycemic outcomes, including time in range (TIR), than those who used fewer. More extensive engagement with CLARITY notifications was associated with higher TIR. Frequent use of the virtual assistant feature was associated with higher TIR and lower mean SGV. Conclusions: Optional features of the G6 CGM system are acceptable to and appear to benefit patients who use them. Different levels of engagement suggest that demographics and personal circumstances play a role in how patients and caregivers use CGM features to help manage diabetes.

Longitudinally Extensive Transverse Myelitis (LETM) Following Varicella Infection in a 13-Year Immunocompetent Child.

Varicella infection in children is a common self-limited illness with neurological complications in less than 0.1% of cases. Longitudinally extensive transverse myelitis (LETM) is uncommon in children following infection with varicella-zoster virus. We describe a case of 13-yr immunocompetent girl with LETM following varicella infection shown a dramatic clinical response to a combination of acyclovir and pulse steroids.