Global Thrombosis Test: Occlusion by Coagulation or SIPA?

The global thrombosis test (GTT) is a point of care device that tests thrombotic and thrombolytic status. The device exposes whole blood flow to a combination of both high and low shear stress past and between ball bearings potentially causing thrombin and fibrin formation. The question arises as to whether thrombosis in the GTT is dominated by coagulation-triggered red clot or high shear-induced white clot.

Negatively charged nanoparticles of multiple materials inhibit shear-induced platelet accumulation.

Platelet accumulation by VWF under high shear rates at the site of atherosclerotic plaque rupture leads to myocardial infarction and stroke. Current anti-platelet therapies remain ineffective for a large percentage of the population, while presenting significant risks for bleeding. We explore a novel way to inhibit arterial thrombus formation.

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.

Inhibition of high shear arterial thrombosis by charged nanoparticles.

Platelet accumulation under high shear rates at the site of atherosclerotic plaque rupture leads to myocardial infarction and stroke. Current antiplatelet therapies remain ineffective within a large percentage of the population, while presenting significant risks for bleeding. We explore a novel way to inhibit arterial thrombus formation by biophysical means without the use of platelet inactivating drugs.

Hemodynamic studies of platelet thrombosis using microfluidics.

Platelets contribute to thrombus formation in a variety of ways. Platelet adhesion, activation, and thrombus growth depend greatly on the type of hemodynamic environment surrounding an inciting event. Microfluidic systems may be used to explore these relationships.

Ex Vivo Assessment of a Parabolic-Tip Inflow Cannula for Pediatric Continuous-Flow VADs.

To address the challenge of unloading the left ventricle during pediatric mechanical circulatory support using next-generation rotary blood pumps, a novel inflow cannula was developed. This unique inflow cannula for pediatric, continuous-flow, left ventricular assist devices (VADs) with a parabolic-shaped inlet entrance was evaluated alongside a bevel-tip and fenestrated-tip cannula via an ex vivo, isolated-heart experimental setup. Performance was characterized using two clinical scenarios of over-pumping and hypovolemia, created by varying pump speed and filling preload pressure, respectively, at ideal and off-axis cannula placement to assess ventricular unloading and positional sensitivity.

Evolution of multi-well pad development and influence of well pads on environmental violations and wastewater volumes in the Marcellus shale (USA).

A majority of well pads for unconventional gas wells that are drilled into the Marcellus shale (northeastern USA) consist of multiple wells (in some cases as many as 12 wells per pad), yet the influence of the evolution of well pad development on the extent of environmental violations and wastewater production is unknown. Although the development of multi-well pads (MWP) at the expense of single well pads (SWP) has been mostly driven by economic factors, the concentrated nature of drilling activities from hydraulic fracturing and horizontal drilling operations on MWP suggests that MWP may create less surface disturbance, produce more volumes of wastewater, and generate more environmental violations than SWP. To explore these hypotheses, we use geospatial techniques and statistical analyses (i.

Muscarinic agonists and antagonists: effects on gastrointestinal function.

Muscarinic agonists and antagonists are used to treat a handful of gastrointestinal (GI) conditions associated with impaired salivary secretion or altered motility of GI smooth muscle. With regard to exocrine secretion, the major muscarinic receptor expressed in salivary, gastric, and pancreatic glands is the M₃ with a small contribution of the M₁ receptor. In GI smooth muscle, the major muscarinic receptors expressed are the M₂ and M₃ with the M₂ outnumbering the M₃ by a ratio of at least four to one.

Analysis of functional responses at G protein-coupled receptors: estimation of relative affinity constants for the inactive receptor state.

We describe a modification of receptor theory that enables the estimation of relative affinity constants for the inactive state of a G protein-coupled receptor. Our approach includes the traditional parameters of observed affinity (K(obs)) and efficacy (fraction of ligand-receptor complex in the active state, ε) and introduces the concept of the fraction of the ligand-receptor complex in the inactive state (intrinsic inactivity, ε(i)). The relationship between receptor activation and the ligand concentration is known as the stimulus, and the operational model expresses the response as a logistic function of the stimulus.

Analysis of agonism and inverse agonism in functional assays with constitutive activity: estimation of orthosteric ligand affinity constants for active and inactive receptor states.

We describe a modification of receptor theory for the estimation of observed affinities (K(obs)) and relative efficacies of orthosteric ligands in functional assays that exhibit constitutive activity. Our theory includes parameters for the fractions of the occupied receptor population in the active (intrinsic efficacy, ε) and inactive (ε(i)) states and analogous parameters for the fractions of the free receptor population in the active (ε(sys)) and inactive (ε(i-sys)) states. The total stimulus represents the summation of the active states of the free and occupied receptor populations.

The guinea pig ileum lacks the direct, high-potency, M(2)-muscarinic, contractile mechanism characteristic of the mouse ileum.

We explored whether the M(2) muscarinic receptor in the guinea pig ileum elicits a highly potent, direct-contractile response, like that from the M(3) muscarinic receptor knockout mouse. First, we characterized the irreversible receptor-blocking activity of 4-DAMP mustard in ileum from muscarinic receptor knockout mice to verify its M(3) selectivity. Then, we used 4-DAMP mustard to inactivate M(3) responses in the guinea pig ileum to attempt to reveal direct, M(2) receptor-mediated contractions.

Selectivity of agonists for the active state of M1 to M4 muscarinic receptor subtypes.

We measured the intrinsic relative activity (RA(i)) of muscarinic agonists to detect possible selectivity for receptor subtypes and signaling pathways. RA(i) is a relative measure of the microscopic affinity constant of an agonist for the active state of a GPCR expressed relative to that of a standard agonist. First, we estimated RA(i) values for a panel of agonists acting at the M(4) muscarinic receptor coupled to three distinct G-protein pathways: G(i) inhibition of cAMP accumulation, G(s) stimulation of cAMP accumulation, and G alpha(15) stimulation of phosphoinositide hydrolysis.

Two-state models and the analysis of the allosteric effect of gallamine at the M2 muscarinic receptor.

We measured the influence of gallamine on the functional responses and binding properties of selected agonists at the M(2) muscarinic receptor and analyzed the data within the context of the allosteric ternary complex model. Our analysis showed that gallamine modified agonist affinity without influencing efficacy. To explain this behavior, we investigated the allosteric ternary complex model at a deeper level of analysis to assess allosterism in terms of the differential affinity of gallamine for ground and active states of the receptor.

Estimation of agonist activity at G protein-coupled receptors: analysis of M2 muscarinic receptor signaling through Gi/o,Gs, and G15.

We developed novel methods for analyzing the concentration-response curve of an agonist to estimate the product of observed affinity and intrinsic efficacy, expressed relative to that of a standard agonist. This parameter, termed intrinsic relative activity (RA(i)), is most applicable for the analysis of responses at G protein-coupled receptors. RA(i) is equivalent to the potency ratios that agonists would exhibit in a hypothetical, highly sensitive assay in which all agonists behave as full agonists, even those with little intrinsic efficacy.

The M2 muscarinic receptor mediates contraction through indirect mechanisms in mouse urinary bladder.

We investigated the contractile role of M2 muscarinic receptors in mouse urinary bladder. When measured in the absence of other agents, contractions elicited to the muscarinic agonist oxotremorine-M exhibited properties consistent with that expected for an M3 response in urinary bladder from wild-type and M2 knockout (KO) mice. Evidence for a minor M2 receptor-mediated contraction was revealed by a comparison of responses in M3 knockout and M2/M3 double knockout mice.

Comparison of the antimuscarinic action of p-fluorohexahydrosiladifenidol in ileal and tracheal smooth muscle.

We investigated the ability of the muscarinic antagonist p-fluorohexahydrosiladifenidol to inhibit muscarinic agonist-induced contractions and phosphoinositide hydrolysis in the guinea pig ileum and trachea. This antagonist displayed higher potency at blocking oxotremorine-M-induced contractions of the ileum compared with those of the trachea. When estimated using a simple model for competitive antagonism, the observed dissociation constant of p-fluorohexahydrosiladifenidol exhibited approximately 12-fold higher potency in the ileum compared with the trachea.

Muscarinic agonist-mediated heterologous desensitization in isolated ileum requires activation of both muscarinic M2 and M3 receptors.

We investigated the subtypes of the muscarinic receptor mediating short-term heterologous desensitization in the isolated ileum. Treatment of the ileum from C57BL/6 mice with acetylcholine (30 microM) for 20 min caused a subsequent decrease in contractile sensitivity to both prostaglandin F2alpha (PGF2alpha) and the muscarinic agonist, oxotremorine-M. This subsensitivity was characterized by 7- and 3-fold increases in the EC50 values of the agonists, respectively, with no significant effect on the maximal response.

Comparison of the pharmacological antagonism of M2 and M3 muscarinic receptors expressed in isolation and in combination.

We compared the binding properties of selective muscarinic antagonists with their potencies for antagonizing muscarinic responses in Chinese hamster ovary (CHO) cells expressing M(2) and M(3) muscarinic receptors in combination and in isolation. When measured by the competitive displacement of [3H]N-methylscopolamine binding to CHO cells expressing both M(2) and M(3) muscarinic receptors (CHO M(2)+M(3) cells), the competition curves of the subtype-selective muscarinic antagonists were consistent with a two-site model. One site exhibited binding properties identical to those of CHO M(2) cells, whereas the other site exhibited properties like those of CHO M(3) cells.

Increased relaxant action of forskolin and isoproterenol against muscarinic agonist-induced contractions in smooth muscle from M2 receptor knockout mice.

The ability of forskolin and isoproterenol to inhibit the contractile action of the muscarinic agonist, oxotremorine-M, was investigated in smooth muscle from wild-type and M(2) muscarinic receptor knockout mice. Forskolin (5.0 micro M) caused a significant reduction in the contractile activity of oxotremorine-M in ileum, trachea, and urinary bladder from both wild-type and M(2) muscarinic receptor knockout mice.